Fermentation methods

Abstract

The present disclosure relates to methods, devices and systems for exposing microorganisms or compositions comprising functional proteins to an oscillating electric field substantially free of a magnetic component in order to improve stress responses, survival and metabolite or protein production. In embodiments, a controller drives one or more insulated emitters with a true AC signal or pulsed DC signal, in stepped, swept or shuffled patterns of an electric field oscillating at one or more frequencies in the range 210 Hz to 99 kHz. Exposure to the field provides surmounting energy that enhances protein breathing, catalysis, and fundamental functions of multiprotein complexes including transcription factors and ribosomes, thereby improving stress resistance of microorganisms, their viability during fermentation, drying, storage, transport, and / or increasing production of functional or therapeutic proteins, metabolites or microbial lysates. The technology is applicable to manufacture of probiotics, therapeutics, metabolites, food, alcohol, feed and bioenergy, cosmetics, nutraceuticals and biopharmaceuticals..

Description

FERMENTATION METHODSPRIORITY DOCUMENT

[0001] The present application claims priority from Australian Provisional Patent Application No. 2024903969 titled “FERMENTATION METHODS” and filed on 29 November 2024, the content of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates to the use of electric fields to modify the activity of microorganisms and functional proteins.BACKGROUND

[0003] Enzymes and microorganisms act as catalysts in industries such as biotechnology, pharmaceuticals, and food production. Enzymes accelerate chemical reactions by lowering activation energy. In alcohol production — like fermentation for beer, wine, and sake — they convert sugars into ethanol and flavour compounds. Fermentation relies on both enzyme activity and the growth of microorganisms like yeast and bacteria to produce ethanol and desired flavours.

[0004] In bioethanol production, enzymes break down biomass into fermentable sugars, which microorganisms convert into ethanol. Enhancing enzyme efficiency and microbial growth can lead to higher yields and cost-effective processes. Similarly, in probiotic production, the enzymes within microorganisms and their ability to multiply are important for growth and metabolic activity, affecting the quality of the final product.

[0005] Enzymes and microorganisms are used in food production processes such as baking, cheese making, and meat tenderisation to improve texture, flavour, and nutritional value. In pharmaceuticals, they are essential for antibiotic production, assisting in synthesising molecules that combat bacterial infections. An enzyme may be produced in an incubator and has utility in its raw or purified form when applied to a task post-incubation.

[0006] Traditionally, optimising the catalytic rate of enzymes and enhancing microbial growth across these diverse applications has involved adjusting various reaction conditions. Common strategies include altering the concentration of substrates, enzymes, and microorganisms; modifying temperature and pH levels; changing solvent or buffer systems; and adjusting atmospheric pressure, such as by adjusting total depth of an incubator, by adjusting pressure using a compressed gas, or by removing gases from the headspace of the incubator. Efforts have also been made to enhance enzyme performance by modifyingtheir amino acid sequences or manipulating their folding states to improve stability and activity, as well as genetically modifying microorganisms to enhance their growth and productivity.

[0007] While these methods can enhance enzyme activity and microbial proliferation, they can come with limitations and drawbacks. Adjusting substrate, enzyme, or microorganism concentrations can be cost-prohibitive, especially on an industrial scale. Altering temperature or pH may lead to enzyme denaturation, microbial stress, or unwanted side reactions that compromise product quality. Modifying the enzyme's amino acid sequence or the genetic makeup of microorganisms requires complex genetic engineering techniques and can raise regulatory concerns, particularly in food and pharmaceutical applications where safety and compliance are important.

[0008] Despite efforts to enhance enzyme activity and microbial proliferation, there remains a need for methods that can maximise the output of an enzyme's catalytic action and / or enhance the growth of microorganisms without altering these conventional parameters. Such advancements would provide benefits by enhancing efficiency and productivity without the associated costs and complexities of traditional optimisation methods. Addressing this unmet need would represent a contribution to the industry, offering sectors involved in alcohol production, bioethanol generation, biofuel production, soluble fibre production, insoluble fibre production, partially soluble fibre production, digestible fibre production, lignocellulose-derivative production, enzyme production, probiotic manufacturing, prebiotic manufacturing, food production, and antibiotic production the ability to improve enzyme-mediated and microorganism -driven processes in a more practical and economically viable manner.

[0009] There is thus a need to provide improved methods to modify the activity of microorganisms and functional proteins.SUMMARY

[0010] The present disclosure arises from research into the use of an electric field to modify the activity of functional proteins and microorganisms. Specifically, the inventors have found that the activity of functional proteins and microorganisms may be affected by an electric field, that is essentially free of a magnetic component, in which the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0011] In an aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0012] In an aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, during a fermentation process of the microorganisms to produce one or more of: dairy products, yoghurt, cheese, a probiotic, a feed, an encapsulated probiotic, probiotic manufacturing, and a probiotic packaged with excipients, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0013] In an aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, during a fermentation process of the microorganisms, and of improving survival of the microorganisms during storage or transport, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0014] In an aspect, there is provided a method of treating a composition to provide surmounting energy to improve protein breathing during a fermentation of microorganisms to increase production of one or more of: a functional protein, a therapeutic protein, a bacteriocin, an antibiotic protein, an insulin, a growth hormone, a hormone replacement, a functional part of an antibody, and other functional protein in the microorganisms, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0015] In an aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz.

[0016] In certain embodiments of the methods of the disclosure, the generated electric field is generated by an AC signal, or a pulsed DC signal with a peak-to-peak voltage and a duty cycle; and wherein the stress resistance, or the longevity, or both, of the microorganisms is used to reduce loss of viable cell count, or loss of stress resistance or longevity during one or more of: a fermentation, a freeze- drying, a packaging and a long-term storage. In certain embodiments of the methods of the disclosure, the AC signal, or the pulsed DC signal, with a waveform, is applied to at least one voltage terminal or antenna, the AC signal or the pulsed DC signal comprising a single frequency, a set of frequencies, afrequency sweep, or a stochastic or shuffled progression of frequencies in the range of about 210 Hz to 99 kHz; and the AC signal or pulsed DC signal operating at a peak-to-peak voltage between 1 V and 45 kV. In certain embodiments of the methods of the disclosure, an integrated monitor is embodied in or connected to the controller to monitor a presence of a signal in the antenna or antennae, comprising monitoring a waveform of the signal, an amplitude of the signal, or a frequency of the signal.

[0017] In certain embodiments of the methods of the disclosure, the progression of frequencies is: a stepped quartic sweep defined by a formula F(t)=at4+bt+ct2+dt+e that expresses frequency as a function of time t, and with a, b, c, d, e being coefficients; or a curved sweep, or a linear sweep, or is a stepped non-quartic sweep progressing upward in frequency or downward in frequency, or is a non-stepped sweep progressing upward in frequency or downward in frequency, the sweep taking a fixed period time before rebeginning. In certain embodiments of the methods of the disclosure, the progression of frequencies is defined using a lookup table or subsets of a lookup table.

[0018] In certain embodiments of the methods of the disclosure, the microorganisms are cultivated in a medium optimised to maintain a selected dissolved oxygen concentration, with or without pH control by an intermittent compensatory dosing with alkali or acid. In certain embodiments of the methods of the disclosure, the microorganisms are selected from one or more of: eubacteria, fungi, actinomycetes, probiotic yeast, probiotic bacteria, Lactobacilli, Lactobacillus species, Lactobacillus-\i e species, Bifidobacterium species, Bifidobacierium-\\ c species, Saccharomyces spp., and other probiotic species, strains, and genera, which together comprise the probiotic group, being a non-taxonomic grouping.

[0019] In certain embodiments of the methods of the disclosure, each of the at least one voltage terminal or antenna comprises one or more of (i) insulated conductive material helically wound around a non-conducting material; (ii) an insulated conductive terminus material of any shape such as an insulated conductive plate; or (iii) two or more insulated conductive plates separated by a non-conducting material or by the composition; to provide surmounting energy to facilitate increased protein breathing in the cells’ functional proteins. In certain embodiments of the methods of the disclosure, the electric field sweeps through sequential frequencies in a range of about 1400 Hz to about 4540 Hz or sequential frequencies in the range of about 210 Hz to about 99 kHz.

[0020] In another aspect, there is provided an electric field-generating device to improve a stress resistance, a longevity, or both, of microorganisms, the device comprising: a controller to generate an electric field; wherein the generated electric field is oscillating at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is substantially free of a magnetic field; and wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0021] In certain embodiments, the controller is configured to generate a waveform and supply a modulated voltage to at least a first and a second voltage terminals or antennae, wherein at least the first and second voltage terminals or antennae are controlled by an one or more output channels configured to supply at least a first and a second signal that are of the same phase as each other or are phase-shifted relative to each other; or to control the at least a first and a second voltage terminals to increase field exposure and uniformity of exposure to microbial cells; or both. In certain embodiments, the modulated voltage supplied to at least the first voltage terminal or antenna is in a range of 1 V RMS to 40 kV RMS, and wherein the controller or at least the first voltage terminal or antenna, or both, comprise a means to prevent formation of the magnetic component to the generated electric field.

[0022] In another aspect, there is provided an electric field-generating device configured to perform the methods of the disclosure.

[0023] In another aspect, there is provided an electric field-generating device configured to perform the methods of the disclosure comprising one or more reradiation emitter or emitters, that is or are separate from the at least one electrically -insulated voltage terminal or antenna and separate from any other electrically-insulated voltage terminals or antennae.

[0024] In another aspect, there is provided an electric field-generating system for treating a composition, comprising: a controller configured to generate a waveform and supply a modulated voltage to at least a first voltage terminal or antenna of an emitter; wherein the modulated voltage is supplied to at least the first electrically -insulated voltage terminal at one frequency or more frequency in a range of about 210 Hz to 99 kHz, to generate an electric field substantially free of a magnetic component; and wherein the controller is configured to supply an AC signal or a pulsed DC signal; and a container configured to contain the composition, wherein at least the first electrically -insulated voltage terminal positioned within the container to optimise exposure of the composition to the generated electric field.

[0025] In certain embodiments, the container is configured to perform a batch culture, a fed-batch, a continuous culture, or a repeated fed-batch or semi -continuous culture, a perfusion process or a continuous or non-continuous feeding process and, in use, supplies a nutrient at a selected time within a culturing process, to augment the nutritional status of microorganisms to increase or maximise obtainable viable cell density per gram of dry cell. In certain embodiments, the container is separate from a fermenter vessel but connected by liquid flow to a fermenter vessel by a conduit, and wherein the container or conduit facilitates recirculation of a composition past an electrically -insulated electric field emitter and then back to a fermenter vessel for continued fermentation, such that the composition is exposed to a near electric field.

[0026] In another aspect, there is provided an electric field-generating system configured to perform the methods of the disclosure or configured to employ the device of the disclosure.

[0027] In another aspect, there is provided an electric field of the device of the disclosure, the methods of the disclosure, or the system of the disclosure, used in conjunction with a combined-semi-electric field such as the combined-semi-electric field emitted from a reradiation coil or a combined-semi-electric field emitted from another type of field emission technology, to achieve an industrial benefit for stress resistance and viability of a microorganism, specifically by means of the electric component of the field providing surmounting energy to influence and improve protein breathing in a microorganism.

[0028] In another aspect, there is provided an electric field of the device of the disclosure, the methods of the disclosure, or the system of the disclosure, used in conjunction with a combined-semi-electric field such as emitted from another type of field emission technology, to achieve an industrial benefit for stress resistance and viability of a microorganism without ohmic heating, where the improvement in stress resistance and viability is specifically by means of the electric component of the field influencing protein breathing in a microorganism without involvement of ohmic heating.

[0029] In another aspect, there is provided an n electric field of the device of the disclosure, the methods of the disclosure, or the system of the disclosure, used in conjunction with a combined-semielectric field such as emitted from another type of field emission technology such as an electrode or electrodes, or an uninsulated current carrying loop or an insulated current carrying loop, to achieve an industrial benefit for stress resistance and viability of a microorganism without electrochemical damage, where the improvement in stress resistance and viability is specifically by means of the electric component of the near electric field influencing protein breathing in a microorganism despite the presence of potentially damaging field emission technology.

[0030] In another aspect, there is provided a nutritional supplement, a food, a supplement supporting gut health, a supplement supporting digestive health, a supplement containing beneficial therapeutic proteins derived from a microorganism, or a supplement containing beneficial therapeutic metabolites derived from a microorganism, prepared by fermenting with the device of the disclosure, the methods of the disclosure, or the system of the disclosure,.

[0031] In another aspect, there is provided a skin health or beauty supplement, a bioenergy compound, a pharmaceutical molecule, an enzyme, a prebiotic, an oligosaccharide, a cofactor, a fibre or a biologically derived product prepared by fermenting with the device of the disclosure, the methods of the disclosure, or the system of the disclosure, for the purpose of producing a microbial lysate having skin health or beauty properties.

[0032] In another aspect, there is provided the device of the disclosure, the methods of the disclosure, or the system of the disclosure, for manufacturing a product from microorganisms in a fermenter, or for manufacturing a product being microorganisms themselves using a fermenter, wherein the controller is configured to execute a deterministic sequencing algorithm, and be continuously monitored or data-logged for internal auditing purposes or external auditing purposes, thereby providing a highly repeatable and verifiable exposure regime suitable for GMP (Good Manufacturing Practice) compliance.

[0033] In another aspect, there is provided a metabolite manufactured using the device of the disclosure, the methods of the disclosure, or the system of the disclosure, wherein the metabolite is of cellular origin, and is increased in amount or activity by the electric field providing surmounting energy to a functional protein or to a set of functional proteins that work together for increased production of the metabolite.

[0034] In another aspect, there is provided the device of the disclosure, the methods of the disclosure, or the system of the disclosure, where the emitter is inserted within a bioreactor housing that is in turn inserted or emplaced into a fermenter or container.

[0035] In another aspect, there is provided the device of the disclosure, the methods of the disclosure, or the system of the disclosure, wherein the rise and fall respectively of the waveform are defined using a quartic formula of the form V(t)=at4+bt+ct2+dt+e that expresses voltage V as a function of time t, and with a, b, c, d, e being coefficients that may differ in sign or quantity between the rise and fall equations.BRIEF DESCRIPTION OF DRAWINGS

[0036] Embodiments of the present disclosure will be discussed with reference to the accompanying drawings wherein:

[0037] Figure 1 is a flowchart of the series of experiments. (A) The flowchart for Experiments 1 and 2 shows that, in each case, an untreated wort was divided into two fermenter vessels, Fvl and Fv2. The "treated" fermenter vessel contained the device and method described in this disclosure, while the "control" fermenter vessel did not. All other input and environmental parameters were kept equal between the two vessels. The specifications of the inputs to wort production and the parameters of wort production were consistent across both Experiments 1 and 2. (B) The flowchart for Experiment 3, which differed significantly in design compared to Experiments 1 and 2, illustrates that the wort was not split. Instead, the mash was performed twice under identical inputs and conditions. The only difference was that the treated mash used the device and method of this disclosure, while the control mash did not. Similarly, in Experiment 3a, fermentation was untreated, whereas in Experiment 3b, fermentation was treated using the device and method of this disclosure. Throughout the text, the control part of Experiment 3 is referred to as Experiment 3a, and the treated part as Experiment 3b.

[0038] Figure 2 is a graph showing the progress of fermentation in Experiment 1, indicated by degrees Plato calculated from specific gravity. Degrees Plato varies with sugar content. Data collection and graphing extended to Day 11, representing the active extent of fermentation.

[0039] Figure 3 is a graph showing the progress of fermentation in Experiment 1, indicated by an electrolyte-based pH meter in the brewery. The real-time pH increases when the rate of decrease in specific gravity begins to taper off.

[0040] Figure 4 shows the results of fermentables analysis on Day 8 of Experiment 1. Only fermentables above the organoleptic threshold are presented, and these are above threshold for both control and treated fermentations. Percent reductions (or increases in the case of ethyl -hexanoate) are displayed above each treated bar relative to its control bar. A) High-range concentrations of fermentables. B) Low-range concentrations of fermentables.

[0041] Figure 5 shows the progress of fermentation in Experiment 2, indicated by degrees Plato calculated from specific gravity. Degrees Plato varies with sugar content. Data collection and graphing extended to Day 9, representing the active extent of fermentation.

[0042] Figure 6 shows the progress of fermentation in Experiment 2, as indicated by an electrolytebased pH meter used in the brewery. The real-time pH increases when the rate of decrease in specific gravity begins to taper off. Therefore, pH data collection was continued until Day 14 to detect this pH uptick.

[0043] Figure 7 presents a graph of suspended yeast counts in Experiment 2, determined using a haemocytometer. Cell counts were obtained for both control and treated fermentations. The control fermentation exhibits the typical phases of yeast fermentation: lag phase, exponential growth phase, diauxic shift, respiration, and then a drop-off as stationary -phase cells flocculate. In contrast, the treated fermentation shows elevated suspended yeast counts from Day 4 to Day 5. This demonstrates that cell division may continue unabated during the period that would otherwise be defined as the diauxic shift. The treated fermentation also shows more rapid flocculation at day 7 than the control fermentation.

[0044] Figure 8 shows alcohol production in Experiment 2. Both the treated and control fermentations produce alcohol approximately equally during the exponential growth phase (Days 2 to 4) and during the diauxic shift (Days 4 to 5). Therefore, differences in alcohol production cannot reasonably explain the cell count differences between Days 4 and 5 observed in the preceding figure (Figure 7). The treated fermentation produces slightly more alcohol than the control during the anaerobic respiration period (Days 5 to 6).

[0045] Figure 9 is a graph depicting total sugar content in Experiment 3 for both control and treated samples. The first pair of columns represents the total sugars in the wort, while subsequent columns represent total sugars during fermentation on Days 5 and 8. The treated wort contained 10% more total sugars than the control wort. By Day 5 of fermentation, the total amount of sugars was 7% less in the treated sample compared to the control. On Day 8, the total sugars were 16% less in the treated samplethan in the control. Specifically, the total sugar concentration on Day 8 was 6.3 g / L for the control beer and 5.3 g / L for the treated beer.

[0046] Figure 10 presents a quantitative analysis of selected sugars — specifically saccharides, disaccharides, and a trisaccharide — in the wort from Experiment 3 (both control and treated). In the treated wort, a greater proportion of maltose and maltotriose has been converted to glucose compared to the control. The percent reductions in the treated wort relative to the control were: maltose decreased by 21%, maltotriose by 41%, fructose by 20%, and sucrose by 5%. Conversely, glucose increased by 50% in the treated wort relative to the control.

[0047] Figure 11 presents a graph showing the quantitative analysis of multi -nitrogen amino acids in wort from Experiment 3, comparing the control (3a) and treated (3b) conditions. The three multi -nitrogen amino acids analysed — asparagine, arginine, and glutamine — were significantly elevated in the treated wort (3b) compared to the control (3a). Specifically, asparagine was elevated by 37%, arginine by over 900%, and glutamine by over 300%.

[0048] Figure 12 illustrates the actual increase or decrease (in mg / L) of standard amino acids in treated wort compared to the control in Experiment 3. The data represents the difference between treated and control conditions for each amino acid (treated minus control). See Table 5 and Figure 11 for full values. Only arginine (Arg), asparagine (Asn), and glutamine (Gin) were elevated in the treated wort. Derivation of each bar in the graph above is: treated minus control in each case.

[0049] Figure 13 shows the increase in wort metabolites, analysed via GC-MS, in treated wort compared to control wort in Experiment 3. Fermentable sugars derived from mannans and other valueadding compounds were higher in treated wort.

[0050] Figure 14 displays a graph of suspended yeast cell counts during fermentation in the control (3a) and treated (3b) conditions of Experiment 3, plotted against hours after mashing completion. Cell counts, measured with a hemocytometer, were higher in the treated fermentation (3b) and peaked differently compared to the control (3a). The control fermentation showed distinct phases: Exponential (E), Diauxic Shift (DS), and Respiration (R). The diauxic shift in the control occurred between 48 and 72 hours, consistent with typical anaerobic fermentation. In contrast, the treated fermentation did not exhibit the same plateau in cell counts during this period.

[0051] Figure 15 provides quantitative analysis of selected sugar components in beer, including a monosaccharide, two disaccharides, and a trisaccharide, from Experiment 3. On day 8, yeast in the treated fermentation consumed glucose more efficiently than in the control, despite starting with higher glucose levels due to mash treatment. Glucose was undetectable in both treated and control beers by day 5.Fructose was undetectable on both days 5 and 8i n both treated and control. Percentage reductions on day5 for the treated beer compared to the control were: maltose -19%, maltotriose -26%, glucose 0%, and sucrose 0%. On day 8, reductions were: maltose -33%, maltotriose -33%, glucose -50%, and sucrose 0%. Maltose and maltotriose levels varied initially between treated and control mashes but were utilised at similar rates in both conditions.

[0052] Figure 16 depicts the increase in primary metabolites of treated yeast in Experiment 3, analysed via GC-MS. Data are shown for growth completion (day 5) and stationary phase (day 8) as ratios to control yeast. (A) Metabolites of high-range concentration that were increased in the treated yeast relative to control yeast. (B) Metabolites of low -range concentration that were increased in the treated yeast relative to control yeast. DHAP is dihydroxy acetone phosphate.

[0053] Figure 17 illustrates the increase in fermentable compounds in Experiment 3 beers, exceeding taste or odour thresholds. (A) High-range, (B) Mid-range, and (C) Low-range metabolites are presented. All metabolites, except 2-methylbutyl acetate, were within perceptible taste thresholds. 2-methylbutyl acetate and ethyl hexanoate were within aqueous odour threshold. All metabolites shown were within air odour threshold. Flavour and aroma descriptors for each metabolite are noted in brackets, with percentage increases in the treated compared to the control provided in square brackets.

[0054] Figure 18 shows a functional analysis of the genome-wide upregulation of gene categories in the treated fermentation on day 5 of Experiment 3. The figure, resembling a pie chart (in that the contributions sum to 100), allows for a 0.02 to 0.11 error rate in manual assignment of functional class in Table 11, which comprises the data used to generate the figure. The relative contribution metric is calculated as: 100 x contribution / sum of contributions, which is: 100 x [SQRT (of number of genes in category) x (Avg score of all genes in the category)] / sum of contributions. The square root function takes into account that a functional category may involve many genes, and yet that the contributions of multiple genes to a functional class are rarely additive, rather are each positive yet fractional.

[0055] Figure 19 shows a functional analysis of the genome-wide upregulation of gene categories in the treated fermentation on day 8 / 9 of Experiment 3. The figure, resembling a pie chart (in that the contributions sum to 100), allows for a 0.02 to 0.13 error rate in manual assignment of functional class in Table 12, which comprises the data used to generate the figure. The formula for the relative contribution metric is given beneath Figure 18.

[0056] Figure 20 illustrates beta amylase response to treatment. (A) Demonstrates that beta-amylase generated maltose and maltotriose from dextrin beyond the initial sugar content in the sourced dextrin, or the "No Enzyme No Treatment" sample, the maltose bar represents an LOR of <0.2 g / 100ml rather than a measured value of 0.2 g / 100 ml. The other bars all represent measured amounts. (B) Depicts the percentage increase in maltose and maltotriose due to the treatment, showing the effect of the treatment on the catalytic activity of beta amylase via a 30 minute. Graph B shows the effect of treatment on themaltose and maltotriose concentration following the action of beta amylase on dextrin in percentage relative to control.

[0057] Figure 21 is a schematic diagram of an embodiment of the device of the disclosure.

[0058] Figure 22 is a schematic diagram of another embodiment of the device of the disclosure.

[0059] Figure 23 panel A is a graphical representation of the voltage at a first voltage terminal in an embodiment of the disclosure. Figure 23 panel B is a graphical representation of the voltage at a second voltage terminal in an embodiment of the disclosure.

[0060] Figure 24 is a diagram of an embodiment of the device of the disclosure having spaced apart first and second voltage terminals.

[0061] Figure 25 is a diagram of an embodiment of the device of the disclosure having spaced apart first and second voltage terminals, which are separated by a non-conducting material.

[0062] Figure 26 is a diagram of an embodiment of the device of the disclosure in which the first and second voltage terminals comprise conductive material (e.g., insulated or uninsulated) helically wound around a non-conducting material.

[0063] Figure 27 is a diagram of an embodiment of the device of the disclosure, in which a housing is shown.

[0064] Figure 28 is a diagram of an embodiment of the system of the disclosure.

[0065] Figure 29 is a diagram of an embodiment of the system of the disclosure, in which the container is a flow cell.

[0066] Figure 30 is a diagram of another embodiment of the system of the disclosure, in which the container is a flow cell.

[0067] Figure 31 is a schematic diagram of an embodiment of the system of the disclosure utilising a single voltage terminal for treating a composition.

[0068] Figure 32 is a schematic diagram of an embodiment of the device of the disclosure having the core controller used in Experiments 5 and 6.

[0069] Figure 33 is a graph illustrating the definitive carrier waveform pulse shape, anchored to the 81.3 microsecond transition time and 98 V amplitude.

[0070] Figure 34 is a schematic diagram of an embodiment of the device of the disclosure, in which the controller utilises an Arbitrary Waveform Generator (AWG) to physically simulate the signal supplied to the emitter in Experiments 5 & 6.

[0071] Figure 35 is a schematic diagram of an embodiment of the device of the disclosure having integrated monitoring capability.

[0072] Figure 36 is a schematic diagram of another embodiment of the device of the disclosure having the circuit topology of the LC fdters in Experiment 7.

[0073] Figure 37 is a diagram of another embodiment of the system of the disclosure having a flow module for continuous treatment of a composition.

[0074] In the following description, like reference characters designate like or corresponding parts throughout the figures.DESCRIPTION OF EMBODIMENTS

[0075] As mentioned above, the present disclosure arises from research into the use of an electric field to modify the activity of functional proteins and microorganisms. Specifically, the inventors have found that the activity of functional proteins and microorganisms may be affected by an electric field, that is essentially free of a magnetic component, in which the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0076] The terms “free”, “essentially free” and “substantially free” are used interchangeably to denote that any magnetic component is minimised and negligible in effect.

[0077] The methods, device and system of the present disclosure find application in a vast array of fields. The methods, device and system of the present disclosure may address rate-limiting steps across multiple industries, including brewing, bioenergy, pharmaceuticals, agriculture, and biotechnology. The methods, device and system of the present disclosure may enhance production efficiency, product quality, and reliability by optimising microbial processes and enzymatic reactions. The person skilled in the art would understand that this might be termed a bioenergetic accelerator (“BioEncel™”).

[0078] For example, the inventors have discovered that during the production of low -carbohydrate, low-sugar beer, yeasts experience significant stresses that adversely affect the flavour of the final product. These stresses alter yeast metabolism in ways that diminish the beer's taste quality compared to traditional high-carbohydrate, high-sugar beers. Using the methods, device and system of the present disclosure, the inventors have found a way to produce low -carbohydrate, low-sugar beer with a flavour profile similar toits high-carbohydrate counterparts. The inventors carried out experiments disclosed here using a low- pressure system of limited depth yet, while in the process, they discovered ways to allow microbes to cope with stresses that occur in much larger systems of much greater depth and much higher pressure.

[0079] In the brewing and alcohol industries, the rate at which flavours and aromas are produced is an important determinant of a producer's competitiveness. The inventors have identified rate-limiting steps that hinder the efficient production of these sensory attributes. The methods, device and system of the present disclosure increase both the measurable and perceived flavours and aromas from a given amount and quality of input material.

[0080] The diauxic shift is a known rate-limiting adaptive step in fermentation processes, during which microbial cell division slows as cells adapt to depleting oxygen and sugars. This period can lead to stuck fermentations, where the process halts and fails to produce the desired levels of alcohol, sugars, flavours, and aromas. This may be monitored by aseptically sampling from a fermentation above the level of flocculated cells and performing cell density measurements. The methods, device and system of the present disclosure may prevent fermentations from stalling during this adaptive phase and the subsequent respiration period.

[0081] In winemaking and the production of spirituous liquors, there are inherent limitations in generating desirable flavours and aromas that affect the palate and overall sensory experience. The methods, device and system of the present disclosure may enrich wines and spirits with these desirable components. The methods, device and system of the present disclosure also find application in improving yield without requiring additional input materials.

[0082] Without wishing to be bound by theory, it is thought that the provision of an electric field oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz enhances protein breathing by affecting the charged moieties within the liquid that contains the protein. Protein breathing allows substrates and cofactors to react and therefore to synthesise products. The methods, device and system of the present disclosure are thought to stimulate or power protein breathing, and also stimulate substrates and cofactors to react and synthesise products, which also facilitates cell division.

[0083] In embodiments, there is provided an electric field, generated to provide surmounting energy that enables a protein or functional protein complex to overcome the activation energy barrier specific for their function. While not bound by theory, it is known that each biological or biochemical action of a functional protein or function protein complex of several proteins, is limited by the energy available in random brownian motion, and it is believed that the targeted non-random energy provided by the electric field of this disclosure provides surmounting energy through improved protein breathing as Resonance - Enhanced Conformational Dynamics (RECD) to overcome such activation energy barriers. RECD describes the underlying physical and biological theory that accounts for the effect of the electric fieldsignal. The methods, device and system disclosed herein is tuned to specific, non-arbitrary resonance frequencies in the 210 Hz to 99 kHz range that maximize energy transfer into the biological system, thus enhancing that energy transfer. Conformational dynamics refers to the shape -shifting, or "protein breathing" of enzymes and functional proteins. While not bound by theory, the disclosure posits that the precise electric field oscillation provides surmounting energy (kinetic energy) to the charged / polar domains of proteins, driving these conformational changes and thereby increasing the rates of catalysis, metabolic activity, RNA transcription, protein translation and other biological reactions.

[0084] As such, in certain embodiments, the generated electric field provides surmounting energy to improve protein breathing and is generated by a controller.

[0085] The defined one or more frequency may maximise the number of iterations of an alternating field per second while still within the effective window for protein breathing. That maximisation may be important when using large volumes of liquid as required by industry. In industry, as a body of liquid travels at speed past an emitter that provides the oscillating electric field, each parcel of water making up that body of liquid gets a small amount of time in close proximity to the field. During that proximity, the number of field-polarity reversals experienced by the in vivo and ex vivo enzymes in the liquid and experienced by the enzyme complexes in the liquid, affects the achieved activity of those enzymes or enzyme complex.

[0086] This contrasts with devices and methods that set up an alternating field via two electrodes placed into contact with the liquid itself, in which case the liquid acts as a conductor closing the circuit and drawing high power. Using an electrode in a liquid intended as a beverage also alters the flavours or metabolites in the liquid, perhaps undesirably. Further, use electrodes may be limited by the degree of contact between the passing liquid and the electrode or electrodes. Some prior research on the effects of different fields on proteins and microorganisms have focussed on electromagnetic fields or magnetic fields, provided by, e.g., a closed circuit or fixed magnets, respectively. However, an electromagnetic field at 50Hz may negatively affect adjacent devices operating on AC power at the same frequency. Similarly, an electromagnetic field at frequencies of communication channels (522 kHz and up) can disrupt radio communications because it interferes with wireless signals operating in that frequency range — such as Wi-Fi, Bluetooth, and certain cellular networks — by causing electromagnetic interference that degrades signal quality, leads to dropped connections, and can overload receivers.

[0087] As such, the methods, device and system of the present disclosure are based on the concept of using an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0088] In certain embodiments, the frequency is in the range of about 210 Hz to about 80 kHz, about 210Hz to about 70kHz, about 210Hz to about 60kHz, about 210Hz to about 50kHz, about 210Hz to about40kHz, about 210Hz to about 30kHz, about 210Hz to about 200kHz, or about 210 Hz to about 10 kHz. In particular embodiments, the frequency is in the range of about 210 Hz to about 8 kHz, about 500Hz to about 5kHz or about 1kHz to about 5kHz. As would be appreciated by the person skilled in the art, the frequency may be modified within the disclosed ranges depending upon the required effect. The person skilled in the art would understand that as the frequency increases, electric fields penetrate water less effectively, which reduces the ability to deliver sufficient voltage to influence the charged moieties in the liquid.

[0089] The electric field may be of a defined strength. As would be appreciated by the person skilled in the art, various factors determine the strength of an electric field. These factors include the magnitude of the voltage applied. In certain embodiments, the voltage is in the range of about 1 V to about 450 V. In certain embodiments, the voltage is in the range of about 1 V to about 450 V, about 2 V to about 400 V, about 5 V to about 350 V, about 10V to about 300 V, about 20 V to about 250 V, or about 30V to about 200 V. In particular embodiments, the voltage is in the range of about 50 V to about 150 V.

[0090] As would be appreciated by a person skilled in the art, the strength of an electric field experienced by, e.g., a protein or microorganism is inversely proportional to the distance from the source of the electric field. When a protein or microorganism is closer to the source, such as a voltage terminal, it experiences a stronger electric field, which may range up to about 450 V / cm, depending on the specific application and setup. Conversely, as the distance increases, the field strength weakens. In certain embodiments, the electric field has a strength in the range of about 0.01 V / cm to about 450 V / cm. In certain embodiments, the electric field has a strength in the range of about 0.01 V / cm to about 450 V / cm, about 0.1 V / cm to about 450 V / cm, about 0.1 V / cm to about 400 V / cm, about 0.1 V / cm to about 350 V / cm, about 0.1 V / cm to about 300 V / cm, about 0. 1 V / cm to about 250 V / cm, or about 0.1 V / cm to about 200 V / cm, about 0.1 V / cm to about 150 V / cm, about 0.1 V / cm to about 100 V / cm, about 0.1 V / cm to about 80 V / cm, or about 0.1 V / cm to about 50 V / cm. The electric field, and the absence of a magnetic field, may be measured using an EMF meter or oscilloscope. The person skilled in the art would also understand that a voltage terminal may alternatively be referred to as a terminal, an antenna, an antenna terminal or an emitter.

[0091] In particular embodiments, frequencies of modulation are selected that are known to optimally stimulate protein breathing by providing surmounting energy and thus to enhance cellular processes without causing cellular damage. These preferred frequencies may range from about 210 Hz to about 99 kHz. The voltage applied across any two terminals is in the range of about 1 V to about 45,000 V, with specific embodiments employing voltages between about 30 V and about 220 V, chosen to optimize the electric field strength for stimulating probiotic activity without detrimental effects. In specific embodiments voltages between about 30 V and about 220 V are also selected to maintain electrical safety.

[0092] The Root Mean Square (RMS) Voltage across the terminals is in the range of about 1 V to about 40,000 V, with specific embodiments employing RMS voltages between about 15 V and about 85 V, chosen to optimize the electric field strength for stimulating probiotic activity without detrimental effects. In specific embodiments RMS voltages between about 15 V and about 85 V are also selected to maintain electrical safety.

[0093] The electric field strength, measured at the location of the microorganism -inoculated growth medium, is typically in the range of about 0.01 V / cm to about 4,500 V / cm, and more between about 0.1 V / cm and about 10 V / cm to stimulate probiotic activity without detrimental effects. In specific embodiments electric field strengths between about 0.1 V / cm and about 10 V / cm are also selected to maintain electrical safety. This precise control over field strength may also be important for ensuring the effective penetration of the electric near-field into the cellular environment while remaining safely below the threshold for cellular electroporation or thermal damage.

[0094] Precise control over field strength enables the triggering of advantageous physiological responses, such as enhanced metabolic activity and improved stress tolerance, rather than inhibitory effects. A heightened stress tolerance is a direct consequence of the specific non-random kinetic energy input by the technology, which provides surmounting energy to facilitate the overcoming of activation energy (Ea) barriers that play a part in in stress-response mechanisms, longevity and natural cellular repair, so effectively maintaining homeostasis through increasing the functional rate of functional proteins, and hence increasing cell viability as predicted by the Arrhenius principle. The operational status of the emitter, particularly the strength and dominance of the low -frequency electric near-field, may be professionally verified using a calibrated measurement system, such as a Narda Field Probe and Spectrum Analyzer, to confirm that the E-field is present and the B-field component is negligible.

[0095] Charged loops are elements of protein primary structure that are naturally present on proteins surfaces, and in the context of this disclosure the charged loops act as sails, efficiently collecting kinetic energy from the externally applied electric field of the disclosure. This collected energy is then transferred through the internal protein secondary structures, which are comparatively rigid and act as levers, transferring the collected kinetic energy inwards to in turn enable the reaction machinery inside proteins to surmount the natural activation barrier of the protein’s evolved function. The surmounting of an activation barrier in many individual proteins and protein-complexes increases the functional rate and efficiency of the cell's natural processes, leading directly to gains in the function of organelles and other internal cell architectures that orchestrate cellular processes. Since proteins are the main proactive generative elements in cells, the gain of function across many proteins that is provided by this disclosure provides an ability for cells to resist stress, live longer and gain in viability.

[0096] As would be appreciated by the person skilled in the art, various other factors determine the strength of an electric field, such as the distance between terminals, and the properties of the mediumthrough which the field propagates — such as its permittivity and conductivity. Additionally, the geometry and surface area of the terminals, as well as their orientation relative to each other, can influence the electric field distribution and intensity. These are described elsewhere in this disclosure with reference to embodiments of the device and system.

[0097] The person skilled in the art would understand that there are various means to generate an electric field. In certain embodiments, the electric field is generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at the frequency. Other means to generate an electric field are described elsewhere in this disclosure with reference to embodiments of the device and system.

[0098] The generation of an electric field that is essentially free of a magnetic component may be important, as significant magnetic fields can have unintended and potentially inhibitory effects on microbial physiology. Generation is achieved through specific device configurations that prevent the formation of a closed circuit between any two voltage terminals, thereby minimising current flow and accordingly minimising the resultant magnetic field. Preventing formation of a closed circuit also minimises wear and tear on electronic components, significantly lengthening the operable life of the device used. In a case of even a single terminal, current is not permitted to flow out of the terminal to ground or to the growth medium. The generation of an electric field that is essentially free of a magnetic component also well describes the lack of current flowing into the composition, such current that can induce electrochemical damage in biological systems, and such current that can cause the known adverse occurrence of ohmic heating.

[0099] In certain embodiments, there is provided a means to prevent formation of any significant magnetic component to the electric field. In certain embodiments, the means to prevent formation of any significant magnetic component to the electric field is one or more gyrator circuit, switched-capacitor circuit, inductor configured within a resonant circuit, capacitor or an integrated circuit comprising resistors and active components. As the person skilled in the art would understand, any of these are capable of performing the function of blocking or controlling the free flow of current. In particular embodiments, the means to prevent formation of any significant magnetic component to the electric field is one or more capacitor, such a capacitor connected to ground. As would be appreciated by the person skilled in the art, the degree of impedance of a capacitor or capacitors may be selected so as not to reduce the oscillating voltage signal nor distort the oscillating voltage signal. In some embodiments, a capacitor or an array of capacitors is strategically integrated within the circuit at nodes or elsewhere, to impede current flow and ensure the dominance of the electric field component. The capacitance values arecarefully selected to block or control current effectively without attenuating or distorting the desired oscillating voltage signal.

[0100] As mentioned above, the concept of using an electric field may be applied to a vast range of fields. These are described below in different contexts and form the first through thirteenth aspects of the disclosure. The described frequency ranges, voltage, electric field strength, means to generate an electric field and means to prevent formation of any significant magnetic component to the electric field apply to these aspects and further aspects.

[0101] As such, in a first aspect, there is provided a method of modulating the activity of a functional protein, for example to improve protein breathing and thereby increase production of a functional protein, a therapeutic protein, a bacteriocin, an antibiotic protein, an insulin, a functional part of an antibody or another functional protein in microorganisms, the method comprising exposing the functional protein or a composition comprising the functional protein, to an electric field, that is essentially or substantially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0102] In some embodiments of the first aspect, the method is a method of treating a composition to provide surmounting energy to improve protein breathing during a fermentation of microorganisms so as to increase production of one or more of: a functional protein, a therapeutic protein, a bacteriocin, an antibiotic protein, an insulin, a functional part of an antibody and another functional protein in the microorganisms. The method comprises subjecting the microorganisms to a generated electric field that is substantially or essentially free of a magnetic component, wherein the generated electric field is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to about 99 kHz, and wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter positioned to expose the composition to the electric field. Electrically-insulated is simply defined as any property whereby the conductive part of the emitter does not contact the composition. Such insulation can comprise the wall of a housing or a container or a conduit.

[0103] In some embodiments of the first aspect, the microorganisms are production hosts that express the functional protein, a therapeutic protein, a bacteriocin, an antibiotic protein, an insulin, a functional part of an antibody or other functional protein of interest. The microorganisms may include eubacteria such as Escherichia coli. Bacillus species and Corynehacterium glutamicunr, actinomycetes such as Streptomyces species; Gram-positive bacteria such as Lactococcus lactis and Lactobacillus species; yeasts such as Saccharomyces cerevisiae. Pichia pasioris. Kluyveromyces lactis, Kluyveromyces marxianus and Yarrowia lipolyliccr. and filamentous fungi such as Aspergillus niger, Aspergillus oryzae and Trichoderma reesei. In other embodiments, the microorganisms include probiotic bacteria or yeasts that produce functional proteins such as enzymes, bioactive peptides or antibody fragments intended fornutritional, veterinary or therapeutic applications. In other embodiments, the microorganisms are as described elsewhere in this disclosure.

[0104] In some embodiments, the composition is a fermentation broth comprising the microorganisms, a culture medium and the expressed functional protein. The fermentation may be conducted in batch, fed- batch, repeated fed-batch, semi-continuous or continuous mode, and may include one or more of: an initial growth phase; an induction or expression phase; and a production or maintenance phase. The electric field treatment may be applied during all or part of any of these phases, for example during an induction phase to support folding and assembly of the functional protein, or during a late production phase to maintain activity and yield of the functional protein prior to harvest.

[0105] As would be appreciated by a person skilled in the art, exposing the functional protein to an electric field refers to positioning the protein within the influence of the field. In certain embodiments, the functional protein may be exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for an entire reaction process, which may last up to 1, 2, or even 3 weeks. In certain embodiments, the generated electric field is applied intermittently or continuously throughout the fermentation. For example, the field may be applied in cycles comprising treatment periods and rest periods, or at different frequencies or amplitudes during different stages of the fermentation. In some embodiments, a first treatment regime is applied during an early growth or induction phase, and a second treatment regime, for example with different frequencies, waveforms or duty cycles, is applied during a production or stationary phase. Such regimes may be selected to improve protein breathing and support increased production of correctly folded and functional proteins in the microorganisms.

[0106] The method may be applied to any functional protein to modulate the activity of the protein. In certain embodiments, the method may be applied to increase the activity of the protein. In certain embodiments, the method may increase the activity of a functional protein by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%; by at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%; or may increase the activity by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold. In certain embodiments, the method may increase the activity of a functional protein by about 2% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%. In certain embodiments, the method may increase the activity of a functional protein by about 2-fold to about 4- fold, about 2-fold to about 6-fold, or about 2-fold to about 8-fold.

[0107] In some embodiments, the controller that generates the electric field comprises a waveform generator, amplifier and control logic configured to produce an AC signal or a pulsed DC signal oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz. The controller may beconfigured to deliver a single frequency, a set of discrete frequencies, a frequency sweep or a stochastic or shuffled progression of frequencies to the at least one electrically insulated emitter. The emitter can include one or more insulated voltage terminals or antennae positioned within a vessel or flow module so that the microorganisms in the fermentation broth are exposed to the electric field while the emitter remains electrically insulated from the bulk liquid. In some embodiments, the emitter forms part of a removable bioreactor module or cartridge that can be installed into or removed from standard fermentation equipment. In such embodiments, the container is separate from a fermenter vessel but connected by liquid flow to a fermenter vessel, and the container facilitates recirculation of a composition past an electrically-insulated electric field emitter and then back to a fermenter vessel for continued fermentation, such that the composition is exposed to a near electric field. The electrically-insulated emitter is such that the conductive part of the emitter does not contact the composition. Such insulation can comprise the wall of a housing or a container or a conduit. Near electric field refers to the much greater strength of an electric field near to an electric field emitter of low frequency, than at a distance from such emitter.

[0108] In further embodiments, the composition treated according to the first aspect may include, in addition to the fermentation broth, one or more downstream intermediates such as a concentrated cell slurry, a cell-free extract, a partially purified protein fraction or an immobilised preparation of the microorganisms. The electric field may be applied to these intermediates to modulate protein breathing and thereby improve stability, activity or recovery of the functional protein, a therapeutic protein, a bacteriocin, an antibiotic protein, an insulin, a growth hormone, a hormone replacement, a functional part of an antibody or other functional protein during downstream processing, formulation or storage.

[0109] A person skilled in the art would understand the increase the activity of a functional protein to mean enhancing the specific functional capabilities of the protein in accordance with its biological role. Overall, increasing the activity of a functional protein encompasses any measurable enhancement of the protein’s inherent functions that contribute to improved biological or biochemical outcomes in the specified applications.

[0110] For example, in the case of an enzymatic protein, this could involve increasing its catalytic efficiency or turnover rate. For anabolic and catabolic proteins, it may refer to accelerating metabolic pathway fluxes. Intramembrane, transporter, pump, and channel proteins might exhibit enhanced rates or capacities for molecule transport across membranes. Motor proteins could show improved movement or force generation, while signalling proteins may have heightened signal transduction capabilities. Electron transport proteins might exhibit increased electron flow, and subunits of ATPases could demonstrate enhanced ATP hydrolysis rates. Mitochondrial and organellar proteins could show improved functions in energy production or metabolite shuttling, respectively. Nuclear proteins, including DNA-binding and transcription factors, may have increased efficiency in gene transcription and regulation. Polypeptide-import proteins might transport proteins more effectively into organelles, and biosynthetic or glycoproteins could produce their respective products at higher rates. Stress response proteins, proteins involved in processes like flocculation, endocytosis, pinocytosis, exocytosis, cell wall maintenance, cytoskeletal dynamics, proteasomal degradation, thermotolerance, mating, meiosis, alcohol and ester production, beta-oxidation of fatty acids, sugar utilisation, metabolite shuttling, alcohol utilisation, and homeostasis would all exhibit enhanced activities relevant to their specific functions. Additionally, proteins involved in DNA synthesis, replication, chromatin remodelling and modification, ribosomal biogenesis, cell division, resistance to inhibitors, and regulatory functions would demonstrate increased effectiveness in their respective biological processes and by that increased effectiveness - an acceleration of cellular functions and cellular activities.

[0111] In certain embodiments, the functional protein is an enzymatic protein, an anabolic protein, a catabolic protein, an intramembrane protein, a transporter protein, a pump protein, a channel protein, a motor protein, a signalling protein, an electron transport protein, a subunit of an ATPase, a mitochondrial protein, an organellar protein, a nuclear protein, a polypeptide -import protein, a transcription factor, a biosynthetic protein, a glycoprotein, a stress response protein, a protein involved in flocculation, a protein involved in endocytosis, a protein involved in pinocytosis, a protein involved in exocytosis, a cell wall protein, a cytoskeletal protein, a proteasomal protein, a thermotolerance protein, a protein involved in mating, a protein involved in meiosis, a protein involved in alcohol production, a protein involved in ester production, a protein involved in beta oxidation of fatty acids, a protein involved in sugar utilisation, a protein involved in shuttling metabolites between organelles, a protein involved in alcohol utilisation, a protein utilising a source of reduced carbon, a protein requiring cofactors, a protein producing cofactors, a protein involved in homeostasis, a DNA-binding protein, a protein involved in RNA transcription, a protein involved in gene transcription, a protein involved in mRNA processing, a protein involved in polypeptide synthesis, a protein involved in DNA synthesis, a protein involved in DNA replication, a protein involved in chromatin remodelling, a protein involved in chromatin modification, a protein involved in ribosomal biogenesis, a protein promoting cell division, a protein involved in resistance to inhibitors, or a regulatory protein.

[0112] In certain embodiments, the functional protein is a therapeutic protein or functional part of an antibody produced by the microorganisms. Examples of therapeutic proteins include, without limitation, antibiotic proteins, bacteriocins, insulin and insulin analogues, growth hormone, erythropoietin, interferons, interleukins, colony stimulating factors, clotting factors, enzyme -replacement proteins, and fusion proteins. Examples of functional parts of antibodies include antibody fragments such as Fab, F(ab')2, scFv, single-domain antibodies and nanobodies, as well as Fc-fusion proteins and other engineered binding proteins. In some embodiments, the functional protein is an industrial or diagnostic protein, such as a recombinant enzyme used in food processing, biofuel production, bioremediation or analytical assays.

[0113] In certain embodiments, the functional protein is an enzymatic protein selected from the group consisting of amylases (including alpha-amylase, beta-amylase, glucoamylase, maltogenic amylase, and cyclodextrin glucanotransferase), debranching enzymes (including isoamylase and pullulanase), proteases (including endo-protease and exo -protease), hydrolases, lyases, isomerases, transferases, oxidoreductases, reductases, dehydrogenases, peroxidases, sulfotransferases, aminoacyl -tRNA synthetases, kinases, phosphorylases, adenosine deaminase, carbonic anhydrase, aldolases, esterases, lipases, peptidases, ribonucleases, and nucleases. In certain embodiments, the functional protein is involved in starch hydrolysis or carbohydrate metabolism and may include enzymes such as beta-glucanase, cellulase, hemicellulase, pectinase, invertase, fructosyltransferase, xylose isomerase, trehalase, and phosphorylase. Additional enzymes relevant to brewing processes include lipase, esterase, xylanase, laccase, glutathione peroxidase, and alpha-acetolactate decarboxylase (ALDC). In certain embodiments, these enzymes function to enhance mash efficiency, improve wort clarity, optimize fermentation, and influence the flavour and aroma profiles of the final product. Proteins involved in sugar utilization, beta-oxidation of fatty acids, alcohol and ester production, and thermotolerance may also include specific hydrolases, oxidoreductases, and dehydrogenases that enhance cellular activities and fermentation processes. In certain embodiments, the functional protein is involved in metabolic pathways such as beta-oxidation of fatty acids, glycolysis, or gluconeogenesis. In certain embodiments, the functional protein is an enzyme participating in metabolism, selected from the group consisting of pyruvate decarboxylase, lactate dehydrogenase, malate dehydrogenase, malic enzyme, fumarase, succinate dehydrogenase, transketolase, aldolase, acetyl-CoA carboxylase, pyruvate kinase, phosphofructokinase, phosphoglucose isomerase, glucose-6-phosphatase, hexokinase, components of the pyruvate dehydrogenase complex, glutathione reductase, glucose-6-phosphate dehydrogenase, enolase, triose phosphate isomerase, dihydrolipoyl acetyltransferase, phosphoglycerate kinase, and glyceraldehyde-3-phosphate dehydrogenase. In certain embodiments, the functional protein is an anabolic protein selected from the group consisting of cytochrome P450 enzymes, phosphopantetheinyl transferases, lipoxygenases, and acetyl-CoA carboxylases. In certain embodiments, the functional protein is involved in catabolic processes and is selected from the group consisting of lactate dehydrogenase, aldolase, pyruvate kinase, triose phosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase, malate dehydrogenase, phosphoglycerate kinase, and glucose-6-phosphatase.

[0114] In certain embodiments, the functional protein is an intramembrane protein selected from the group consisting of voltage-gated ion channels, ligand-gated ion channels, transporters, receptors, and membrane-bound enzymes. In certain embodiments, the functional protein is a transporter protein selected from the group consisting of sodium-potassium pumps (Na+ / K+-ATPases), amino acid / sugar transport proteins, and secondary active transporters. In certain embodiments, the functional protein is a pump protein selected from the group consisting of ATPases and pyrophosphatases. In certain embodiments, the functional protein is a channel protein selected from the group consisting of potassium channels, calcium channels, sodium channels, proton channels, chloride channels, anion channels, andtwo-pore domain potassium channels (K2P channels). In certain embodiments, the functional protein is a motor protein selected from the group consisting of kinesins, dyneins, myosins, and proteins involved in cilia and flagella motility. In certain embodiments, the functional protein is a signalling protein selected from the group consisting of G-protein coupled receptors (GPCRs), signal transduction proteins, and proteins containing nuclear localization signals (NLS) or nuclear export signals (NES). In certain embodiments, the functional protein is an electron transport protein selected from the group consisting of components of the cytochrome b-Ci complex, cytochrome c oxidase, cytochrome c reductase, cytochrome ce, and cytochrome aas. In certain embodiments, the functional protein is a subunit of an ATPase selected from the group consisting of F-type ATPases, V-type ATPases, Na+ / K+-ATPases (a type of P-type ATPase), P-type ATPases, and AAA+ATPases (ATPases associated with diverse cellular activities). In certain embodiments, the functional protein is a mitochondrial protein selected from the group consisting of cytochrome b, NADH dehydrogenase (complex I subunits), succinate dehydrogenase (complex II subunits), coenzyme Q reductase, cytochrome c oxidase, and ATP synthase components.

[0115] In certain embodiments, the functional protein is an organellar protein selected from the group consisting of chloroplast proteins, plastoglobule proteins, thylakoid proteins, stroma proteins, photosystem I (PSI) proteins, photosystem II (PSII) proteins, ATP synthase subunits, and ferredoxin. In certain embodiments, the functional protein is a nuclear protein selected from the group consisting of histones, DNA repair proteins, chromatin remodelling proteins, transcription factors, histone acetyltransferases (HATs), histone deacetylases (HDACs), and co-activators. In certain embodiments, the functional protein is involved in polypeptide import and is selected from the group consisting of translocases, import receptors, and proteins facilitating protein translocation across membranes. In certain embodiments, the functional protein is a transcription factor selected from the group consisting of general transcription factors, specificity factors, activators, repressors, and regulatory proteins. In certain embodiments, the functional protein is involved in DNA synthesis and is selected from the group consisting of DNA polymerases, DNA ligases, DNA gyrases, topoisomerase I (topo I), topoisomerase II (topo II), and helicases. In certain embodiments, the functional protein is involved in DNA replication and is selected from the group consisting of DNA polymerases, DNA primases, DNA helicases, DNA gyrases, topoisomerase I (topo I), topoisomerase II (topo II), and single -stranded DNA-binding proteins. In certain embodiments, the functional protein is involved in chromatin remodelling and is selected from the group consisting of histone acetyltransferases (HATs) and histone deacetylases (HDACs). In certain embodiments, the functional protein is involved in chromatin modification and is selected from the group consisting of nucleosome assembly proteins, ATP -dependent chromatin remodelling enzymes, histone chaperones, and chromatin assembly factors (CAFs). In certain embodiments, the functional protein is involved in ribosomal biogenesis and is selected from the group consisting of RNA polymerase I, RNA helicases, RNA methyltransferases, and ribonuclease H. In certain embodiments, the functional protein is involved in cell division and is selected from the group consisting of tubulin, actin, cyclins, cyclin- dependent kinases (CDKs), microtubule -associated proteins, and septin proteins. In certain embodiments,the functional protein is involved in resistance to inhibitors and is selected from the group consisting of GTPase -activating proteins (GAPs), guanine nucleotide exchange factors (GEFs), and phospholipases. In certain embodiments, the functional protein is a regulatory protein selected from the group consisting of transcription factors, RNA-binding proteins, DNA-binding proteins, histone acetyltransferases (HATs), histone deacetylases (HDACs), kinases, phosphatases, GTP -binding proteins, and cytoskeletal proteins.

[0116] As the person skilled in the art would understand, measuring a modification (e.g. an increase) in the activity of a functional protein involves selecting methods appropriate to the protein's role and function. For anabolic and catabolic proteins, increased activity can be evaluated using metabolic flux analysis with isotope-labelled substrates, enzyme activity assays, or metabolomics to assess pathway intermediates. Intramembrane, transporter, pump, and channel proteins may be assessed via transport assays with radiolabelled or fluorescent substrates, electrophysiology techniques such as patch-clamping, or ATPase activity assays. Motor protein activity can be measured using in vitro motility assays, force generation studies with optical or magnetic tweezers, or ATP turnover analysis. For signalling proteins, phosphorylation assays, reporter systems, or protein-protein interaction studies may be used. Electron transport proteins can be analysed through oxygen consumption rate measurements, redox state assays, or cytochrome activity tests. ATPases and mitochondrial proteins may demonstrate increased activity through ATP hydrolysis assays, mitochondrial membrane potential assessments, or ATP production rate measurements. Nuclear proteins, including DNA-binding and transcription factors, may be evaluated via electrophoretic mobility shift assays, chromatin immunoprecipitation, or transcriptomic analysis.Polypeptide -import proteins may show enhanced activity through import efficiency assays or subcellular fractionation. Biosynthetic and glycoprotein activity can be monitored using mass spectrometry or by measuring pathway-specific products. Stress response proteins can be evaluated through survival assays under stress conditions, molecular stress markers, or functional adaptation tests. Cytoskeletal dynamics and motor protein activities can be observed through live-cell imaging or polymerization assays, while proteasomal proteins may be analysed using ubiquitin turnover or peptide hydrolysis assays. DNA synthesis and replication proteins can be assessed via BrdU incorporation assays or replication origin mapping. Chromatin remodelling proteins may demonstrate increased activity through nucleosome remodelling or chromatin accessibility studies, while ribosomal biogenesis proteins can be evaluated via rRNA quantification or polysome profiling. Finally, regulatory proteins may show heightened activity through transcriptomic or proteomic studies and functional assays to measure specific cellular responses. The person skilled in the art would understand which techniques to use, individually or in combination, to evaluate modified functional protein activity.

[0117] As would be appreciated by a person skilled in the art, the activity of the functional proteins described, including enzymatic proteins, anabolic and catabolic proteins, and other specialised protein types, depends on appropriate medium or buffer conditions tailored to their specific biochemical requirements. These conditions may include optimal pH, typically ranging from about 4.0 to 9.0,depending on the protein's active site stability, with acidic buffers (e.g., citrate or acetate buffers) for hydrolases and neutral to slightly alkaline buffers (e.g., phosphate or Tris buffers) for oxidoreductases and transferases. The buffer may also require specific ionic strength, often maintained using salts such as NaCl or KC1, to stabilise the protein structure. Essential cofactors such as Mg2+, Mn2+, Zn2+, or NADVNADH may be included for catalytic activity. Proteins involved in electron transport, such as components of the cytochrome b-Ci complex or ATPases, may require buffers supplemented with reducing agents like dithiothreitol (DTT) or oxidising agents depending on the redox state needed. Mitochondrial proteins and those involved in organellar processes may require sucrose, glycerol, or polyethylene glycol to maintain activity in vitro. For intramembrane proteins, such as transporters and ion channels, detergents like Triton X-100 or specific lipids may be necessary to preserve membrane-like environments. The medium comprising the functional protein may be temperature controlled, for example, between 20°C and 40°C, depending upon the specific protein. The medium may include stabilisation agents, such as bovine serum albumin (BSA), to prevent denaturation. Typical buffers include, e.g., MES, Tris-HCl, CHES, TES, HEPES, MOPS, PIPES, sodium phosphate buffer, sodium citrate buffer, sodium acetate buffer, and glycine-HCl buffer. Typical concentrations of buffers are known in the art and from manufacturer’s instructions.

[0118] In some embodiments, the methods of the first aspect are implemented using any of the electric field-generating devices and systems described elsewhere in this specification. For example, the controller that generates the electric field may correspond to a controller of an electric field-generating device as described for the device embodiments, configured to supply a waveform or modulated voltage to one or more electrically insulated emitters or voltage terminals. The composition containing the functional protein, microorganisms or both may be contained in a vessel, bioreactor module or flow module of an electric field-generating system as described for the system embodiments, such that the electrically insulated emitter is positioned within or adjacent to the composition to expose it to the electric field that is essentially or substantially free of a magnetic component. The container of the composition being treated by the electric field provided may be separate from a fermenter vessel, but connected by liquid flow to a fermenter vessel, and the container so connected may thus facilitate recirculation of a composition past an electrically-insulated electric field emitter and then back to a fermenter vessel for continued fermentation, such that the composition is exposed to a near electric field. The electrically- insulated emitter is such that the conductive part of the emitter does not contact the composition. Such insulation can comprise the wall of a housing or a container or a conduit.

[0119] In a second aspect, there is provided a method of at least one of: modulating the speed of microbial cell division, affecting microbial cell density, improving a stress resistance, or a longevity of microorganisms, comprising exposing one or more microorganism to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0120] In some embodiments of the second aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0121] In some embodiments of the second aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, during a fermentation process of the microorganisms to produce one or more of: dairy products, yoghurt, cheese, a probiotic, a feed, an encapsulated probiotic, probiotic manufacturing, and a probiotic packaged with excipients, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0122] In some embodiments of the second aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, during a fermentation process of the microorganisms, and of improving survival of the microorganisms during storage or transport, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0123] In some embodiments of the second aspect, there is provided a method of improving a stress resistance, a longevity, or both, of microorganisms, comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz.

[0124] As such, in certain embodiments, the method comprises subjecting the microorganisms, or a composition comprising the microorganisms, to a generated electric field that is essentially or substantially free of a magnetic component, wherein the generated electric field is produced by a controller configured to generate a signal oscillating at one or more frequency in a range of about 210 Hz to about 99 kHz and to supply the signal to at least one electrically insulated emitter. In other embodiments, the method comprises subjecting the microorganisms to a generated electric field that is essentially or substantially free of a magnetic component, wherein the generated electric field is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to about 99 kHz, as described elsewhere in this disclosure.

[0125] In certain embodiments, the method of the second aspect is carried out during a fermentation process of the microorganisms to produce one or more of: dairy products, yoghurt, cheese, a probiotic, a feed, an encapsulated probiotic, a probiotic manufactured for subsequent formulation, or a probiotic packaged with excipients. In such embodiments, the microorganisms are subjected during fermentation to the generated electric field that is essentially or substantially free of a magnetic component, wherein the generated electric field is produced by the controller as described above, and is supplied to at least one electrically insulated emitter positioned to expose the fermentation to the field.

[0126] In other embodiments, the method of the second aspect is carried out during a fermentation process of the microorganisms and also to improve survival of the microorganisms during storage or transport. For example, the microorganisms may be subjected to the generated electric field during one or more of: fermentation to produce biomass or product; concentration of biomass; formulation into a dried, encapsulated or otherwise stabilised product; and subsequent storage or transport of that product. The generated electric field is in these embodiments produced by the controller to oscillate at one or more frequency in a range of about 210 Hz to about 99 kHz and is supplied to at least one electrically insulated emitter, such that stress resistance, longevity, or both, are improved relative to untreated controls.

[0127] As would be appreciated by a person skilled in the art, exposing the one or more microorganism to an electric field refers to positioning the one or more microorganism within the influence of the field. In certain embodiments, the one or more microorganism may be exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for an entire fermentation process, which may last up to 1, 2, or even 3 weeks. Shorter exposure times, such as 1 to 10 minutes, may be employed to achieve specific effects, such as accelerating activity or growth.

[0128] In embodiments, the method may be applied to any microorganism to modulate the speed of cell division or affect microbial cell density. Specifically, the method may be applied to any microorganism to increase the speed of cell division. In certain embodiments, the method may increase the division rate by at least 2%. In certain embodiments, the method may increase the speed of cell division from about 2% to about 100%, for example, about 2% to about 10%, about 10% to about 25%, about 25% to about 50%, and about 50% to about 100%. Increases in cell division speed may be measured by evaluating parameters such as the reduction in doubling time, optical density (OD) at specific wavelengths (e.g., ODeoo) to assess cell density, conducting cell counts using flow cytometry or hemocytometers, performing colony -forming unit (CFU) assays to quantify viable cell proliferation, or measuring the amount of trub at the end of a fermentation.

[0129] In some embodiments, the generated electric field of the second aspect is generated by an AC signal or by a pulsed DC signal having a peak-to-peak voltage and a duty cycle selected for the microorganism and process. The improved stress resistance, longevity, or both, can be used to reduce lossof viable cell count or loss of stress resistance or longevity during one or more of: a fermentation; a freeze-drying, a packaging or other dehydration process; and a long-term storage period of the microorganisms or a product comprising the microorganisms.

[0130] In certain embodiments, the AC signal or pulsed DC signal has a waveform that is applied to at least one voltage terminal or antenna of an emitter. The AC signal or the pulsed DC signal may comprise a single frequency, a set of frequencies, a frequency sweep, or a stochastic or shuffled progression of frequencies in the range of about 210 Hz to about 99 kHz. In certain embodiments, the electric field sweeps through sequential frequencies in a range of about 1400 Hz to about 4540 Hz or sequential frequencies in the range of about 210 Hz to about 99 kHz. In some embodiments, the AC signal or pulsed DC signal operates at a peak-to-peak voltage between about 1 V and about 45 kV, for example within the voltage ranges described elsewhere in this disclosure for generating electric fields having strengths in the range of about 0.01 V / cm to about 450 V / cm.

[0131] In some embodiments, each of the at least one voltage terminal or antenna comprises one or more of (i) insulated conductive material helically wound around a non-conducting material; (ii) an insulated conductive terminus material of any shape such as an insulated conductive plate; or (iii) two or more insulated conductive plates separated by a non-conducting material or by the composition; to provide surmounting energy to facilitate increased protein breathing in the cells’ functional proteins. In some embodiments, each of the at least one voltage terminal or antenna are as described elsewhere in this disclosure.

[0132] In some embodiments, an integrated monitor is embodied in or connected to the controller to monitor the presence of signal in the antenna or antennae, such as the waveform of the signal, the amplitude of the signal, and the frequency of the signal. The integrated monitor may include one or more sensors and a user interface or data logging module, and may optionally provide feedback to the controller to adjust one or more of the frequency, amplitude, duty cycle or waveform shape during operation. Where an integrated monitor is embodied in or connected to the controller to monitor the presence of signal in the antenna or antennae, the parameters of the signal or signals that may be monitored may include waveform, amplitude and frequency.

[0133] In certain embodiments, the one or more frequencies of the generated electric field follow a stochastic or shuffled progression or other programmed sweep within the range of about 210 Hz to about 99 kHz. For example, the stochastic progression of frequencies may be implemented as a quartic sweep defined by a formula F(t) = a t4+ b t3+ c t2+ d t + e, expressing frequency as a function of time t, where a, b, c, d and e are coefficients selected to define the sweep profile. In other embodiments, the sweep is a curved sweep or a substantially linear sweep or is a stepped non-quartic sweep , progressing upward in frequency or downward in frequency , or is a non-stepped sweep progressing upward in frequency or downward in frequency, the sweep taking a fixed period time before rebeginning.

[0134] In certain embodiments, the controller is configured to implement a discrete stepwise sweep of the carrier frequency. For example, the carrier frequency may be varied over a sweep window of about 664 seconds using a fixed number of steps, such as 64 steps of substantially equal duration (for instance, about 10.37 seconds per step). The sequence of carrier frequencies can be generated by evaluating a quartic sweep progression formula, such as Frequency(t) = A t4+ B t3+ C t2+ D t + E, where t is time and A, B, C, D and E are coefficients selected so that the base frequency at the start of the sweep is in the lower part of the preferred band (for example, about 1398 Hz) and the final frequency at the end of the sweep is in the upper part of the preferred band (for example, about 4539 Hz). The controller can evaluate this quartic expression at the start time of each step and hold the resulting frequency constant for the full step duration, optionally allowing minor jitter from the waveform generator and amplifier circuitry. The discrete frequencies may be stored in a lookup table for efficient implementation.

[0135] In some embodiments, rather than evaluating a sweep equation in real time for every instant of the applied waveform, the progression of frequencies over time is implemented by a lookup table stored in or accessible to the controller. The lookup table defines a finite set of discrete frequency values which, when applied in sequence and for defined step durations, together approximate a desired underlying sweep function such as the quartic sweep defined by Equation {3}. In such embodiments, the controller steps through the entries of the lookup table in order and configures the waveform generator so that, for each entry, the corresponding frequency is applied to the at least one emitter for a defined period, for example for about 8 to 12 seconds or about 10.37 seconds.

[0136] In one illustrative embodiment corresponding to Experiments 5 and 6, the lookup table contains 64 entries derived from Equation {3}. Each entry includes a start time and a corresponding frequency value (see Table 14). The quartic sweep equation is evaluated only at the start of each 10.37 second interval, with time t progressing from 0 seconds to about 653.625 seconds in steps of about 10.37 seconds. The resulting set of 64 frequencies, each associated with its start time, is stored as a lookup table. During operation, the controller determines the current elapsed time since the beginning of the sweep, identifies the entry in the lookup table whose start time corresponds to that elapsed time, and holds the waveform generator at the associated frequency for the full 10.37 second duration of that step, subject only to small variations arising from jitter in the waveform generator or amplifier circuitry. In this way, the sweep is implemented by successive fixed-frequency steps determined by the lookup table, rather than by continuously re-evaluating Equation {3}.

[0137] In some embodiments, the lookup table implements an approximation to Equation {3} that is within a predetermined tolerance. In the embodiment of Experiments 5 and 6, the lookup table of Table 14 provides an average standard deviation of about 0.22% from the ideal values generated by Equation {3} across the full sweep. Such a small deviation is acceptable in practice, and still results in a progression of frequencies that provides surmounting energy sufficient to influence protein breathing inthe treated microorganisms as described herein. Other embodiments may employ lookup tables with more or fewer entries, different step durations, or different tolerances, provided that the overall progression of frequencies remains within a range suitable for generating the desired electric field effects in the microorganisms.

[0138] In further embodiments, the progression of frequencies is not strictly sequential in time, but is still defined using one or more lookup tables. For example, in a “stochastic” or “shuffled” section corresponding to Experiments 5 and 6, a lookup table (Table 15) is provided that specifies a set of discrete frequency values without explicit start times. At run-time, the controller selects, for each time interval of between 8 and 12 seconds or about 10.37 seconds, a frequency from the lookup table, and configures the waveform generator to output that selected frequency for the duration of that interval. The next interval is then supplied with a frequency selected from either the same table or from a defined subset of the frequencies contained in the table. The selection is carried out according to predetermined rules governing which subsets of the lookup table may be used as the source of the next frequency, such that over a long operating period, for example about 15 hours, each of the frequencies in the lookup table is represented approximately an equal number of times.

[0139] In such stochastic or shuffled embodiments, the lookup table thus may define the set of allowed frequencies, whilst the controller’s selection rules and any defined subsets of the lookup table together define the progression of frequencies over time. Subject to the particular electronic architecture, empirically realised frequencies in the stochastic section may vary from the nominal frequencies listed in the lookup table by not more than a predetermined fraction, for example by no more than about 7% of the nominal frequency dictated by the table. For the purposes of the present disclosure, the expression “lookup table or subsets of a lookup table” encompasses both embodiments where the controller steps through the entire table in a predefined order (for example, the sequential quartic sweep of Table 14), and embodiments where the controller selects from the table or from subsets of the table according to an algorithmic rule (for example, the stochastic or shuffled progression defined using Table 15), provided that the sequence of frequencies applied to the emitter over time is determined from data stored in the lookup table.

[0140] In some embodiments, the lookup table is stored in a non -transitory electronic memory associated with the controller, and each entry of the lookup table comprises at least a frequency value in Hertz, and optionally an associated nominal start time and / or nominal duration for which that frequency is to be applied. The controller may maintain an internal time counter or step counter and, on the basis of that counter, index into the lookup table to retrieve the next frequency value or the next pair of (start time, frequency) values. In embodiments where subsets of the lookup table are employed, the controller may designate one or more subsets of entries (for example, sub-ranges of frequencies or groups associated with different parts of a fermentation phase) and may select the next frequency only from entriesbelonging to a currently -active subset. The subset itself can be changed over time according to one or more rules, such as after a predetermined number of steps, or when a process variable (for example fermentation time, viable cell density, or dissolved oxygen level) reaches a threshold, thereby providing a deterministic or semi -stochastic progression of frequencies defined via the lookup table and its subsets.

[0141] Thus, in certain embodiments, the progression of frequencies supplied to the waveform generator and hence to the at least one emitter is governed by one or more stored tables of discrete frequency values, optionally with associated timing information, and optionally with defined subsets and selection rules. The use of the lookup table or its subsets enables the progression of frequencies supplied to the emitter to be accurately defined, reproduced, audited and, where desired, shuffled in a controlled manner, thereby facilitating generation of a repeatable or controlled-random exposure regime that can be optimised for enhancing stress resistance, longevity and metabolite production by the microorganisms being treated.

[0142] In some embodiments, a stepped quartic sweep is defined by a formula F(t)=at4+bt+ct2+dt+e that expresses frequency as a function of time t, and with a, b, c, d, e being coefficients, or is defined by: a curved sweep, or a linear sweep, or a stepped non-quartic sweep progressing upward in frequency or downward in frequency, or a non-stepped sweep progressing upward in frequency or downward in frequency; the sweep taking a fixed period time before rebeginning. In some embodiments the time that a given step of frequency lasts for is 10.37 seconds.

[0143] In some embodiments, the same discrete frequency set is reused in a stochastic, randomised, or shuffled mode of operation. For example, after a sweep window of a selected duration (such as about 25 hours of repeated sweeps) the controller may transition to a randomised mode in which frequencies are selected from the previously defined discrete set according to a probability distribution. The probability of selecting a particular frequency can be defined by a quartic probability function P(f) of the carrier frequency f, chosen so that lower frequencies within a preferred range (for example, about 1398-4539 Hz) are selected more frequently than higher frequencies. A table may be used to determine how many steps forward or backward in the ordered frequency list the controller moves when choosing the next frequency, thereby avoiding repeated selection of the same frequency bin while remaining within the overall band. In such embodiments, the controller applies each selected frequency for a step duration that is nominally constant (for example, about 10.37 seconds, optionally with a small standard deviation), so that a controlled but stochastic progression of carrier frequencies is obtained within the 210 Hz-99 kHz range.

[0144] In some embodiments a quartic carrier waveform is used being a waveform in which the rise and fall respectively of the waveform are defined using a quartic formula of the form V(t)=at4+bt+ct2+dt+e that expresses voltage V as a function of time t, and with a, b, c, d, e being coefficients that may differ in sign or quantity between the rise and fall equations.

[0145] In some embodiments, the quartic carrier waveform is further amplitude -modulated to add a lower-frequency structure. For example, the controller may generate a double -sideband full-carrier (DSB- FC) amplitude-modulated signal in which a low-frequency modulation component (for instance, about 50 Hz) serves as an envelope for the higher-frequency carrier. The modulation envelope may itself be trapezoidal or pseudo-trapezoidal, optionally using quartic rise and fall curves related to those used for the carrier rise and fall. The modulation depth can be set so that the composite waveform retains a desired peak-to-peak carrier amplitude while introducing upper and lower sidebands at the carrier frequency plus and minus the modulation frequency. The combination of the quartic carrier, low-frequency envelope and any equalising elements or filters in the post-amplifier circuit can be selected such that the dominant spectral components lie in the preferred range of about 210 Hz to about 99 kHz, and so that the electric near-field is enhanced while the magnetic component remains negligible.

[0146] The method may be applied to any microorganism to affect microbial cell density through increasing the speed of flocculation. In certain embodiments, the method further comprises accelerating the speed of flocculation. In certain embodiments, the method further comprises accelerating the speed of flocculation following on from accelerated cell division. As the person skilled in the art would understand, flocculation rate may be measured as either a function of suspended cell counts, or as a function of the amount of trub. In certain embodiments, an increase in flocculation may be observed by a decrease in suspended cell counts or in increase in the amount of trub. In certain embodiments, the method may increase the speed of flocculation from about 2% to about 100%, for example, about 2% to about 10%, about 5% to about 15%, about 10% to about 20%, about 10% to about 25%, about 25% to about 50%, and about 50% to about 100%. If using suspended cell counts, this may be observed by, e.g., a decrease of any of the above percentages, such as about 10% to about 20%, relative to an untreated control. If using the amount of trub, this may be observed by, e.g., an increase of any of the above percentages, such as about 10% to about 20%, relative to an untreated control. The increase in the speed of flocculation is typically observed from about day 7, 8 or 9.

[0147] Depending upon the species of microorganism, the method may be applied to microorganisms that undergo a diauxic shift, so as to accelerate the diauxic shift. A diauxic shift is often observed as a cell count plateau between a distinct phase of rapid exponential growth, and a subsequent secondary exponential growth phase, and this shift occurs as the organism switches to a secondary carbon source, while the environment is typically also depleting in oxygen. The types of organisms include, but are not limited to, Escherichia coli. Saccharomyces cerevisiae. Bacillus subtilis, Lactobacillus spp.. Pseudomonas aeruginosa, and Candida albicans. Other organisms are mentioned further below. These microorganisms are commonly employed in various fermentation applications where a diauxic shift is observed, typically characterised by the sequential utilisation of multiple carbon sources. For example, Escherichia coli may initially consume glucose before shifting to lactose, and Saccharomyces cerevisiae often uses glucose followed by ethanol during alcoholic fermentation processes. The fermentation typesthat exhibit diauxic shifts encompass batch fermentations, where nutrients are provided all at once; fed- batch fermentations, which involve the gradual addition of substrates; and continuous fermentations, where fresh medium is continuously supplied, and culture liquid is continuously removed. Applications of these fermentation processes include, for example, the production of alcohol, biofuels, pharmaceuticals, antibiotics, organic acids, amino acids, and other valuable bioproducts. Controlling diauxic shifts in these organisms may facilitate improved industrial fermentation performance. The person skilled in the art would understand that an accelerated, reduced or avoided diauxic shift can be observed through, e.g., monitoring growth using optical density measurements (e.g., ODeoo) and observing a reduced or absent lag phase following the initial phase of rapid exponential growth. Additionally, an accelerated, reduced or avoided diauxic shift can be observed through, e.g., depletion of primary and secondary substrate, which may be quantified using techniques such as high-performance liquid chromatography (HPLC), enzymatic assays to measure the concentrations of specific substrates and byproducts in the culture medium, or conducting gene expression analysis.

[0148] In certain embodiments, the microorganisms comprise dairy or cheese starter or adjunct cultures, for example Lactococcus lactis, Streptococcus thermophilus, Lactobacillus species, Leuconostoc species, Penicillium camemberti, Penicillium roqueforii. Geotrichum candidum. Debaryomyces hansenii. Kluyveromyces lactis and Kluyveromyces marxianus. In such embodiments, the treated cultures may be concentrated and subsequently subjected to freeze-drying or other dehydration processes, optionally with cryoprotectants or encapsulating matrices, to produce shelf-stable starter or adjunct preparations with improved viable cell counts and stress resistance relative to untreated controls.

[0149] In certain embodiments, the microorganisms treated according to the second aspect are selected from one or more of: eubacteria, actinomycetes, probiotic yeast, probiotic bacteria, Lactobacilli, Lactobacillus species, Lactobacillus-\d e species, Bifidobacterium species, Bifidobacierium-\\ c species Saccharomyces spp. and other probiotic species, strains and genera, which together comprise a probiotic group that is a non-taxonomic grouping. In other embodiments, the microorganisms are selected from among the microorganisms described elsewhere in this disclosure for use in probiotic, dairy, beverage, feed, biofuel, bioenergy, enzyme, or therapeutic production.

[0150] In certain embodiments, the method may be applied to any Ascomycetes species utilised in various alcohol production processes, including fermentation for beverages such as beer, wine, and sake, as well as bioethanol production and traditional brewing methodologies. The Ascomycetes species to which the method may be applied include, but are not limited to, Saccharomyces cerevisiae.Saccharomyces pastorianus, Saccharomyces bayanus, Saccharomyces uvarum, Saccharomyces paradoxus, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces eubayanus, Saccharomyces mikatae, Saccharomyces arboricola, Saccharomyces castellii, Saccharomyces jurei, Saccharomyces mikadoi, Saccharomyces cariocanus, Saccharomyces rouxii, Saccharomyces paradoxusvar. balsamiferae, Saccharomyces ludwigii, Saccharomyces fibuligera, Saccharomyces exiguus (formerly Candida milleri), Starmerella bacillaris (formerly Candida stellata), Candida versatilis, Candida tropicalis, Candida shehatae, Candida utilis, Aspergillus niger. Aspergillus oryzae. Aspergillus sojae. Aspergillus fimigaius. Aspergillus davaius. Aspergillus ierreus. Aspergillus nididans. Aspergillus tubingensis, Aspergillus flavus, Aspergillus wentii, Aspergillus iamarii. Aspergillus brasiliensis, Aspergillus chevalieri, Aspergillus carbonarius. Aspergillus foe Ildus. Aspergillus glaucus, Aspergillus me Ileus. Aspergillus versicolor, Aspergillus ustus, Aspergillus niveus, Aspergillus pseudoglaucus , Aspergillus puccini, Aspergillus sulphureus, Neosartorya fischeri (teleomorph of Aspergillus fischerij. Neosartorya udagawae, Neosartorya siamensis, Neosartorya pseudofischeri , Neosartorya glabra, Neosartorya spinosa, Neosartorya hiratsukae, Neosartorya fisheri, Neosartorya argillacea, Neosartorya canadensis, Neosartorya quadrilateralis , Neosartorya paulistensis, Neosartorya himalayensis, Neosartorya Candida, Neosartorya fischeri var. japonica, Neosartorya hamata, Neosartorya minuta, Neosartorya bipola, Neosartorya clavigera, Neosartorya fumigata, Neosartorya leptoceras, Neosartorya parahiratsukae , Neosartorya saskatchewanensis , Neosartorya Stella, Neosartorya tetrasperma, Neosartorya webbii, Neosartorya biformis, Neosartorya cylindrospora, Neosartorya echinulata, Neosartorya granulosa, and Neosartorya lornensis. The selection of specific species can be tailored to the type of alcohol production, desired flavour profiles, sugar profiles and efficiency of fermentation.

[0151] In certain embodiments, the method may be applied to any edible Ascomycetes species utilised in various food and beverage production processes, including culinary applications such as morels (Morchella spp.) and truffles (Tuber spp.), as well as other fungi employed in food fermentations and production. The edible Ascomycetes species to which the method may be applied include, but are not limited to, Morchella esculenta, Morchella conica, Morchella deliciosa, Morchella vulgaris, Tuber melanosporum, Tuber magnatum, Tuber aestivum, Tuber borchii, Tuber indicum, Tuber uncinatum, Aspergillus oryzae, Aspergillus sojae, Aspergillus niger (used in citric acid production), Aspergillus fumigatus (in controlled fermentation processes), Penicillium camemberti (used in Camembert cheese production), Penicillium roqueforti (used in Roquefort and other blue cheeses), Penicillium nalgiovense (used in salami curing), Saccharomyces cerevisiae (used in baking, brewing, and winemaking), Saccharomyces pastorianus, Saccharomyces bayanus, Saccharomyces uvarum, Kluyveromyces lactis (used in dairy fermentations), Kluyveromyces marxianus, Debaryomyces hansenii (used in cheese and meat fermentations), Pichia pastoris (now Komagataella phaffii, used in enzyme production), Yarrowia lipolytica (used in specialty food ingredient production), Geotrichum candidum (used in cheese and dairy product fermentations), Neurospora crassa, Neurospora intermedia (used in traditional fermentations), Candida utilis (used as a protein source in food products), Metschnikowia pulcherrima (used in wine fermentation and biocontrol), and Rhizopus oryzae, Aspergillus oryzae, Saccharomycopsis fibuligera, Issatchenkia orientalis, Pichia anomala (used in jiuqu, koji or baijiu).

[0152] In certain embodiments, the method may be applied to any Ascomycetes species utilised in the production of antibiotics, encompassing both established and emerging fungal sources. The Ascomycetes species to which the method may be applied include, but are not limited to, Penicillium chrysogenum (producer of penicillin), Penicillium notatum (another penicillin producer), Acremonium chrysogenum (producer of cephalosporins), Fusarium solani (source of fusaric acid derivatives), Aspergillus terreus (producer of terreic acid derivatives with antibiotic properties), Neosartorya fischeri (teleomorph of Aspergillus fischeri, involved in secondary metabolite production), Eurotium ruhrum (producer of rubratoxins with antibiotic activity), Aspergillus fumigatus (source of fumagillin and related compounds), Aspergillus nidulans (used in genetic studies for antibiotic compound production), Aspergillus clavatus (producer of clavine alkaloids with antimicrobial properties), Aspergillus flavus (source of aflatoxins with potential antibiotic applications), Aspergillus oryzae (used in fermentation processes that can yield antibiotic compounds), Aspergillus sojae (similar applications as A. oryzae), Aspergillus versicolor (producer of sterigmatocystin derivatives with antimicrobial activity), Aspergillus carbonarius (source of ochratoxins with potential antibiotic uses), Aspergillus tamarii (involved in the production of bioactive secondary metabolites), Aspergillus brasiliensis (producer of bioactive compounds with antimicrobial properties), Aspergillus chevalieri (source of novel antimicrobial agents), Aspergillus wentii (producer of wentilactones with antibiotic activity), Aspergillus tubingensis (involved in the synthesis of antimicrobial metabolites), Aspergillus sydowii (source of sydonic acid derivatives with potential antibiotic applications), Aspergillus restrictus (producer of restrictin with antimicrobial properties), Aspergillus glaucus (source of glaucocin with antibiotic activity), Aspergillus melleus (producer of mellein derivatives with antimicrobial properties), Aspergillus ustus (source of ustatic acids with antibiotic activity), Aspergillus niveus (producer of niveicin with antimicrobial properties), Aspergillus pseudoglaucus (involved in the production of bioactive antibiotic compounds), Aspergillus puccini (source of pucciniastatin with potential antibiotic applications), Aspergillus sulphureus (producer of sulfur-containing antibiotic metabolites), and Neosartorya udagawae (teleomorph involved in the synthesis of antibiotic secondary metabolites).

[0153] In certain embodiments, the method may be applied to any commercially important Ascomycetes species utilised across various industries, including food production, biotechnology, agriculture, pharmaceuticals, nutraceuticals, and beauty product manufacture. The Ascomycetes species to which the method may be applied include, but are not limited to, Trichoderma reesei, renowned for its exceptional cellulase and hemicellulase production used in biofuel, textile, and paper industries; Fusarium venenatum, the primary source of mycoprotein for Quom® meat substitutes; Claviceps purpurea, known for producing ergot alkaloids essential in pharmaceuticals for treating migraines and managing psychiatric disorders; Saccharomyces boulardii, a probiotic yeast strain employed in dietary supplements for gastrointestinal health; Trichoderma harzianum, utilised as a biocontrol agent in agriculture to protect crops from fungal pathogens; Aspergillus cristatus, used in the fermentation of traditional teas such as Pu-erh to enhance flavour profdes; Saccharomyces stipitis, distinguished by itsability to ferment pentose sugars for bioethanol production from lignocellulosic biomass; Fusarium oxysporum, utilised in biofuel production and bioremediation processes due to its metabolic versatility; Penicillium chrysogenum. significant in both antibiotic production and the food industry for certain cheeses and enzyme production; Neurospora cras a. employed in biotechnology for industrial enzyme production and metabolic pathway studies; Yarrowia lipolytica, extensively used for producing specialty lipids, organic acids, and as a host for recombinant protein expression; and Geotrichum candidum. essential in the dairy industry for cheese production, contributing to flavour development, texture modification, and rind formation.

[0154] In addition to the abovementioned Ascomycetes species, the method of the disclosure is able to enhance the growth of microorganisms generally, including but not limited to Schizosaccharomyces pombe (utilised in brewing and research applications), Hansenula polymorpha (employed in recombinant protein production and industrial biotechnology), Torulaspora delbrueckii (used in winemaking and baking for flavour enhancement), and Eurotium cristatum (utilised in traditional tea fermentation processes such as Fu brick tea).

[0155] Moreover, the method may be applied to enhance the growth of other Ascomycetes species, including but not limited to Ashbya gossypii (utilised in the industrial production of riboflavin), Wickerhamomyces anomalus (applied in biocontrol and fermentation processes), Thermoascus aurantiacus (produces thermostable enzymes for industrial applications), and Pichia methanolica (used in the synthesis of specialty chemicals and bioconversion processes). Additionally, Botrytis cinerea (employed in the production of enzymes and as a model organism in plant pathology studies) and Eurotium amstelodami (utilised in enzyme production and biotransformation processes) are included.

[0156] Beyond Ascomycetes, the method of the disclosure extends to enhancing the growth of various eubacteria, archaebacteria, and other commercially important microorganisms. This includes Escherichia coli (widely used for recombinant protein and biologic production such as insulin), Bacillus subtilis (employed in enzyme production and as a host for industrial biotechnology applications), Pseudomonas putida (utilised in bioremediation and the synthesis of biodegradable plastics), Corynebacterium glutamicum (used in amino acid production), Streptomyces coelicolor (a model organism for antibiotic discovery), and Lactobacillus acidophilus (employed in probiotic supplements and dairy fermentations). Archaebacteria such as Methanococcus jannaschii (used in studies of methane production and biogas applications) and Halobacterium salinarum (employed in high-salt fermentation processes) are also included. Additionally, cyanobacteria like Synechocystis sp. (utilised in biofuel production and carbon capture technologies) and Chlamydomonas reinhardtii (employed in biohydrogen production and as a model organism for photosynthesis research) are encompassed. Many bacteria in the Lactobacilli group produce proteins with antibiotic properties such as bacteriocin proteins, collectively bacteriocins, thathave beneficial effects for gut health. Microorganisms in the probiotic group can also be lysed to produce a beauty supplement for application on human skin, to benefit skin ecology and skin health.

[0157] Other commercially important microorganisms to which the method may be applied include Bacillus licheniformis (used in enzyme and antibiotic production), Mycobacterium smegmatis (employed in bioremediation and pharmaceutical research), and Gluconobacter oxydans (utilised in the production of vitamin C and other biochemicals). Additionally, Thermomyces lanuginosus (produces lipases for industrial applications) and Rhodotorula glutinis (used in carotenoid and lipid production) are included.

[0158] As would be appreciated by the person skilled in the art, the method may use a diverse array of substrates to support the growth and metabolic activity of the microorganism. Suitable substrates include simple sugars such as glucose, fructose, sucrose, maltose, and lactose for fermentation and biomass production; complex carbohydrates like starch, cellulose, and lignocellulosic biomass for sustained energy and structural components; nitrogen sources including ammonium sulfate, yeast extract, peptones, and amino acids for protein synthesis and cellular metabolism; essential vitamins and minerals such as biotin, thiamine, magnesium, potassium, calcium, and trace elements for enzymatic functions and cellular health; specialised substrates like molasses, com steep liquor, glycerol, and ethanol to optimise fermentation conditions and product yields; precursors for secondary metabolite biosynthesis in antibiotic production; substrates such as dairy whey, soy extract, fruit juices, and vegetable infusions for food production processes; biomass-derived materials like spent grains, agricultural residues (e.g., com stover, sugarcane bagasse, rice husks, cereal husks, lucerne, spent cropping plants, unused stems and leaves, inedible plant materials, hard or woody materials, soft decaying organic matter), municipal solid waste, and other lignocellulosic by-products for biofuel production; and algae biomass or aquatic plants supplying lipids and carbohydrates for biodiesel and biogas generation. The person skilled in the art would understand how to select and tailor these substrates to meet the specific requirements of the microorganism and the intended industrial application.

[0159] As would be appreciated by a person skilled in the art, determining the appropriate culture conditions for a microorganism to meet specific industrial applications involves optimising parameters such as temperature, pH, aeration, agitation, humidity (if not submerged), and fermentation time. Temperature requirements typically range from about 10°C to 50°C, with specific organisms thriving within narrower ranges, such as 25-30°C for yeast fermentation or 37°C for bacterial cultures like Escherichia coli. pH levels are generally maintained between 4.0 and 8.0, with acidic conditions (pH 4.0- 6.0) favoured in lactic acid fermentations and near-neutral pH (6.5-7.5) in bacterial production systems. Aeration can vary from minimal to high, measured in volumes of air per volume of culture per minute (e.g., 0.1-1.0 volumes of air per volume of liquid per minute (vvm)), while agitation speeds typically range from 50 to 500 rpm, depending on the reactor size and the organism's oxygen demand. For solid- state fermentations, relative humidity levels are often maintained at 60-90%, while submergedfermentations rely on dissolved oxygen concentrations tailored to the microorganism’s needs. Fermentation time may vary widely, from a few hours for rapid bacterial processes to several weeks or months for complex fermentations such as cheese maturation or wine aging. In some embodiments, the pH is maintained at the selected value by intermittent or continuous addition of an alkali or acid to the medium.

[0160] In some embodiments, the electric field in these probiotic-specific applications may be generated by applying a voltage to at least one terminal. The electric field in these probiotic-specific applications may be generated by applying a voltage to a first terminal and a second terminal, or more terminals, which are spaced apart by at least milllimetres and not in contact with each other, and in the case of two terminals the voltages supplied to the two terminals may have opposite polarities or the same polarities.

[0161] In some embodiments, the terminal to which the voltage is supplied may be electrically insulated so that current does not flow from the terminal into the growth medium. The applied voltage and waveform may be either of an alternating current (AC) or a direct current (DC) type. The polarity of the voltage in any one terminal may be iteratively reversed at one or more frequencies in the range of about 210 Hz to about 99 kHz. This comprises an AC modulation. The on / off status of the voltage in any one terminal may be toggled at one or more frequencies in the range of about 210 Hz to about 99 kHz. This comprises a DC modulation.

[0162] The device or system of this disclosure may be used in accordance with any aspect where the enhancement of microbial growth, viability or activity is desired, particularly for probiotic applications.

[0163] In a third aspect, there is provided a method of modifying transcription in a microorganism, comprising: exposing the microorganism to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0164] As would be appreciated by a person skilled in the art, exposing the microorganism to an electric field refers to positioning the microorganism within the influence of the field.

[0165] As would be appreciated by the person skilled in the art, the method may be applied to any microorganism to modify transcription. In certain embodiments, the method may be applied to any microorganism to increase transcription. In certain embodiments, the method may be applied to any microorganism to decrease transcription. The microorganism may be one or more microorganism described in the second aspect, or elsewhere in this disclosure.

[0166] As would be appreciated by the person skilled in the art, transcription in microorganisms involves a coordinated effort by several essential enzymes and protein complexes. The method may modify the activity of one or more of these enzymes and protein complexes. These include RNA polymerase, which catalyses the synthesis of RNA from a DNA template; transcription factors, such as sigma factors, that assist in promoter recognition and initiation of transcription; helicases, which unwind the DNA duplex to provide a single-stranded template for RNA synthesis; topoisomerases, including topoisomerase I and topoisomerase II, which relieve supercoiling ahead of the transcription machinery; and elongation factors, such as NusA and NusG, that stabilise the transcription complex and regulate RNA chain elongation. Additional enzymes include RNA ligases, which repair nicks in RNA molecules, and RNA processing enzymes, such as ribonucleases and RNA methyltransferases, which modify and mature RNA transcripts. In certain microorganisms, transcription is also influenced by histone-like proteins and nucleoid-associated proteins that modulate DNA structure and accessibility. These enzymes and factors collectively ensure the accurate and efficient transcription of genetic material, which is vital for cellular function and adaptation in microbial systems.

[0167] In certain embodiments, the method modifies transcription levels in microorganisms, resulting in either an increase or, in other embodiments, a reduction in transcriptional activity. The controlled modulation of transcription may enhance the expression of target genes for increased production of desired biomolecules or, alternatively, to suppress the transcription of specific genes to reduce the synthesis of unwanted or detrimental products. The genes with modified transcription may include, e.g., any one or more genes encoding any protein mentioned in the first aspect, or elsewhere in this disclosure.

[0168] In certain embodiments, the method comprises increasing the expression of one or more genes. In certain embodiments, the method comprises increasing the expression of one or more genes such as AZFI, MBF1, STP2, IOC4, SNF2, PG1, and MSN4, which regulate transcriptional pathways that enhance glucose transport and utilization, including stress responses and derepression of glucose- repressed genes. In certain embodiments, AZFI is upregulated by between about 15% to about 30%, such as about 23%; MBF1 by between about 12% to about 24%, such as about 18%; STP2 by between about 12% to about 24%, such as about 17%; IOC4 by between about 12% to about 24%, such as about 17%; SNF2 by between about 10% to about 20%, such as about 15%; PG1 by between about 30% to about 45%, such as about 39%; and MSN4 by between about 10% to about 20%, such as about 15%.

[0169] In certain embodiments, the method comprises increasing the expression of one or more genes such as SKY1, YHC1, CEF1, PRP40, HRP1, and REF2, which are involved in pre-mRNA splicing and processing, ensuring correct open reading frames (ORFs) and biological function. In certain embodiments, SKY1 is upregulated by between about 15% to about 21%, such as about 18%; YHC1 by between about 12% to about 16%, such as about 14%; CEF1 by between about 12% to about 16%, such as about 14%; PRP40 by between about 14% to about 18%, such as about 16%; HRP1 by between about25% to about 37%, such as about 31%; and REF2 by between about 12% to about 16%, such as about 14%.

[0170] In certain embodiments, the method comprises increasing the expression of one or more genes such as HXT4 and HXT3, which encode hexose transporters directly responsible for glucose uptake into the cell, allowing efficient utilization of glucose as a primary carbon source. In certain embodiments, HXT4 is upregulated by between about 180% to about 240%, such as about 209%; and HXT3 by between about 130% to about 175%, such as about 153%.

[0171] In certain embodiments, the method comprises increasing the expression of one or more genes such as SMP1, STF2, SIP18, PBS2, and SOD1, which encode stress response proteins. In certain embodiments, SMP1 is upregulated by between about 65% to about 85%, such as about 75%; STF2 by between about 50% to about 65%, such as about 57%; SIP18 by between about 40% to about 55%, such as about 48%; PBS2 by between about 40% to about 55%, such as about 47%; and SOD1 by between about 40% to about 55%, such as about 46%.

[0172] In certain embodiments, the method comprises increasing the expression of one or more genes such as TH 12. ALD4, CRC1, CAT2, and POTI, which support growth on ethanol as an energy source. In certain embodiments, THI2 is upregulated by between about 20% to about 35%, such as about 26%;ALD4 by between about 20% to about 35%, such as about 26%; CRC1 by between about 25% to about 45%, such as about 35%; CAT2 by between about 35% to about 50%, such as about 41%; and POTI by between about 25% to about 45%, such as about 34%.

[0173] In certain embodiments, the method comprises increasing the expression of one or more genes such as ADH4 and EEB1, which support alcohol and ester production during fermentation. In certain embodiments, ADH4 is upregulated by between about 65% to about 85%, such as about 74%; and EEB1 by between about 25% to about 37%, such as about 31%.

[0174] In certain embodiments, the method comprises increasing the expression of one or more genes such as CITI, IDH1, IDH2, MDH1, and PGM2, which are involved in glycogen synthesis, gluconeogenesis, and the TCA cycle. In certain embodiments, CITI is upregulated by between about 10% to about 25%, such as about 17%; IDH1 by between about 10% to about 25%, such as about 17%; IDH2 by between about 10% to about 20%, such as about 14%; MDH1 by between about 20% to about 35%, such as about 29%; and PGM2 by between about 20% to about 30%, such as about 25%.

[0175] In certain embodiments, the method comprises increasing the expression of one or more genes such as QCR7, QCR2, CYC7, NDE2, ATP4, ATP5, SER3, and IDP2, which are involved in respiratory chain function and ATP production. In certain embodiments, QCR7 is upregulated by between about 20% to about 28%, such as about 24%; QCR2 by between about 35% to about 45%, such as about 40%; CYC7by between about 50% to about 70%, such as about 60%; NDE2 by between about 55% to about 70%, such as about 62%; ATP4 by between about 12% to about 20%, such as about 16%; ATP5 by between about 10% to about 18%, such as about 14%; SER3 by between about 35% to about 47%, such as about 41%; and IDP2 by between about 55% to about 70%, such as about 62%.

[0176] In certain embodiments, the method comprises increasing the expression of one or more genes such as HXK1, GLK1, and PFK1, which are involved in glycolysis and glucose utilization. In certain embodiments, HXK1 is upregulated by between about 50% to about 70%, such as about 59%; GLK1 by between about 20% to about 32%, such as about 26%; and PFK1 by between about 10% to about 18%, such as about 14%.

[0177] In certain embodiments, the method comprises increasing the expression of one or more genes such as CLN3, CLN2, CDC55, CDC4, MCM10, SLD7, and SLD3, which regulate cell division. In certain embodiments, CLN3 is upregulated by between about 30% to about 40%, such as about 34%; CLN2 by between about 10% to about 20%, such as about 15%; CDC55 by between about 15% to about 25%, such as about 21%; CDC4 by between about 15% to about 25%, such as about 19%; MCM10 by between about 20% to about 28%, such as about 24%; SLD7 by between about 18% to about 24%, such as about 21%; and SLD3 by between about 10% to about 18%, such as about 14%.

[0178] In certain embodiments, the method comprises increasing the expression of one or more genes such as RRN3, MRP1, MRPS9, MRPS12, MRPS16, MRPL7, PUF6, NOG2, IFH1, and RRT14, which are involved in ribosomal biogenesis and translation. In certain embodiments, RRN3 is upregulated by between about 35% to about 50%, such as about 44%; PUF6 by between about 35% to about 55%, such as about 45%; NOG2 by between about 50% to about 70%, such as about 61%; IFH1 by between about 25% to about 45%, such as about 34%; and RRT14 by between about 45% to about 55%, such as about 49%.

[0179] In certain embodiments, the method comprises increasing or decreasing the expression of one or more genes selected from Table 9 (Full list of 392 genes differentially expressed in the treated fermentation of experiment 3 on day 5), Table 10 (Full list of 169 genes differentially expressed in the treated fermentation of experiment 3 on day 8 / 9), Table 11 (List of 607 genes upregulated in the treated fermentation of experiment 3 on day 5) or Table 12 (List of 682 genes upregulated in the treated fermentation of experiment 3 on day 8 / 9), as appropriate. In certain embodiments, the method comprises increasing the expression of one or more genes shown to be upregulated in Tables 9, 10, 11 and 12. In certain embodiments, the method comprises decreasing the expression of one or more genes shown to be downregulated in Tables 9, 10, 11 and 12. While specific levels of upregulation or downregulation has been observed, the disclosure contemplates variations in the changes of expression level, for example, expression levels that are 2-fold to 4-fold lower, or 2-fold to 4-fold higher than recorded. The personskilled in the art would understand that variations in gene expression may occur due to variations in microorganisms, substrates or culture conditions.

[0180] In certain embodiments, the method may increase or decrease the expression of one or more genes by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%; by at least 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%; or may increase or decrease the expression by 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or at least 10-fold, as appropriate. In certain embodiments, the method may increase gene expression by about 2% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about90%, or about 90% to about 100%. In certain embodiments, the method may increase gene expression by about 2-fold to about 4-fold, about 2-fold to about 6-fold, or about 2-fold to about 8-fold. In certain embodiments, the method may decrease gene expression by about 2% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%. In certain embodiments, the method may decrease gene expression by about 2-fold to about 4-fold, about 2-fold to about 6-fold, or about 2-fold to about 8-fold. As would be appreciated by a person skilled in the art, increases in gene expression can be monitored using techniques such as quantitative PCR (qPCR) or RNA sequencing (RNA-seq) to measure mRNA levels, and reporter assays using luciferase or GFP to assess transcriptional activity. Protein-level changes, as indirect indicators, can be detected via Western blotting or ELISA.1. In certain embodiments, the microorganism has increased levels of primary metabolites, relative to an untreated microorganism. In certain embodiments, 3 -hydroxybutyric acid increases by between about 10% to about 14%, such as about 12%; 4 -aminobutyric acid increases by between about 15% to about 19%, such as about 17%; palmitic acid increases by between about 14% to about 18%, such as about 16%; 2-hydroxyglutaric acid increases by between about 11% to about 15%, such as about 13%; fumaric acid increases by between about 6% to about 10%, such as about 8%; pyruvic acid increases by between about 13% to about 17%, such as about 15%; and dihydroxyacetone phosphate increases by between about 4% to about 6%, such as about 5%. A person skilled in the art would understand that a metabolite of cellular origin, manufactured using the device method and system of this disclosure, is increased in amount or activity by the electric field providing surmounting energy to a functional protein or to a set of functional proteins that work together for increased production of the metabolite.

[0181] The method may use a diverse array of substrates to support the growth and metabolic activity of the microorganism as described in the second aspect, or elsewhere in this disclosure. The method may use a range of culture conditions as described in the second aspect, or elsewhere in this disclosure.

[0182] In a fourth aspect, there is provided a method of enhancing the flavour of a fermented food, beverage or wash, comprising: contacting a food, beverage or wash substrate with a microorganism to provide a slurry, wherein the microorganism is adapted to metabolise the substrate, exposing the slurry to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0183] As would be appreciated by a person skilled in the art, exposing the slurry to an electric field refers to positioning the slurry within the influence of the field.

[0184] As would be appreciated by a person skilled in the art enhancing the flavour of a fermented food, beverage, or wash refers to increases in the presence or balance of desirable flavour compounds produced during fermentation. These compounds, which contribute to the sensory profile of the product, may include primary metabolites, such as ethyl hexanoate, pyruvic acid, dihydroxyacetone phosphate, 3- hydroxybutyric acid and 4-aminobutyric acid, which add complexity and depth to the flavour.Additionally, specific acids, such as fumaric acid, 2 -hydroxyglutaric acid, and palmitic acid, may enhance mouthfeel, acidity, or richness. In certain embodiments, the desirable flavour compounds increase by at least 2%, such as between about 3% and about 20%, between about 4% and 15%, or about 5% and 13%, compared to an untreated control and when normalised against glucose. In other embodiments, the desirable flavour compounds increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, or about 10% and 70%, compared to an untreated control. The method may also involve promoting the production of volatile compounds associated with fruit and floral aromas, such as those imparting notes of banana (e.g. methylbutyl acetate), cherry (e.g. ethyl acetate), green apple (e.g. ethyl hexanoate), and floral / rose (e.g. 2-phenyl ethanol). In certain embodiments, the volatile compounds associated with fruit and floral aromas increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, about 10% and 70%, or about 10% and 60%, compared to an untreated control. Enhancing these flavour compounds may improve the overall sensory characteristics of the food, beverage, or wash for its intended application.

[0185] In certain embodiments, enhancing the flavour of a fermented food, beverage, or wash refers to decreasing in the presence or balance of undesirable flavour compounds produced during fermentation, such as acetate esters, e.g. ethyl acetate or 3-methylbutyl acetate; or fusel alcohols, e.g. 3-methyl butanol or 2-phenyl ethanol. In certain embodiments, the undesirable flavour compounds decrease by at least 2%, such as between about 3% and about 20%, between about 4% and 15%, or about 5% and 13%, compared to an untreated control.

[0186] The person skilled in the art could select a suitable substrate based on the intended fermented food, beverage or wash. The microorganism being adapted to metabolise the substrate refers to the ability of the microorganism to utilise the specific substrate as a source of carbon, nitrogen, or other essential nutrients to sustain its growth, reproduction, or metabolic activity. In certain embodiments, the food,beverage, or wash substrate comprises one or more of simple sugars such as glucose, fructose, sucrose, maltose, and lactose for fermentation and biomass production; complex carbohydrates like starch for sustained energy release; molasses to enhance fermentation conditions and product yields; dairy whey for dairy-based beverages; soy extract for plant-based products; fruit juices for fermented beverages; vegetable infusions for specialised food or drink formulations; or spent grains from brewing processes, which provide residual nutrients and organic matter for wash production. The person skilled in the art can select these and other substrates to meet the specific requirements of the microorganism and the intended application, such as food or beverage fermentation or the production of a wash for alcoholic spirits.

[0187] The microorganisms may be any suitable microorganism used in fermenting food, beverage, or wash substrates. For example, one or more of Saccharomyces cerevisiae, Saccharomyces pastoricinus, Saccharomyces bayanus, Saccharomyces uvarum, widely used in brewing, winemaking, and baking, as well as Aspergillus oryzae and Aspergillus sojae. essential for soy-based fermentations and sake production. Penicillium camemberti and Penicillium roqueforti are critical for cheese production, while Penicillium nalgiovense supports salami curing. Dairy fermentations often use Kluyveromyces lactis, Kluyveromyces marxianus, and Geotrichum candidum. with Debaryomyces hansenii enhancing cheese and meat fermentation. Less common species include Metschnikowia pulcherrima for wine fermentation, Torulaspora delbrueckii for aroma enhancement in baking and winemaking, Wickerhamomyces anomalus for biocontrol and niche applications, Schizosaccharomyces pombe used in brewing and research, or Yarrowia lipolytica and Neurospora intermedia used in specialty and traditional fermentations. In other embodiments, the microorganism is one or more microorganism described in the second aspect, or elsewhere in this disclosure.

[0188] The method may use a range of culture conditions as described in the second aspect, or elsewhere in this disclosure.

[0189] As above, the method comprises contacting a food, beverage or wash substrate with a microorganism to provide a slurry, that is, inoculating the substrate. The typical inoculation rate is within the range of 1% to 10% (v / v) of the slurry volume, depending on the type of microorganism and the specific fermentation application. Based on this, in certain embodiments, the concentration of the microorganism in the slurry may be in the range of about 2 milligrams dry weight per millilitre of slurry (mg / mL) to about 200 mg / mL, for example, about 5 to 150 mg / mL, about 10 to 100 mg / mL, about 15 to 50 mg / mL, or about 25 mg / mL. The person skilled in the art could routinely determine the appropriate inoculum size or microorganism concentration based on experimentally determined fermentation parameters, such as substrate utilisation or metabolite production.

[0190] In a fifth aspect, there is provided a method of disinhibiting cell division in a microorganism, comprising: exposing the microorganism to an electric field, that is essentially free of a magneticcomponent, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0191] As would be appreciated by a person skilled in the art disinhibiting cell division in a microorganism refers to removing or reducing the factors inhibiting the normal progression of the microbial cell cycle, thereby allowing or enhancing cell division. This may involve counteracting the effects of internal or external stressors, nutrient limitations, osmotic limitations, pressure limitations, pH limitations, oxidative limitations, accumulation of inhibitory metabolites, or genetic regulatory mechanisms that prevent cell division under certain conditions. For example, disinhibition may include modulating the availability of key nutrients, such as carbon or nitrogen sources, recycling cellular components from dead cells, or stimulating autophagy to provide essential nutrients through the degradation and repurposing of internal cellular materials. Additionally, the removal of toxic by-products, such as ethanol or lactic acid, which can accumulate during fermentation, can further support optimal conditions for cell division. It may also involve genetic or biochemical interventions, such as upregulating genes involved in the synthesis of essential cell division proteins or downregulating genes that encode division inhibitors. Chemical agents, such as quorum -sensing inhibitors or specific enzyme activators, may also be used to disinhibit division. By disinhibiting cell division, the method can improve microbial growth rates and biomass production, enhancing the efficiency of industrial processes, such as fermentation, biofuel production, or bioproduct synthesis.

[0192] Disinhibiting cell division in a microorganism may also be used to accelerate the diauxic shift, thereby maintaining exponential growth and preventing or reducing the lag phase associated with the shift to a secondary carbon source. This may be achieved by promoting cellular processes such as protein recycling, endocytosis, and selective autophagy prior to and / or during the time when the diauxic shift would typically occur. These mechanisms enable the microorganism to efficiently utilise internal resources, compensating for the depletion of primary nutrients like glucose and oxygen. By activating genes involved in these pathways, the microorganism sustains growth and metabolic activity, avoiding the metabolic slowdown and inhibition of division associated with the diauxic shift. This approach enhances microbial productivity and supports continuous fermentation processes in industrial applications.

[0193] In certain embodiments, the method comprises increasing the expression of one or more genes as described in the third aspect. For example, increasing or decreasing the expression of one or more genes selected from AZFI, MBF1, STP2, IOC4, SNF2, PG1, MSN4, SKY1, YHC1, CEF1, PRP40, HRP1, REF2, HXT4, HXT3, SMP1, STF2, SIP18, PBS2, SOD1, THI2, ALD4, CRC1, CAT2, POTI, ADH4, EEB1, CITI, IDH1, IDH2, MDH1, PGM2, QCR7, QCR2, CYC7, NDE2, ATP4, ATP5, SER3, IDP2, HXK1, GLK1, PFK1, CLN3, CLN2, CDC55, CDC4, MCM10, SLD7, SLD3, RRN3, MRP1,MRPS9, MRPS12, MRPS16, MRPL7, PUF6, N0G2, IFH1, and RRT14. The changes in expression may be as described in the third aspect, or elsewhere in this disclosure.

[0194] In certain embodiments, the method comprises increasing or decreasing the expression of one or more genes selected from Table 9 (Full list of 392 genes differentially expressed in the treated fermentation of experiment 3 on day 5), Table 10 (Full list of 169 genes differentially expressed in the treated fermentation of experiment 3 on day 8 / 9), Table 11 (List of 607 genes upregulated in the treated fermentation of experiment 3 on day 5) or Table 12 (List of 682 genes upregulated in the treated fermentation of experiment 3 on day 8 / 9), as appropriate. In certain embodiments, the method comprises increasing the expression of one or more genes shown to be upregulated in Tables 9, 10, 11 and 12. In certain embodiments, the method comprises decreasing the expression of one or more genes shown to be downregulated in Tables 9, 10, 11 and 12. While specific levels of upregulation or downregulation has been observed, the disclosure contemplates variations in the changes of expression level, for example, expression levels that are 2-fold to 4-fold lower, or 2-fold to 4-fold higher than recorded. The person skilled in the art would understand that variations in gene expression may occur due to variations in microorganisms, substrates or culture conditions.

[0195] In certain embodiments, the method may increase or decrease the expression of one or more genes by at least 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%; by at least 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%; or may increase or decrease the expression by 2-fold, 3 -fold, 4-fold, 5 -fold, 6-fold, 7-fold, 8 -fold, 9-fold, or at least 10-fold, as appropriate. In certain embodiments, the method may increase gene expression by about 2% to about10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about90%, or about 90% to about 100%. In certain embodiments, the method may increase gene expression by about 2-fold to about 4-fold, about 2-fold to about 6-fold, or about 2-fold to about 8-fold. In certain embodiments, the method may decrease gene expression by about 2% to about 10%, about 10% to about 20%, about 20% to about 30%, about 30% to about 40%, about 40% to about 50%, about 50% to about 60%, about 60% to about 70%, about 70% to about 80%, about 80% to about 90%, or about 90% to about 100%. In certain embodiments, the method may decrease gene expression by about 2-fold to about 4-fold, about 2-fold to about 6-fold, or about 2-fold to about 8-fold. As would be appreciated by a person skilled in the art, increases in gene expression can be monitored using techniques such as quantitative PCR (qPCR) or RNA sequencing (RNA-seq) to measure mRNA levels, and reporter assays using luciferase or GFP to assess transcriptional activity. Protein-level changes, as indirect indicators, can be detected via Western blotting or ELISA.

[0196] As would be appreciated by a person skilled in the art, exposing the microorganism to an electric field refers to positioning the microorganism within the influence of the field. The microorganismmay be one or more microorganism described in the second aspect, or elsewhere in this disclosure. The method may use a range of culture conditions as described in the second aspect, or elsewhere in this disclosure. The method may use a diverse array of substrates to support the growth and metabolic activity of the microorganism as described in the second aspect, or elsewhere in this disclosure.

[0197] In a sixth aspect, there is provided a method of brewing beer, comprising: contacting a grist, grain or cereal with water to provide a mash; exposing the mash to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz, to provide a treated mash; obtaining a wort from the treated mash; and using the wort to brew beer.

[0198] The method of the sixth aspect relates to a series of steps in the process of brewing beer. The person skilled in the art could readily incorporate the method into the process of brewing beer, which may optionally comprise the steps of malting, wherein grains such as barley are soaked, germinated, and dried to develop enzymes necessary for starch conversion; milling, wherein the malt is ground into a coarse powder (grist) to expose the starches; mashing, which involves mixing the grist with hot water to activate enzymes that convert starches into fermentable sugars; lautering, wherein the mash is transferred to a vessel to separate the liquid wort from the spent grain; boiling, wherein the wort is heated with hops to sterilise it, extract hop flavours and bitterness, and concentrate the sugars; cooling, wherein the boiled wort is rapidly cooled to fermentation temperature to prevent contamination; and fermentation, during which yeast is added to the cooled wort to convert the sugars into alcohol and carbon dioxide, thereby producing beer.

[0199] The method is useful for producing a treated wort to brew beer. Without wishing to be bound by theory, it is thought that the application of the electric field enhances protein breathing by affecting the charged moieties within the mash liquid that contains enzymes. Protein breathing allows substrates and cofactors to react through the action of the enzymes to synthesise products.

[0200] In certain embodiments, the method comprises increasing the activity of one or more enzymes involved in producing glucose, mannose, galactose, allose, nonanoic acid, sorbitol, mannitol, glycerol, monostearin, or arabitol. As such, the wort obtained from the treated mash may comprise increased levels of one or more of glucose, mannose, galactose, allose, nonanoic acid, sorbitol, mannitol, glycerol, monostearin, or arabitol. As would be appreciated by the person skilled in the art, the increase would be relative to an untreated control.

[0201] In certain embodiments, the treated wort has greater proportion of the maltose and maltotriose converted to glucose, compared to untreated wort. In certain embodiments, the level of glucose is about 10% to about 100% higher in the treated wort. In certain embodiments, the level of glucose is about 10% to about 90%, about 10% to about 70%, about 20% to about 60% or about 30% to about 50% higher inthe treated wort. In certain embodiments, the level of either or both of maltose or maltotriose is reduced by about 10% to about 90%, about 10% to about 70%, about 20% to about 60% or about 30% to about 40% in the treated wort. As would be appreciated by the person skilled in the art, the increase or decrease would be relative to an untreated control.

[0202] In certain embodiments, the treated wort has reduced levels of specific sugars and carbohydrates relative to the control wort. In certain embodiments, the level of maltose is reduced by about 10% to about 30%, such as about 21%. In certain embodiments, the level of maltotriose is reduced by about 31% to about 51%, such as about 41%. In certain embodiments, the level of fructose is reduced by about 10% to about 30 %, such as about 20%. In certain embodiments, the level of sucrose is reduced by about 2% to about 10%, such as about 5%.

[0203] In certain embodiments, the treated wort has between about 5% and about 20% less total amino acids compared to an untreated control. For example, between about 5% and about 18%, about 8% and about 16% or about 13% less total amino acids compared to an untreated control.

[0204] In certain embodiments, the treated wort has an increased level of arginine, asparagine and glutamine compared to an untreated control. In certain embodiments, the level of asparagine is elevated by between about 5% and about 60%, between about 10% and about 50% or by about 37%. In certain embodiments, the level of arginine is elevated by between about 100% and 15 -fold, or between 5 -fold and 12-fold, or by more than 9-fold. %. In certain embodiments, the level of glutamine is elevated by between about 100% and about 10-fold, between about 2-fold and 6-fold, or by more than 3-fold. In certain embodiments, the treated wort has modified levels of amino acids as shown in Table 5 , Figure 11 or Figure 12. While specific increases or decreases in amino acid levels has been observed, the disclosure contemplates variations in these increases or decreases, for example, levels that are 2-fold to 4-fold lower, or 2-fold to 4-fold higher than recorded. The person skilled in the art would understand that variations in amino acid levels may occur due to variations in the grist, grain or cereal or mash conditions.

[0205] In certain embodiments, the treated wort has an increased level of total sugars as measured by Brix. In certain embodiments, the level of total sugars in the treated wort as measured by Brix is increased by between about 5% and about 25%, between about 7% and about 13% or by about 10%, relative to an untreated control.

[0206] In certain embodiments, the treated wort has increased levels of malted grain metabolites, when normalised to glucose, with specific compounds showing notable percentage increases relative to control wort. In certain embodiments, the level of 3 -hydroxybutyric acid is increased by at least 3%, between about 3% to about 12%, or more specifically by between about 5% to about 10%, such as about 7%. In certain embodiments, the level of mannose is increased by at least 35%, between about 35% to about 65%, or more specifically by between about 45% to about 55%, such as about 51%. In certainembodiments, the level of galactose is increased by at least 35%, between about 35% to about 65%, or more specifically by between about 45% to about 55%, such as about 51%. In certain embodiments, the level of glucose is increased by at least 35%, between about 35% to about 65%, or more specifically by between about 45% to about 55%, such as about 50%. In certain embodiments, the level of allose is increased by at least 35%, between about 35% to about 65%, or more specifically by between about 45% to about 55%, such as about 50%. In certain embodiments, the level of nonanoic acid is increased by at least 2%, between about 2% to about 8%, or more specifically by between about 3% to about 7%, such as about 5%. In certain embodiments, the level of sorbitol is increased by at least 35%, between about 35% to about 65%, or more specifically by between about 45% to about 55%, such as about 50%. In certain embodiments, the level of mannitol is increased by at least 35%, between about 35% to about 65%, or more specifically by between about 45% to about 55%, such as about 50%. In certain embodiments, the level of glycerol is increased by at least 10%, between about 10% to about 20%, or more specifically by between about 14% to about 18%, such as about 16%. In certain embodiments, the level of monostearin is increased by at least 2%, between about 2% to about 6%, or more specifically by between about 3% to about 5%, such as about 4%. In certain embodiments, the level of arabitol is increased by at least 180%, between about 180% to about 230%, or more specifically by between about 200% to about 210%, such as about 205%.

[0207] In certain embodiments, the grist, grain, or cereal comprises any suitable plant material containing starches, carbohydrates, or other fermentable or processable components. This may include barley, wheat, rye, oats, maize (com), sorghum, millet, rice, or triticale, as well as any varieties, hybrids, or genetically modified strains thereof. Additionally, it may encompass processed forms such as malted grains, cracked grains, rolled grains, flour, meal, or grits. By-products from grain processing, such as bran, husks, or spent grains, may also be included, as well as alternative sources like pseudocereals such as quinoa, amaranth, or buckwheat. The term may further extend to mixtures or blends of different grains and cereals to achieve specific nutritional, structural, or fermentation properties, tailored to the intended application in brewing.

[0208] The person skilled in the art would understand that a typical mash process involves heating water to a temperature of between 65°C and 80°C, usually between 72°C and 78°C. The heated water is then mixed with the grist, grain, or cereal, at a ratio of around 1: 1 to 1:2 (water-to-grain) to provide a mash. The mash is left to rest for a period of time, typically between 30 minutes to 60 minutes, allowing the enzymes present in the grain to convert starches into fermentable sugars. The person skilled in the art could readily adjust the mash temperature and duration based on factors such as the type of grist, grain, or cereal, water chemistry, and desired beer style. After the mash, the liquid is drained off, known as wort, and used to brew beer.

[0209] As would be appreciated by a person skilled in the art, exposing the mash to an electric field refers to positioning the mash within the influence of the field. The mash may be exposed to the electric field for a portion or all of the mashing process. For example, for a period of at least 1 minute. In certain embodiments, the mash is exposed to the electric field for a duration of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 55 or up to 60 minutes. In other embodiments, the mash is exposed to the electric field for at least 60 minutes or the entire mashing process. The person skilled in the art may modify the exposure time to optimise the mashing processes and tailor the treatment of the grist, grain, or cereal to specific beer styles or brewing goals.

[0210] In a seventh aspect, there is provided a method of brewing beer, comprising: adding yeast to a boiled cooled wort to provide a yeast-inoculated wort; fermenting the yeast-inoculated wort under conditions and for a period of time to produce beer, wherein the conditions comprise; exposing the yeast- inoculated wort to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0211] The method of the seventh aspect relates to a series of steps in the process of brewing beer. The person skilled in the art could readily incorporate the method into the process of brewing beer, which may optionally comprise the steps of malting, wherein grains such as barley are soaked, germinated, and dried to develop enzymes necessary for starch conversion; milling, wherein the malt is ground into a coarse powder (grist) to expose the starches; mashing, which involves mixing the grist with hot water to activate enzymes that convert starches into fermentable sugars; lautering, wherein the mash is transferred to a vessel to separate the liquid wort from the spent grain; boiling, wherein the wort is heated with hops to sterilise it, extract hop flavours and bitterness, and concentrate the sugars; cooling, wherein the boiled wort is rapidly cooled to fermentation temperature to prevent contamination; and fermentation, during which yeast is added to the cooled wort to convert the sugars into alcohol and carbon dioxide, thereby producing beer.

[0212] The method is useful for treating a fermentation to provide a beer. Without wishing to be bound by theory, it is thought that the application of the electric field enhances protein breathing in the enzymes located within the yeast, by affecting the charged moieties within the fermentation. Protein breathing allows substrates and cofactors to react through the action of the enzymes in the yeast to synthesise products.

[0213] The yeast may be any suitable yeast used in brewing beer, such as Saccharomyces cerevisiae. commonly used for ales, stouts, and porters; Saccharomyces pastoriamis. used for lagers and pilsners; Brettanomyces spp., used for sour ales and farmhouse beers; Torulaspora delbrueckii, used for specialty ales; or hybrid or genetically engineered yeast strains, used for various beer styles. The yeast selection is tailored to the style of beer being brewed. Alternatively, the yeast may be any other suitable yeast described in this disclosure.

[0214] The method involves fermenting the yeast-inoculated wort under conditions and for a period of time to produce beer. As would be appreciated by a person skilled in the art, the conditions comprise regulating, e.g., inputs, steps, times, temperatures, aeration, and pH conditions. In certain embodiments, the conditions comprise one or more of: maintaining temperature within the range of about 7°C to 24°C depending on the yeast strain and beer style; maintaining pH within the range of about 4.0 to 5.5 to optimise yeast metabolism and inhibit undesirable microorganisms; providing aeration during the initial stages of fermentation to achieve dissolved oxygen levels of about 8 to 10 ppm for yeast propagation; regulating sugar concentration, measured as original gravity, to control alcohol production and fermentation kinetics; providing nutrients, such as amino acids and trace elements, to support yeast health and activity; and, where applicable, providing agitation to ensure uniform distribution of nutrients and temperature. In certain embodiments, carbon dioxide levels may also be monitored and vented or retained to influence beer carbonation. Typically, for ale yeast such as Saccharomyces cerevisiae. the fermentation temperature is generally maintained between 15 °C and 24°C, while for lager yeast such as Saccharomyces pasiorianus. a lower range of 7°C to 13°C may be preferred.

[0215] In certain embodiments, the period of time is between about 7 and about 21 days. The person skilled in the art would understand that fermentation times may be modified depending upon the beer style, yeast strain, fermentation temperature, desired final sugar content and desired flavour profile. In certain embodiments, ales may undergo primary fermentation for about 5 to 7 days at temperatures ranging from 15 °C to 24°C, followed by conditioning or maturation for about 1 to 2 weeks. Lagers may have a longer fermentation process, with primary fermentation lasting about 1 to 3 weeks at cooler temperatures of 7°C to 13°C, followed by lagering or cold conditioning for about 4 to 12 weeks. Sours may ferment for about 1 to 2 weeks and then age for about 6 months to several years to develop desired acidity and complexity. High-alcohol beers, such as Imperial Stouts or Barleywines, may undergo primary fermentation for about 1 to 2 weeks, with extended conditioning or maturation for about 2 to 6 months or longer to allow flavours to integrate. Wheat beers, such as Hefeweizen or Witbier, may ferment more quickly, with primary fermentation lasting about 3 to 5 days and minimal conditioning for about 1 to 2 weeks. IPAs may undergo primary fermentation for about 5 to 7 days, followed by conditioning for about 1 to 2 weeks, with dry hopping incorporated during this period in some styles.

[0216] In certain embodiments, the method comprises increasing the levels of certain compounds in the yeast. Such compounds may have the effect of enhancing the flavour of the beer. In certain embodiments, the level of 3 -hydroxybutyric acid is increased by at least 5%, between about 5% to about 70%, and more specifically by between about 10% to about 60%, such as about 50%. 3 -hydroxybutyric acid enhances the flavour of the beer by contributing to its creamy and buttery notes. In certain embodiments, the level of 4-aminobutyric acid is increased by at least 40%, between about 40% to about 90%, and more specifically by between about 60% to about 80%, such as about 69%. 4-aminobutyric acid enhances the beer's flavour by adding subtle umami characteristics. In certain embodiments, the level ofpalmitic acid is increased by at least 40%, between about 40% to about 90%, and more specifically by between about 60% to about 80%, such as about 67%. Palmitic acid contributes to the mouthfeel and overall richness of the beer. In certain embodiments, the level of 2-hydroxyglutaric acid is increased by at least 40%, between about 40% to about 70%, and more specifically by between about 35% to about 60%, such as about 52%. 2-hydroxyglutaric acid enhances the beer's flavour by adding complexity and depth. In certain embodiments, the level of fumaric acid is increased by at least 20%, between about 20% to about 50%, and more specifically by between about 25% to about 40%, such as about 33%. Fumaric acid contributes to the beer's acidity and balance. In certain embodiments, the level of pyruvic acid is increased by at least 40%, between about 40% to about 80%, and more specifically by between about 50% to about 70%, such as about 59%. Pyruvic acid enhances the beer's flavour by providing fruity and tart notes. In certain embodiments, the level of dihydroxyacetone phosphate is increased by at least 15%, between about 15% to about 40%, and more specifically by between about 20% to about 30%, such as about 22%. Dihydroxyacetone phosphate contributes to the beer's sweetness and overall flavour profile.

[0217] In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 3 -hydroxybutyric acid is increased by at least 5%, between about 5% to about 25%, and more specifically by between about 10% to about 20%, such as about 12%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 4 -aminobutyric acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 15% to about 25%, such as about 17%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of palmitic acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 14% to about 25%, such as about 16%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 2-hydroxyglutaric acid is increased by at least 10%, between about 10% to about 25%, and more specifically by between about 12% to about 20%, such as about 13%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of fumaric acid is increased by at least 5%, between about 5% to about 20%, and more specifically by between about 6% to about 15%, such as about 8%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of pyruvic acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 12% to about 25%, such as about 15%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of dihydroxyacetone phosphate is increased by at least 3%, between about 3% to about 15%, and more specifically by between about 4% to about 10%, such as about 5%.

[0218] The method may also involve promoting the production of volatile compounds associated with fruit and floral aromas, such as those imparting notes of banana (e.g. methylbutyl acetate), cherry (e.g. ethyl acetate), green apple (e.g. ethyl hexanoate), and floral / rose (e.g. 2-phenyl ethanol). In certain embodiments, the volatile compounds associated with fruit and floral aromas increase by at least 2%,such as between about 3% and about 100%, between about 5% and 80%, about 10% and 70%, or about10% and 60%, compared to an untreated control. In certain embodiments, the level of methylbutyl acetate (associated with banana aromas) is increased by at least 35%, between about 35% to about 65%, and more specifically by between about 47% to about 57%, such as about 52%. In certain embodiments, the level of ethyl acetate (associated with cherry aromas) is increased by at least 15%, between about 15% to about 35%, and more specifically by between about 20% to about 24%, such as about 22%. In certain embodiments, the level of ethyl hexanoate (associated with green apple aromas) is increased by at least 8%, between about 8% to about 12%, and more specifically by between about 8% to about 10%, such as about 9%. In certain embodiments, the level of 2-phenyl ethanol (associated with floral and rose aromas) is increased by at least 8%, between about 8% to about 12%, and more specifically by between about 8% to about 10%, such as about 9%. In certain embodiments, the level of ethyl decanoate is increased by at least 15%, between about 15% to about 25%, and more specifically by between about 15% to about 19%, such as about 17%. Enhancing these flavour compounds may improve the overall sensory characteristics of the beer.

[0219] In certain embodiments, the method comprises increasing the levels of certain compounds in the produced beer. The compounds may comprise one or more of, e.g., maltose, maltotriose, or glucose. Higher levels of these compounds may be important in a low carb beer. In certain embodiments, the levels of one or more of maltose, maltotriose, or glucose increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, about 10% and 70%, or about 10% and 60%, compared to an untreated control.

[0220] In certain embodiments, the method comprises decreasing the level of total sugars in the produced beer as measured by Brix. In certain embodiments, the level of total sugars in the produced beer as measured by Brix is decreased by between about 5% and about 25%, between about 10% and about 20% or by about 16%.

[0221] In certain embodiments, the produced beer has a reduced total amino acid concentration relative to an untreated control. For example, on a day when fermentation has completed, such as day 8. In certain embodiments, the produced beer has a reduced total amino acid concentration of between about 5% and 20%, such as about 5% to about 10%, or about 10% to about 15%, compared to an untreated control. The person skilled in the art would understand that this reduction in total amino concentration may indicate that yeast in the treated fermentation was scavenging amino acids more effectively than yeast in the control fermentation on that day. In certain embodiments, the produced beer has modified levels of amino acids as shown in Table 5. While specific increases or decreases in amino acid concentration has been observed, the disclosure contemplates variations in these increases or decreases, for example, levels that are 2-fold to 4-fold lower, or 2-fold to 4-fold higher than recorded. The person skilled in the art wouldunderstand that variations in amino acid concentration may occur due to variations in the wort, microorganism or fermentation conditions.

[0222] In certain embodiments, the boiled cooled wort may comprise the wort from the sixth aspect of the disclosure. In other embodiments, the boiled cooled wort may have been prepared from any grist, grain, or cereal as described in the sixth aspect or elsewhere in the disclosure.

[0223] As would be appreciated by a person skilled in the art, exposing the fermenting wort to an electric field refers to positioning the wort within the influence of the field. The wort may be exposed to the field for a portion or all of the fermentation process. For example, the exposure may be for a period of at least 1 minute. In certain embodiments, the wort is exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for the entire fermentation process, which may last up to 1, 2, or even 3 weeks depending on the beer style. Shorter exposure times, such as 1 to 10 minutes, may be employed to achieve specific effects, such as accelerating yeast activity or enhancing flavour compound production. The person skilled in the art may adjust the exposure time to optimise the fermentation process and tailor the treatment to specific beer styles or production goals.

[0224] In an eighth aspect, there is provided a method of brewing beer, comprising: contacting a grist, grain or cereal with water to provide a mash; treating the mash under a first set of conditions and for a first period of time to provide a treated mash; obtaining a wort from the treated mash; boiling and cooling the wort; adding yeast to a boiled cooled wort to provide a yeast-inoculated wort; fermenting the yeast- inoculated wort under a second set of conditions and for a second period of time to produce beer; wherein the first set of conditions comprise; exposing the mash to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz; and wherein the second set of conditions comprise; exposing the fermenting beer to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0225] The eighth aspect effectively combines the sixth and seventh aspects. As such, both the mash and fermentation are exposed to the electric field and a beer is produced. The treated wort may have the characteristics as described in the sixth aspect. The produced beer may have the characteristics described in the seventh aspect. Without wishing to be bound by theory, it is thought that the application of the electric field enhances protein breathing in the enzymes in the mash and located within the yeast during fermentation, by affecting the charged moieties within the mash and fermentation. Protein breathing allows substrates and cofactors to react through the action of the free enzymes and those in the yeast to synthesise products.

[0226] The effect of exposing the mash to the electric field may comprise increasing the activity of one or more enzymes involved in producing glucose, mannose, galactose, allose, nonanoic acid, sorbitol, mannitol, glycerol, monostearin, or arabitol. As such, the wort obtained from the treated mash may comprise increased levels of one or more of glucose, mannose, galactose, allose, nonanoic acid, sorbitol, mannitol, glycerol, monostearin, or arabitol.

[0227] In certain embodiments, the method comprises increasing the levels of certain compounds in the yeast. Such compounds may have the effect of enhancing the flavour of the beer. In certain embodiments, the level of 3 -hydroxybutyric acid is increased by at least 5%, between about 5% to about 70%, and more specifically by between about 10% to about 60%, such as about 50%. 3 -hydroxybutyric acid enhances the flavour of the beer by contributing to its creamy and buttery notes. In certain embodiments, the level of 4-aminobutyric acid is increased by at least 40%, between about 40% to about 90%, and more specifically by between about 60% to about 80%, such as about 69%. 4-aminobutyric acid enhances the beer's flavour by adding subtle umami characteristics. In certain embodiments, the level of palmitic acid is increased by at least 40%, between about 40% to about 90%, and more specifically by between about 60% to about 80%, such as about 67%. Palmitic acid contributes to the mouthfeel and overall richness of the beer. In certain embodiments, the level of 2-hydroxyglutaric acid is increased by at least 40%, between about 40% to about 70%, and more specifically by between about 35% to about 60%, such as about 52%. 2-hydroxyglutaric acid enhances the beer's flavour by adding complexity and depth. In certain embodiments, the level of fumaric acid is increased by at least 20%, between about 20% to about 50%, and more specifically by between about 25% to about 40%, such as about 33%. Fumaric acid contributes to the beer's acidity and balance. In certain embodiments, the level of pyruvic acid is increased by at least 40%, between about 40% to about 80%, and more specifically by between about 50% to about 70%, such as about 59%. Pyruvic acid enhances the beer's flavour by providing fruity and tart notes. In certain embodiments, the level of dihydroxyacetone phosphate is increased by at least 15%, between about 15% to about 40%, and more specifically by between about 20% to about 30%, such as about 22%. Dihydroxyacetone phosphate contributes to the beer's sweetness and overall flavour profile.

[0228] In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 3 -hydroxybutyric acid is increased by at least 5%, between about 5% to about 25%, and more specifically by between about 10% to about 20%, such as about 12%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 4 -aminobutyric acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 15% to about 25%, such as about 17%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of palmitic acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 14% to about 25%, such as about 16%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 2-hydroxyglutaric acid is increased by at least 10%, between about 10% to about25%, and more specifically by between about 12% to about 20%, such as about 13%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of fumaric acid is increased by at least 5%, between about 5% to about 20%, and more specifically by between about 6% to about 15%, such as about 8%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of pyruvic acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 12% to about 25%, such as about 15%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of dihydroxyacetone phosphate is increased by at least 3%, between about 3% to about 15%, and more specifically by between about 4% to about 10%, such as about 5%.

[0229] The method may also involve promoting the production of volatile compounds associated with fruit and floral aromas, such as those imparting notes of banana (e.g. methylbutyl acetate), cherry (e.g. ethyl acetate), green apple (e.g. ethyl hexanoate), and floral / rose (e.g. 2-phenyl ethanol). In certain embodiments, the volatile compounds associated with fruit and floral aromas increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, about 10% and 70%, or about 10% and 60%, compared to an untreated control. In certain embodiments, the level of methylbutyl acetate (associated with banana aromas) is increased by at least 35%, between about 35% to about 65%, and more specifically by between about 47% to about 57%, such as about 52%. In certain embodiments, the level of ethyl acetate (associated with cherry aromas) is increased by at least 15%, between about 15% to about 35%, and more specifically by between about 20% to about 24%, such as about 22%. In certain embodiments, the level of ethyl hexanoate (associated with green apple aromas) is increased by at least 8%, between about 8% to about 12%, and more specifically by between about 8% to about 10%, such as about 9%. In certain embodiments, the level of 2-phenyl ethanol (associated with floral and rose aromas) is increased by at least 8%, between about 8% to about 12%, and more specifically by between about 8% to about 10%, such as about 9%. In certain embodiments, the level of ethyl decanoate is increased by at least 15%, between about 15% to about 25%, and more specifically by between about 15% to about 19%, such as about 17%. Enhancing these flavour compounds may improve the overall sensory characteristics of the beer.

[0230] In certain embodiments, the method comprises increasing the levels of certain compounds in the produced beer. The compounds may comprise one or more of, e.g., maltose, maltotriose, or glucose. Higher levels of these compounds may be important in a low carb beer. In certain embodiments, the levels of one or more of maltose, maltotriose, or glucose increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, about 10% and 70%, or about 10% and 60%, compared to an untreated control.

[0231] The first set of conditions and the first period of time for treating the mash may be as described in the sixth aspect. All other conditions may also be as described in the sixth aspect, or as describedelsewhere in the disclosure. For example, the identity of the grist, grain, or cereal, the steps, time and temperature of the mashing process, and the exposure of the mash to the electric field. The treated mash may be as described in the sixth aspect.

[0232] The second set of conditions and the second period of time for treating the fermentation may be as described in the seventh aspect, or as described elsewhere in the disclosure. All other conditions may also be as described in the seventh aspect, or as described elsewhere in the disclosure. For example, the yeast strains, the conditions of the inputs, steps, times, temperatures, aeration, and pH conditions, and the exposure of the fermentation to the electric field.

[0233] In a ninth aspect, there is provided a method of fermenting a fruit or vegetable juice to make a fermented food, beverage or wash, comprising: providing juice from a fruit or vegetable, adding yeast to the juice to provide a yeast-inoculated juice, fermenting the yeast-inoculated juice under conditions and for a period of time to produce a fermented food, beverage or wash, wherein the conditions comprise: exposing the yeast-inoculated juice to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0234] In certain embodiments, the method of fermenting a fruit or vegetable juice to make a fermented food, beverage, or wash may utilise a wide variety of juices, comprising, but not limited to: apple, orange, grape, pear, cherry, pomegranate, cranberry, mango, peach, apricot, pineapple, kiwi, and banana juices; and vegetable juices such as carrot, beetroot, tomato, celery, cucumber, spinach, kale, pumpkin, sweet potato, or mixed vegetable juices. Additionally, blends of fruit and vegetable juices or concentrates derived from these sources may be used. The selection of juice may be tailored to the desired flavour, nutrient composition, or functional properties of the fermented product.

[0235] As would be appreciated by a person skilled in the art, exposing the fermenting the yeast- inoculated juice to an electric field refers to positioning the juice within the influence of the field. The juice may be exposed to the field for a portion or all of the fermentation process. For example, the exposure may be for a period of at least 1 minute. In certain embodiments, the juice is exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for the entire fermentation process, which may last up to 1, 2, or even 3 weeks depending on the fermented food, beverage or wash. Shorter exposure times, such as 1 to 10 minutes, may be employed to achieve specific effects, such as accelerating yeast activity, enhancing flavour compound production, or affecting sugar content. The person skilled in the art may adjust the exposure time to optimise the fermentation process and tailor the treatment to a specific fermented food, beverage or wash.

[0236] The conditions of exposing the yeast-inoculated juice to an electric field may increase the presence or balance of desirable flavour compounds produced during fermentation. These compounds, which contribute to the sensory profile of the product, may include primary metabolites, such as pyruvic acid dihydroxyacetone phosphate, 3 -hydroxybutyric acid and 4 -aminobutyric acid, which add complexity and depth to the flavour. Additionally, specific acids, such as fumaric acid, 2 -hydroxyglutaric acid, and palmitic acid, may enhance mouthfeel, acidity, or richness. In certain embodiments, the desirable flavour compounds increase by at least 2%, such as between about 3% and about 20%, between about 4% and 15%, or about 5% and 13%, compared to an untreated control and when normalised against glucose. In other embodiments, the desirable flavour compounds increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, or about 10% and 70%, compared to an untreated control. The method may also involve promoting the production of volatile compounds associated with fruit and floral aromas, such as those imparting notes of banana (e.g. methylbutyl acetate), cherry (e.g. ethyl acetate), green apple (e.g. ethyl hexanoate), and floral / rose (e.g. 2-phenyl ethanol). In certain embodiments, the volatile compounds associated with fruit and floral aromas increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, about 10% and 70%, or about 10% and 60%, compared to an untreated control. Enhancing these flavour compounds may improve the overall sensory characteristics of the food, beverage, or wash for its intended application.

[0237] The person skilled in the art could select a suitable substrate based on the intended fermented food, beverage or wash, which may be as described in the fourth aspect, or as described elsewhere in the disclosure. The microorganisms may be any suitable microorganism used in fermenting a juice to provide a fermented food, beverage, or wash, which may be as described in the fourth aspect, or as described elsewhere in the disclosure. The method may use a range of culture conditions as described in the second aspect, or elsewhere in this disclosure. Other features of the fermentation may be as described in the fourth aspect, or as described elsewhere in the disclosure, e.g., inoculation rate.

[0238] In a tenth aspect, there is provided a method of producing a carbonated fermented beverage, comprising: providing juice from a fruit, adding yeast to the juice to provide a yeast-inoculated juice, fermenting the yeast-inoculated juice under conditions and for a period of time to produce a fermented beverage, wherein the conditions comprise: exposing the yeast-inoculated juice to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz; and performing a secondary fermentation to provide a carbonated fermented beverage.

[0239] In certain embodiments, the carbonated fermented beverage is a carbonated white wine, a carbonated red wine, a sparkling rose, a sparkling wine, a carbonated ready to drink beverage, an alcoholic mixer, a cider or beer.

[0240] In certain embodiments, juice from a fruit comprises grape juice (used for carbonated white wine, carbonated red wine, sparkling rose, and sparkling wine); apple juice (used for cider); pear juice (used for perry or mixed ciders); and juices from cherries, blackberries, raspberries, and elderberries (used for fruit wines or blended alcoholic beverages). For carbonated ready -to-drink beverages and alcoholic mixers, additional juices such as lime, lemon, orange, grapefruit, cranberry, pomegranate, and pineapple juice may be used. Beer production may incorporate adjunct juices such as orange, cherry, or raspberry to enhance flavour profdes. The selection of juice depends on the intended beverage type, flavour profile, and carbonation characteristics desired.

[0241] The conditions of exposing the yeast-inoculated juice to an electric field may increase the presence or balance of desirable flavour compounds in the yeast produced during fermentation. In certain embodiments, the method comprises increasing the levels of certain compounds in the yeast. Such compounds may have the effect of enhancing the flavour of the fermented beverage. In certain embodiments, the level of 3 -hydroxybutyric acid is increased by at least 5%, between about 5% to about 70%, and more specifically by between about 10% to about 60%, such as about 50%. 3 -hydroxybutyric acid enhances the flavour of the beer by contributing to its creamy and buttery notes. In certain embodiments, the level of 4-aminobutyric acid is increased by at least 40%, between about 40% to about 90%, and more specifically by between about 60% to about 80%, such as about 69%. 4-aminobutyric acid enhances the beer's flavour by adding subtle umami characteristics. In certain embodiments, the level of palmitic acid is increased by at least 40%, between about 40% to about 90%, and more specifically by between about 60% to about 80%, such as about 67%. Palmitic acid contributes to the mouthfeel and overall richness of the beer. In certain embodiments, the level of 2 -hydroxyglutaric acid is increased by at least 40%, between about 40% to about 70%, and more specifically by between about 35% to about 60%, such as about 52%. 2-hydroxyglutaric acid enhances the beer's flavour by adding complexity and depth. In certain embodiments, the level of fumaric acid is increased by at least 20%, between about 20% to about 50%, and more specifically by between about 25% to about 40%, such as about 33%. Fumaric acid contributes to the beer's acidity and balance. In certain embodiments, the level of pyruvic acid is increased by at least 40%, between about 40% to about 80%, and more specifically by between about 50% to about 70%, such as about 59%. Pyruvic acid enhances the beer's flavour by providing fruity and tart notes. In certain embodiments, the level of dihydroxyacetone phosphate is increased by at least 15%, between about 15% to about 40%, and more specifically by between about 20% to about 30%, such as about 22%. Dihydroxyacetone phosphate contributes to the beer's sweetness and overall flavour profile.

[0242] In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 3 -hydroxybutyric acid is increased by at least 5%, between about 5% to about 25%, and more specifically by between about 10% to about 20%, such as about 12%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 4-aminobutyric acid is increased by at least 10%, between about 10% to about 30%, and more specifically by betweenabout 15% to about 25%, such as about 17%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of palmitic acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 14% to about 25%, such as about 16%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of 2-hydroxyglutaric acid is increased by at least 10%, between about 10% to about 25%, and more specifically by between about 12% to about 20%, such as about 13%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of fumaric acid is increased by at least 5%, between about 5% to about 20%, and more specifically by between about 6% to about 15%, such as about 8%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of pyruvic acid is increased by at least 10%, between about 10% to about 30%, and more specifically by between about 12% to about 25%, such as about 15%. In certain embodiments, when normalized to a glucose analysis of the wort metabolites versus GCMS, the level of dihydroxyacetone phosphate is increased by at least 3%, between about 3% to about 15%, and more specifically by between about 4% to about 10%, such as about 5%.

[0243] The method may also involve promoting the production of volatile compounds associated with fruit and floral aromas, such as those imparting notes of banana (e.g. methylbutyl acetate), cherry (e.g. ethyl acetate), green apple (e.g. ethyl hexanoate), and floral / rose (e.g. 2-phenyl ethanol). In certain embodiments, the volatile compounds associated with fruit and floral aromas increase by at least 2%, such as between about 3% and about 100%, between about 5% and 80%, about 10% and 70%, or about 10% and 60%, compared to an untreated control. In certain embodiments, the level of methylbutyl acetate (associated with banana aromas) is increased by at least 35%, between about 35% to about 65%, and more specifically by between about 47% to about 57%, such as about 52%. In certain embodiments, the level of ethyl acetate (associated with cherry aromas) is increased by at least 15%, between about 15% to about 35%, and more specifically by between about 20% to about 24%, such as about 22%. In certain embodiments, the level of ethyl hexanoate (associated with green apple aromas) is increased by at least 8%, between about 8% to about 12%, and more specifically by between about 8% to about 10%, such as about 9%. In certain embodiments, the level of 2-phenyl ethanol (associated with floral and rose aromas) is increased by at least 8%, between about 8% to about 12%, and more specifically by between about 8% to about 10%, such as about 9%. In certain embodiments, the level of ethyl decanoate is increased by at least 15%, between about 15% to about 25%, and more specifically by between about 15% to about 19%, such as about 17%. Enhancing these flavour compounds may improve the overall sensory characteristics of the beer.

[0244] The microorganisms may be any suitable microorganism used in fermenting a juice to provide a fermented beverage, which may be as described in the fourth aspect, or as described elsewhere in the disclosure. The method may use a range of culture conditions as described in the second aspect, orelsewhere in this disclosure. Other features of the fermentation may be as described in the fourth aspect, or as described elsewhere in the disclosure, e.g., inoculation rate.

[0245] As would be appreciated by a person skilled in the art, exposing the fermenting juice to an electric field refers to positioning the juice within the influence of the field. The juice may be exposed to the field for a portion or all of the fermentation process. For example, the exposure may be for a period of at least 1 minute. In certain embodiments, the juice is exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for the entire fermentation process, which may last up to 1, 2, or even 3 weeks depending on the beer style. Shorter exposure times, such as 1 to 10 minutes, may be employed to achieve specific effects, such as accelerating yeast activity or enhancing flavour compound production. The person skilled in the art may adjust the exposure time to optimise the fermentation process and tailor the treatment to specific styles of carbonated fermented beverage or production goals.

[0246] The person skilled in the art would understand how to perform a secondary fermentation to provide a carbonated fermented beverage. In certain embodiments, the secondary fermentation comprises adding residual sugars and selected yeast strains to a previously fermented alcoholic beverage, such as white or red wine, rose, alcoholic mixer, cider, or beer, to form a mixture. The mixture is then sealed in a pressurised vessel, such as a bottle or fermentation tank, to enable the re -fermentation of remaining sugars. This process generates additional carbon dioxide, which dissolves into the liquid to achieve the desired carbonation level. For sparkling wines and roses, traditional methode champenoise techniques may be used, involving aging on lees to enhance flavour and effervescence. Alternatively, for ciders and beers, tank-based methods like the Charmat process can be employed for rapid carbonation. Parameters such as temperature, pressure, and fermentation duration, may be controlled, which would be readily performed by the person skilled in the art, depending on the beverage.

[0247] In an eleventh aspect, there is provided a method of producing probiotic microorganisms, comprising: providing a growth medium for growing a probiotic microorganism; adding the probiotic microorganism to the growth medium to provide a probiotic microorganism-inoculated growth medium; incubating the probiotic microorganism-inoculated growth medium under conditions and for a period of time to produce a plurality of the probiotic microorganism, wherein the conditions comprise: exposing the probiotic microorganism-inoculated growth medium to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0248] This disclosure provides specific embodiments for enhancing the production and activity of probiotic microorganisms using the electric field technology described herein. As previously detailed and elsewhere in this disclosure, the method of producing probiotic microorganisms comprises providing a growth medium, inoculating it with a probiotic microorganism, and incubating it under specificconditions and for a period of time to produce a plurality of the probiotic microorganism. The enhanced conditions for this production specifically include exposing the probiotic microorganism -inoculated growth medium to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequencies in the range of about 210 Hz to about 99 kHz.

[0249] In some embodiments, the application of this electric field significantly improves the metabolic efficiency, stress resistance and longevity of probiotic microorganisms, leading to higher viable yields and more robust cultures, which represents a key advantage over conventional methods. The growth medium for probiotic microorganisms can be any suitable medium known to a person skilled in the art, based on the nutritional requirements of the microorganisms, and may include feedstocks, substrates, or other nutrient-rich compositions described elsewhere in this disclosure. The conditions for incubation, including temperature, pH, aeration, and agitation, are optimised to support efficient production, generally aligning with the ranges described in this disclosure, but specifically tailored to the selected probiotic strain. For instance, many Lactobacillus and Bifidobacterium species thrive at temperatures between 35 °C and 42°C and pH levels of 4.8 to 6.5, often requiring microaerophilic conditions, or in some cases anaerobic conditions. The duration of incubation can range from about 12 hours to several days (e.g., 12 to 48 hours for many probiotic cultivations), adjusted by a person skilled in the art to maximise the yield and viability of the probiotic microorganisms. Slow growing organisms can be grown from days to weeks.

[0250] As would be appreciated by a person skilled in the art, exposing the microorganism to an electric field refers to positioning the microorganism within the influence of the field. The juice may be exposed to the field for a portion or all of the fermentation process. For example, the exposure may be for a period of at least 1 minute. In certain embodiments, the juice is exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for the entire fermentation process, which may last up to 1, 2, or even 3 weeks depending on the beer style. Shorter exposure times, such as 1 to 10 minutes, may be employed to achieve specific effects, such as accelerating yeast activity or enhancing flavour compound production. The person skilled in the art may adjust the exposure time to optimise the fermentation process and tailor the treatment to specific styles of carbonated fermented beverage or production goals.

[0251] In certain embodiments, the probiotic microorganism is selected from the group consisting of Saccharomyces boulardii. Saccharomyces cerevisiae var. boulardii. Kluyveromyces marxianus. Debaryomyces hansenii. Candida utilis (Torula yeast), Pichia pasioris. Aspergillus oryzae. and Torulaspora delbrueckii . Saccharomyces boulardii and Saccharomyces cerevisiae var. boulardii are utilised for their efficacy in preventing and treating gastrointestinal disorders and are incorporated into dietary supplements and functional beverages. Kluyveromyces marxianus is employed for its thermotolerance and ability to enhance lactose digestion, making it suitable for fermented dairy products.Debaryomyces hansenii is used in cheese fermentation to improve gut barrier function and exhibit antimicrobial activity. Candida utilis serves as a high-protein nutritional supplement, supporting gut health by acting as a nutrient source for beneficial bacteria. Pichia pastoris is leveraged primarily for recombinant protein production with emerging applications in immune modulation. Aspergillus oryzae is integral to traditional fermented foods, enhancing digestive health through the production of bioactive compounds. Torulaspora delbrueckii contributes to microbial diversity and flavour profiles in fermented products, indirectly supporting probiotic benefits. In certain embodiments, the probiotic microorganism is an Ascomycetes species selected based on its Generally Recognized As Safe (GRAS) status. In certain embodiments, the probiotic microorganism may be one or more microorganism described in the second aspect, or elsewhere in this disclosure.

[0252] The method may use a range of culture conditions as described in the second aspect, or elsewhere in this disclosure. The method may use a diverse array of substrates to support the growth and metabolic activity of the microorganism as described in the second aspect, or elsewhere in this disclosure.

[0253] A main advantage of applying the electric field technology to probiotic production is the significant enhancement of microbial viability and stress resistance, allowing probiotics to better withstand the manufacturing stresses of freeze-drying and encapsulation. Similarly, a boost in viable biomass enables a greater percentage of cells to resist environmental challenges during storage and delivery.

[0254] Furthermore, the technology enables higher cell densities and faster fermentation times, leading to increased productivity and reduced operational costs for probiotic manufacturers.

[0255] The improved stress response of the microorganisms, including resistance to osmotic stress, temperature stress, pH stress, and oxidative stress, translates directly into more stable and effective probiotic products with extended shelf-life and greater therapeutic efficacy.

[0256] If used in the manner described in this disclosure, the device, system and methods may not appreciably change a strain or its fundamental qualities, and this benign impact is an advantageous quality for industrial adoption. In an imaginable application, this technology may potentially offer the advantage of stimulating discrete cellular functions in probiotic microorganisms, to further enhance the production of viable biomass or to stabilize the colonisation abilities of the probiotics.

[0257] The person skilled in the art would readily appreciate that the disclosed methods and systems provide a superior approach to the cultivation and enhanced manufacturability of probiotic microorganisms, offering distinct advantages in terms of yield, viability, robustness, and cost-efficiency compared to existing techniques.

[0258] In summary, the modulated electric field technology, leveraging specific oscillating electric field frequencies, provides a novel and highly specific method for revolutionising probiotic production, addressing a wide range of current industry challenges, and opening new avenues for improving probiotic manufacturing procedures.

[0259] In a twelfth aspect, there is provided a method of producing a biofuel or fibre, comprising: providing a biomass; contacting the biomass with a microorganism to provide a microorganism - inoculated biomass; incubating the microorganism-inoculated biomass under conditions and for a period of time to produce a biofuel or fibre; wherein the conditions comprise exposing the microorganism- inoculated biomass to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0260] As would be appreciated by the person skilled in the art, microorganisms are used to convert biomass into biofuel through biochemical processes such as fermentation or anaerobic digestion. These microorganisms, including bacteria, yeast, and fungi, metabolise the sugars, cellulose, or other organic components of the biomass to produce biofuels. The biofuels generated comprise one or more of ethanol, butanol, biodiesel, biogas (methane), hydrogen, isobutanol or biogasoline. The person skilled in the art would appreciate that other biofuels could be produced, which depends on the specific microorganism used. The use of genetically engineered microorganisms may enhance the efficiency and yield of biofuel production. Exposing the microorganism -inoculated biomass to an electric field enhances the production of a biofuel, relative to an untreated control.

[0261] As would be appreciated by the person skilled in the art, products generated during biofuel production include residual fibres derived from lignocellulosic biomass or other organic feedstocks. These fibres, which remain after the extraction of fermentable sugars or other biofuel precursors, include specific components such as cellulose, hemicellulose, lignin, arabinoglycans, xylans, mannans, and pectins. These fibres possess properties suitable for use in the production of textiles, composite materials, paper, or biodegradable packaging. Processing techniques, such as refining, pulping, enzymatic hydrolysis, or chemical modification, may be employed to enhance the quality, mechanical strength, and functionality of the fibres, enabling their integration into sustainable industrial and consumer products. Exposing the microorganism-inoculated biomass to an electric field enhances the production and quality of these fibres, relative to an untreated control.

[0262] In certain embodiments, the biomass comprises one or more of agricultural residues, such as com stover, wheat straw, rice husks, and sugarcane bagasse; forestry residues, including wood chips, sawdust, and bark; dedicated energy crops, such as switchgrass, miscanthus, and poplar; organic waste materials, including food waste, animal manure, and municipal solid waste; brewing by-products, such as spent grain; aquatic biomass, such as algae and seaweed; industrial by-products, including glycerol and pulp sludge; and other cellulosic or lignocellulosic materials derived from plants or organic sourcescapable of being converted into biofuels through thermochemical, biochemical, or hybrid conversion processes.

[0263] In certain embodiments, the microorganism is selected from the group consisting of Saccharomyces cerevisiae. Saccharomyces stipitis, Saccharomyces pastorianus, Saccharomyces bayanus, Saccharomyces uvarum. Saccharomyces paradoxus, Saccharomyces kluyveri, Saccharomyces kudriavzevii, Saccharomyces eubayanus, Saccharomyces mikatae, Saccharomyces arboricola, Saccharomyces castellii, Saccharomyces jurei, Saccharomyces mikadoi, Saccharomyces cariocanus, Saccharomyces rouxii, Saccharomyces paradoxus var. balsamiferae, Saccharomyces ludwigii, Saccharomyces fibuligera, Saccharomyces exiguus, Starmerella bacillaris, Candida versatilis, Candida tropicalis, Candida shehatae, Candida utilis, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus fumigatus, Aspergillus clavatus, Aspergillus terreus, Aspergillus nidulans, Aspergillus tubingensis, Aspergillus flavus, Aspergillus wentii, Aspergillus tamarii, Aspergillus brasiliensis, Aspergillus chevalieri, Aspergillus carbonarius, Aspergillus foetidus, Aspergillus glaucus, Aspergillus melleus, Aspergillus versicolor, Aspergillus ustus, Aspergillus niveus, Aspergillus pseudoglaucus, Aspergillus puccini, Aspergillus sulphureus, Neosartorya fischeri, Neosartorya udagawae, Neosartorya siamensis, Neosartorya pseudofischeri , Neosartorya glabra, Neosartorya spinosa, Neosartorya hiratsukae, Neosartorya fisheri, Neosartorya argillacea, Neosartorya canadensis, Neosartorya quadrilateralis, Neosartorya paulistensis, Neosartorya himalayensis, Neosartorya Candida, Neosartorya fischeri var. japonica, Neosartorya hamata, Neosartorya minuta, Neosartorya bipola, Neosartorya clavigera, Neosartorya fumigata, Neosartorya leptoceras, Neosartorya parahiratsukae , Neosartorya saskatchewanensis, Neosartorya Stella, Neosartorya tetrasperma, Neosartorya webbii, Neosartorya biformis, Neosartorya cylindrospora, Neosartorya echinulata, Neosartorya granulosa, Neosartorya lornensis, Trichoderma reesei, Fusarium oxysporum, Pichia methanolica, Clostridium thermocellum, Pseudomonas putida, Synechocystis sp., and Chlamydomonas reinhardtii. These microorganisms are employed in various biofuel production processes, including the fermentation of lignocellulosic biomass to produce bioethanol, the synthesis of biodiesel through lipid accumulation, and the generation of biogas via anaerobic digestion. Saccharomyces cerevisiae and Saccharomyces stipitis are utilised for their robust ethanol fermentation capabilities, while Trichoderma reesei and Aspergillus niger are leveraged for their efficient cellulase and hemicellulase enzyme production to break down complex carbohydrates into fermentable sugars. Fusarium oxysporum and Pichia methanolica contribute to the bioconversion of biomass into biofuels through their metabolic versatility. Cyanobacteria such as Synechocystis sp. and algae like Chlamydomonas reinhardtii are integrated for their ability to perform photosynthetic biofuel production and biohydrogen generation. Additionally, bacteria including Pseudomonas putida and Clostridium thermocellum are utilised fortheir roles in bioremediation and direct biomass conversion processes. The person skilled in the art could readily select microorganisms based on their metabolic efficiency, tolerance to industrial fermentation conditions, and ability to produce high yields of biofuels.

[0264] In certain embodiments, the conditions comprise optimising parameters such as temperature, pH, aeration, and agitation to support efficient biofuel production. Temperature requirements typically range from about 10°C to 50°C, with narrower ranges depending on the microorganism. For example, Saccharomyces cerevisiae and Saccharomyces stipitis, used for ethanol production, perform optimally at temperatures of about 25°C to 37°C, while cellulase -producing fungi such as Trichoderma reesei and Aspergillus niger perform optimally at temperatures of about 25°C to 35°C. pH levels are typically maintained between about 4.0 and 8.0, with acidic conditions (pH 4.0-6.0) preferred for yeast and fungi and near-neutral pH (6.5-7.5) for bacteria like Clostridium thermocellum. Aeration levels range from minimal to high, for example, 0. 1-1.0 volumes of air per volume of culture per minute (vvm), depending on the oxygen demands of the microorganism. Agitation speeds may vary from 20 to 500 rpm to ensure sufficient mixing and oxygen transfer in submerged fermentations, while solid-state fermentations rely on maintaining humidity levels of 60-90%. These parameters can be tailored to the specific microorganism and biomass to maximise biofuel and fibre yields. In certain embodiments, the method may use a range of culture conditions as described in the second aspect, or elsewhere in this disclosure.

[0265] In certain embodiments, the period of time comprises durations ranging from about 12 hours to several weeks, influenced by the type of microorganism, biomass composition, and the desired biofuel product. Ethanol production using yeast, such as Saccharomyces cerevisiae, may involve fermentation periods of about 1 to 3 days under optimal conditions. Biogas (methane) production via anaerobic digestion typically requires about 7 to 21 days at mesophilic temperatures (20°C to 40°C) and may extend to several weeks at lower temperatures. Butanol production using Clostridium thermocellum or similar species often takes about 3 to 7 days under anaerobic conditions. Hydrogen production through dark or photofermentation may occur over 1 to 5 days, depending on microbial efficiency and biomass characteristics. Processes involving lipid-accumulating microorganisms like Pichia methanolica for biodiesel precursor production may take about 3 to 7 days, while photosynthetic organisms, such as Synechocystis sp. and Chlamydomonas reinhardiii. may require extended cultivation periods of 1 to 4 weeks for biohydrogen or lipid synthesis. The person skilled in the art could adjust these timeframes and conditions to improve the efficiency and yields of biofuels and / or fibres. In certain embodiments, the method may use a range of time periods as described in the second aspect, or elsewhere in this disclosure.

[0266] As would be appreciated by a person skilled in the art, exposing the microorganism-inoculated biomass to an electric field refers to positioning the microorganism-inoculated biomass within the influence of the field. The microorganism-inoculated biomass may be exposed to the field for a portion or all of the fermentation process. For example, the exposure may be for a period of at least 1 minute. In certain embodiments, the juice is exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for the entire fermentation process, which may last up to 1, 2, or even 3 weeks depending on thefermentation. Shorter exposure times, such as 1 to 10 minutes, may be employed to achieve specific effects, such as accelerating activity of the microorganism.

[0267] In a thirteenth aspect, there is provided a method of enhancing a fermentation to produce a bioenergy compound, a food, a supplement, a therapeutic, a pharmaceutical molecule, an enzyme, a probiotic, a prebiotic, an antibiotic, an oligosaccharide, a fibre or a biologically derived product, comprising: providing a growth medium for growing a microorganism; adding the microorganism to the growth medium to provide a microorganism -inoculated growth medium; incubating the microorganism- inoculated growth medium under conditions and for a period of time to produce the bioenergy compound, the food, the supplement, the therapeutic, the pharmaceutical molecule, the enzyme, the probiotic, the prebiotic, the antibiotic, the oligosaccharide, the fibre or the biologically derived product; wherein the conditions comprise: exposing the microorganism -inoculated growth medium to an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0268] In certain embodiments, the bioenergy compound comprises ethanol, butanol, biodiesel, biogas, hydrogen, bio-based jet fuel, bio-oil, syngas, methanol, biomethane, dimethyl ether (DME), biohydrogen, biobutanol, biopropanol, isobutanol, acetone, butyric acid, isobutyric acid, propionic acid, succinic acid, furan derivatives such as furfural and 5 -hydroxymethylfurfural (HMF), fatty acid methyl esters (FAME), hydrotreated vegetable oil (HVO), renewable diesel, advanced biofuels, alcohol -to-jet (ATJ) fuels, gas-to- liquid (GTL) fuels, power-to-liquid (PTL) fuels, bioethylene, bioethanol, biobutanol, biopropanol, isobutanol, dimethyl carbonate (DMC), methyl tert-butyl ether (MTBE), ethylene glycol, glycerol, levoglucosan, methyl lactate, and bio-derived hydrocarbons. In certain embodiments, the bioenergy compound is a biofuel as described elsewhere in the disclosure.

[0269] In certain embodiments, the food comprises fermented foods such as yogurt, kefir, kombucha, miso, tempeh, sauerkraut, kimchi, natto, pickles, sourdough bread, cheese, soy sauce, fermented sausages like salami and pepperoni, fermented beverages including beer, wine, cider, and kvass, and fermented plant-based proteins. Additionally, the food comprises protein isolates derived from plants, algae, or microbial sources; food-grade oils including omega-rich oils such as flaxseed oil, fish oil, and chia seed oil, as well as vegetable-derived lipids like canola oil, sunflower oil, and olive oil; and sweeteners or flavour enhancers such as steviol glycosides, xylitol, erythritol, sorbitol, maltitol, sucralose, aspartame, acesulfame potassium, fermented amino acids like monosodium glutamate (MSG), and natural flavour compounds. In certain embodiments, the food comprises a fermented food as described elsewhere in the disclosure.

[0270] In certain embodiments, the supplement comprises amino acids such as lysine, tryptophan, glutamine, arginine, leucine, isoleucine, valine, phenylalanine, methionine, cysteine, tyrosine, and histidine; omega-3 and omega-6 fatty acids including eicosapentaenoic acid (EP A), docosahexaenoic acid(DHA), linoleic acid (LA), and alpha-linolenic acid (ALA); vitamins including B-complex vitamins (Bl, B2, B3, B5, B6, B7, B9, B12), vitamin C, vitamin D, vitamin E, vitamin K, and vitamin A; mineral- enriched formulations such as calcium, magnesium, zinc, iron, selenium, copper, manganese, chromium, iodine, potassium, and phosphorus supplements; prebiotic fibres like inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), and resistant starches (RS); and bioactive peptides or antioxidants including glutathione, coenzyme Q10, polyphenols, and other antioxidant compounds.

[0271] In certain embodiments, the therapeutic comprises antimicrobial peptides, immunomodulatory proteins, enzymes such as lactase or protease for medical or digestive support, anti-inflammatory molecules, wound-healing agents, growth factors, cytokines, extracellular matrix proteins, neuroprotective agents, cardioprotective compounds, metabolic regulators, hormone therapies, anticancer agents, gene therapy vectors, cellular therapies including stem cells and CAR-T cells, bioconjugates, fusion proteins, peptide therapeutics, nucleic acid-based therapies such as antisense oligonucleotides, siRNA, and mRNA, biosensor-integrated therapeutics, antioxidants, free radical scavengers, biologies, biobetters, multivalent antagonists and agonists, and therapeutic vaccines designed to target specific health conditions and enhance overall health outcomes.

[0272] In certain embodiments, the pharmaceutical molecule comprises one or more of a diverse and extensive range of therapeutic agents and biologically active compounds, including, but are not limited to, insulin, monoclonal antibodies, recombinant proteins, antibiotics, antiviral agents, vaccines, smallmolecule drugs, hormone replacements, gene -editing tools such as CRISPR-based therapeutics, antifungal agents, anti-inflammatory drugs, anticoagulants, chemotherapeutic agents, biologies, peptide therapeutics, nucleic acid-based therapies (including siRNA and mRNA), cellular therapies, biosimilars, nanomedicines, prodrug formulations, ion channel modulators, G-protein coupled receptor (GPCR) ligands, enzyme inhibitors, receptor agonists and antagonists, immune checkpoint inhibitors, angiogenesis inhibitors, metabolic regulators, neurotransmitter modulators, opioid receptor ligands, beta-blockers, statins, antidepressants, antipsychotics, antidiabetic agents, antihypertensives, antivirals, antihistamines, analgesics, antipyretics, antiseptics, antiparasitics, antineoplastics, immunosuppressants, vaccinal adjuvants, drug delivery systems, therapeutic enzymes, fusion proteins, bispecific antibodies, CAR-T cell therapies, bioconjugates, exosomes, peptidomimetics, and biosensor-integrated therapeutics.

[0273] In certain embodiments, the enzyme encompasses a wide array of industrial enzymes utilised in various applications. These include cellulases, amylases, lipases, proteases, and specialised enzymes such as lactase for lactose digestion, invertase for sugar conversion, and glucose oxidase for biosensor development. In certain embodiments, the enzyme is used in the brewing industry, including alphaamylase, beta-amylase, glucoamylase, beta-glucanase, and additional proteases used to break down barley proteins during fermentation. In other embodiments, the enzyme is involved in the synthesis of specific compounds: glucose isomerase for converting glucose to fructose in high -fructose com syrup production;mannose-6-phosphate isomerase for the conversion of mannose -6-phosphate to fructose-6-phosphate in glycolysis; galactose oxidase for oxidising galactose to galactonic acid; allose epimerase for converting allose to other aldoses in specialised carbohydrate synthesis; nonanoic acid decarboxylase for producing nonanoic acid from its derivatives; dehydrogenase converting sorbitol to fructose in polyol metabolism. In other embodiments, the enzyme is mannitol- 1 -phosphate dehydrogenase for biosynthesizing mannitol from mannose, glycerol kinase for phosphorylating glycerol to glycerol -3 -phosphate in glycerol metabolism, monostearin synthase for catalyzing the formation of monostearin from stearic acid and glycerol, and arabitol dehydrogenase for converting arabitol to xylulose in pentose metabolism. In other embodiments, the enzyme is xylanase for breaking down xylan into xylose in the paper and biofuel industries; pectinase for degrading pectin in fruit juice clarification and textile processing; laccase for oxidising phenolic compounds in bioremediation and textile dyeing; phytase for hydrolyzing phytic acid in animal feed to release inorganic phosphorus; transglutaminase for facilitating protein cross-linking in food processing; beta-galactosidase for hydrolyzing lactose into glucose and galactose in dairy products; additional invertase for sucrose hydrolysis in confectionery applications; protease for protein breakdown in detergents, leather processing, and food industries; lipase for fat hydrolysis in biodiesel production, food processing, and pharmaceuticals; and sucrase for converting sucrose into glucose and fructose in various sweetening applications.

[0274] In certain embodiments, the probiotic comprises live microbial strains such as Lactobacillus spp., Bifidobacterium spp., Saccharomyces boulardii, Enterococcus faecium. Streptococcus thermophilus, or other beneficial microbes tailored for gut health, immune support, or metabolic enhancement, as well as next-generation probiotics like Akkermansia muciniphila or Faecalibacterium prausnitzii. In certain embodiments, the probiotic is as described elsewhere in the disclosure.

[0275] In certain embodiments, the prebiotic comprises one or more of inulin, fructooligosaccharides (FOS), galactooligosaccharides (GOS), xylooligosaccharides (XOS), mannooligosaccharides (ManOS), arabinooligosaccharides (AOS), isomaltooligosaccharides (IMO), resistant starches (RS), pectin, betaglucans, lactulose, oligofructose, chitin, chitosan, pullulan, levan, dextran, cellooligosaccharides (CelOS), galactan, raffinose, stachyose, verbascose, neokestose, koelichose, mannitol, sorbitol, fructans, arabinogalactan, polyfructose, beta-galactans, hyaluronic acid oligosaccharides, fucosylated oligosaccharides, soy oligosaccharides, isoquercitrin, pectic oligosaccharides, beta-N-acetyl-glucosamine oligosaccharides, heparin oligosaccharides, mannose oligosaccharides, sialylated oligosaccharides, glucooligosaccharides (GlcOS), chito-oligosaccharides (ChitOS), sophorolipids, levans, tetra-oligosaccharides, hexa-oligosaccharides, raffinose family oligosaccharides, cyclodextrins, synthetic oligosaccharides, galacto-oligosaccharides (GalOS), arabinoxylans, glucooligosaccharides, furanosyl oligosaccharides, manno-oligosaccharides, arabinan oligosaccharides, glucoarabinoxylans, and galactomannans.

[0276] In certain embodiments, the antibiotic comprises one or more naturally derived antibiotic or novel antimicrobial agents. These include [3-lactam antibiotics such as ampicillin, cephalexin, cephalothin, cefazolin, cefuroxime, ceftriaxone, cefepime, cefotaxime, cefixime, cefpodoxime, ceftazidime, cefoperazone, cefotiam, cefdinir, cefditoren, ceftibuten, ceftolozane, and cefiderocol; aminoglycosides including gentamicin, kanamycin, amikacin, tobramycin, neomycin, sisomicin, netilmicin, and spectinomycin; tetracyclines such as doxycycline, minocycline, and tetracycline; macrolides including clarithromycin, azithromycin, roxithromycin, josamycin, tilmicosin, spiramycin, and telithromycin; glycopeptides like vancomycin, teicoplanin, dalbavancin, and telavancin; oxazolidinones such as linezolid; fluoroquinolones including ciprofloxacin, levofloxacin, moxifloxacin, norfloxacin, ofloxacin, and gemifloxacin; sulfonamides like sulfamethoxazole and sulfadiazine; lincosamides including clindamycin and lincomycin; rifamycins such as rifampin and rifabutin; polyketides like erythromycin and pikromycin; nitroimidazoles including metronidazole and tinidazole; streptogramins such as quinupristin and dalfopristin; glycylcy clines like tigecy cline; bacteriostatic agents including chloramphenicol and chloramphenicol succinate; and novel antimicrobial agents like bacteriocins, lantibiotics, and engineered peptide antibiotics.

[0277] In certain embodiments, the oligosaccharide comprises one or more of maltodextrins, raffinose, stachyose, fructooligosaccharides (FOS), isomaltooligosaccharides (IMO), xylooligosaccharides (XOS), arabinooligosaccharides (AOS), gluco-oligosaccharides (GlcOS), galacto-oligosaccharides (GalOS), mannooligosaccharides (ManOS), chito-oligosaccharides (ChitOS), pullulan, levan, cellooligosaccharides (CelOS), stearooligosaccharides (SteaOS), lactooligosaccharides (LacOS), sialooligosaccharides (SiaOS), fucosylated oligosaccharides, sophorolipids, hexa-oligosaccharides, tetra-oligosaccharides, non-digestible oligosaccharides, polymeric oligosaccharides, or synthetic oligosaccharides tailored for specific functional properties.

[0278] In certain embodiments, the fibre comprises cellulose, hemicellulose, lignin, chitosan, pectin, alginate, carrageenan, or other biopolymers suitable for use in textiles, composites, biodegradable packaging, dietary fibre supplements, or industrial applications such as filtration or thickening agents. In certain embodiments, the fibre is as described elsewhere in the disclosure .

[0279] In certain embodiments, the biologically derived product comprises bioplastics such as polylactic acid (PLA) or polyhydroxyalkanoates (PHA); organic acids including lactic acid, citric acid, or succinic acid; biosurfactants such as rhamnolipids or sophorolipids; bio-based adhesives; bioinks for 3D printing; and other renewable materials for industrial, agricultural, or consumer applications.

[0280] The person skilled in the art could readily select a growth medium for growing the microorganism, based on the known nutritional requirements for the microorganism. In certain embodiments, the growth medium is a feedstock, substrate, biomass, grist, grain, or cereal, fruit or vegetable, fruit or vegetable juice as described elsewhere in the disclosure.

[0281] The person skilled in the art could readily select the conditions and period of time for incubating the microorganism -inoculated growth medium. This could be selected based on, e.g., the type of microorganism and the product (e.g. the bioenergy compound, the food, the supplement, the therapeutic, the pharmaceutical molecule, the enzyme, the probiotic, a prebiotic, an antibiotic, an oligosaccharide, the fibre or the biologically derived product). In certain embodiments, the conditions comprise optimising parameters such as temperature, pH, aeration, and agitation to support efficient production of bioenergy compounds, foods, supplements, therapeutics, pharmaceutical molecules, enzymes, probiotics, prebiotics, antibiotics, oligosaccharides, fibres, or biologically derived products. Temperature requirements typically range from about 10°C to 50°C, with narrower ranges depending on the microorganism and the target product. For example, Saccharomyces cerevisiae and Saccharomyces stipitis, used for ethanol production, perform optimally at temperatures of about 25 °C to 37°C. Lactic acid bacteria, used for fermented foods like yogurt or kimchi, thrive at 30°C to 45 °C under slightly acidic pH conditions of 4.0 to 6.5. Aspergillus niger and Trichoderma reesei. used for enzyme production and fibre processing, operate efficiently at temperatures of about 25°C to 35°C and pH 4.0 to 5.5. Lipid- accumulating yeasts like Pichia melhanolica. employed for biodiesel precursors, favor temperatures of 20°C to 30°C and near-neutral pH. Probiotic strains, such as Lactobacillus and Bifidobacterium, typically thrive at 35°C to 42°C and pH 5.0 to 6.5. Aeration levels can range from minimal to high, depending on the oxygen demands of the microorganism; for example, 0.1-1.0 vvm for aerobic cultures, while strictly anaerobic bacteria like Clostridium thermocellum require oxygen-free environments. Agitation speeds may vary from 20 to 500 rpm in submerged fermentations to ensure sufficient mixing and oxygen transfer, while solid-state fermentations rely on maintaining humidity levels of 60-90%. These parameters can be tailored to the specific microorganism and feedstock to maximise yields of the desired product. In certain embodiments, the conditions are as described elsewhere in the disclosure.

[0282] In certain embodiments, the period of time comprises durations ranging from about 12 hours to several weeks, influenced by the type of microorganism, feedstock composition, and the target product. Ethanol production using Saccharomyces cerevisiae may involve fermentation periods of about 1 to 3 days under optimal conditions. Lactic acid fermentation for foods such as sauerkraut or kimchi may take about 3 to 7 days, while probiotic cultivation can be completed in 12 to 48 hours. Biogas (methane) production through anaerobic digestion typically requires about 7 to 21 days at mesophilic temperatures (20°C to 40°C) and may extend to several weeks at lower temperatures. Lipid production by Pichia methanolica for biodiesel precursors may take about 3 to 7 days. Pharmaceutical molecules such as insulin or monoclonal antibodies, produced using genetically engineered microorganisms, may require cultivation times of about 3 to 14 days depending on the scale and system. Hydrogen production through photofermentation using Synechocystis sp. or lipid synthesis using Chlamydomonas reinhardtii may require extended cultivation periods of 1 to 4 weeks. Enzyme production by Trichoderma reesei or Aspergillus niger may take about 5 to 10 days, depending on fermentation conditions. The person skilled in the art could adjust these timeframes and conditions to optimise yields for bioenergy compounds,foods, supplements, therapeutics, pharmaceutical molecules, enzymes, probiotics, prebiotics, antibiotics, oligosaccharides, fibres, or biologically derived products. In certain embodiments, the period of time is described elsewhere in the disclosure.

[0283] As would be appreciated by a person skilled in the art, exposing the microorganism-inoculated growth medium to an electric field refers to positioning the microorganism-inoculated growth medium within the influence of the field. The microorganism-inoculated growth medium may be exposed to the field for a portion or all of the fermentation process. For example, the exposure may be for a period of at least 1 minute. In certain embodiments, the juice is exposed to the field for a duration of 1, 2, 5, 10, 15, 20, 30, 45, or up to 60 minutes. In other embodiments, the exposure may extend for several hours, such as 12, 24, or 48 hours, or for the entire fermentation process, which may last up to 1, 2, or even 3 weeks depending on the fermentation. Shorter exposure times, such as 1 to 10 minutes, may be employed to achieve specific effects, such as accelerating activity of the microorganism.

[0284] The above aspects (first to thirteenth) were described in view of the concept of using an electric field, that is essentially free of a magnetic component, wherein the electric field is oscillating at one or more frequency in the range of about 210 Hz to about 99 kHz. These aspects were unconstrained by embodiments of the structure used to generate the electric field. One such embodiment comprises generating the electric field by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz. The following aspects (fourteenth to twenty-sixth) are described in the context of this structure. However, the person skilled in the art would understand that other structures could be used to generate the electric field. Some embodiments of the structure are described elsewhere in the disclosure with reference to the device, methods and system of the disclosure.

[0285] In certain embodiments, the frequency is in the range as described elsewhere in this disclosure. The electric field may be as described elsewhere in this disclosure. The electric field may have a strength as described elsewhere in this disclosure.

[0286] As would be appreciated by the person skilled in the art, various other factors determine the strength of an electric field, such as the distance between terminals, and the properties of the medium through which the field propagates — such as its permittivity and conductivity. Additionally, the geometry and surface area of the terminals, as well as their orientation relative to each other, can influence the electric field distribution and intensity. These are described elsewhere in this disclosure with reference to embodiments of the device, methods and system.

[0287] In certain embodiments, there is provided a means to prevent formation of any significant magnetic component to the electric field as described elsewhere in this disclosure.

[0288] In a fourteenth aspect, there is provided a method of modulating the activity of a functional protein, comprising: exposing the functional protein to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0289] The method may be performed as described in the first aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context. In certain embodiments, the functional protein is as described elsewhere in this disclosure.

[0290] In a fifteenth aspect, there is provided a method of modulating the rate of microbial cell division or affecting microbial cell density, comprising: exposing one or more microorganism to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0291] The method may be performed as described in the second aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0292] In a sixteenth aspect, there is provided a method of modifying gene transcription in a microorganism, comprising: exposing the microorganism to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0293] The method may be performed as described in the third aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0294] In a seventeenth aspect, there is provided a method of enhancing the flavour of a fermented food, beverage or wash, comprising: contacting a food, beverage or wash substrate with a microorganism to provide a slurry, wherein the microorganism is adapted to metabolise the substrate, exposing the slurry to an electric field, that is essentially free of a magnetic component, generated by applying a voltageacross at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0295] The method may be performed as described in the fourth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0296] In an eighteenth aspect, there is provided a method of disinhibiting cell division in a microorganism, comprising: exposing the microorganism to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0297] The method may be performed as described in the fifth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0298] In a nineteenth aspect, there is provided a method of brewing beer, comprising: contacting a grist, grain or cereal with water to provide a mash; exposing the mash to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz, to provide a treated mash; obtaining a wort from the treated mash; and using the wort to brew beer.

[0299] The method may be performed as described in the sixth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0300] In a twentieth aspect, there is provided a method of brewing beer, comprising: adding yeast to a boiled cooled wort to provide a yeast-inoculated wort; fermenting the yeast-inoculated wort under conditions and for a period of time to produce beer, wherein the conditions comprise; exposing the yeast- inoculated wort to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0301] The method may be performed as described in the seventh aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0302] In a twenty-first aspect, there is provided a method of brewing beer, comprising: contacting a grist, grain or cereal with water to provide a mash; treating the mash under a first set of conditions and for a first period of time to provide a treated mash; obtaining a wort from the treated mash; boiling and cooling the wort; adding yeast to a boiled cooled wort to provide a yeast-inoculated wort; fermenting the yeast-inoculated wort under a second set of conditions and for a second period of time to produce beer; wherein the first set of conditions comprise; exposing the mash to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz; and wherein the second set of conditions comprise; exposing the fermenting beer to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0303] The method may be performed as described in the eighth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0304] In a twenty-second aspect, there is provided a method of fermenting a fruit or vegetable juice to make a fermented food, beverage or wash, comprising: providing juice from a fruit or vegetable, adding yeast to the juice to provide a yeast-inoculated juice, fermenting the yeast-inoculated juice under conditions and for a period of time to produce a fermented food, beverage or wash, wherein the conditions comprise: exposing the yeast-inoculated juice to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0305] The method may be performed as described in the ninth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0306] In a twenty-third aspect, there is provided a method of producing a carbonated fermented beverage, comprising: providing juice from a fruit, adding yeast to the juice to provide a yeast-inoculated juice, fermenting the yeast-inoculated juice under conditions and for a period of time to produce afermented beverage, wherein the conditions comprise: exposing the yeast-inoculated juice to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz; and performing a secondary fermentation to provide a carbonated fermented beverage.

[0307] The method may be performed as described in the tenth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0308] In certain embodiments, the fermented beverage is a carbonated white wine, a carbonated red wine, a sparkling rose, a sparkling wine, a carbonated ready to drink beverage, an alcoholic mixer, a cider or beer.

[0309] In a twenty-fourth aspect, there is provided a method of producing probiotic microorganisms, comprising: providing a growth medium for growing a probiotic microorganism; adding the probiotic microorganism to the growth medium to provide a probiotic microorganism-inoculated growth medium; incubating the probiotic microorganism-inoculated growth medium under conditions and for a period of time to produce a plurality of the probiotic microorganism, wherein the conditions comprise: exposing the probiotic microorganism-inoculated growth medium to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0310] The method may be performed as described in the eleventh aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0311] In a twenty-fifth aspect, there is provided a method of producing a biofuel or fibre, comprising: providing a biomass; contacting the biomass with a microorganism to provide a microorganism - inoculated biomass; incubating the microorganism-inoculated biomass under conditions and for a period of time to produce a biofuel or fibre; wherein the conditions comprise exposing the microorganism- inoculated biomass to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0312] The method may be performed as described in the twelfth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0313] In a twenty-sixth aspect, there is provided a method of enhancing a fermentation to produce a bioenergy compound, a food, a supplement, a therapeutic, a pharmaceutical molecule, an enzyme, a probiotic, a prebiotic, an antibiotic, an oligosaccharide, a fibre or a biologically derived product, comprising: providing a growth medium for growing a microorganism; adding the microorganism to the growth medium to provide a microorganism -inoculated growth medium; incubating the microorganism- inoculated growth medium under conditions and for a period of time to produce the bioenergy compound, the food, the supplement, the therapeutic, the pharmaceutical molecule, the enzyme, the probiotic, the prebiotic, the antibiotic, the oligosaccharide, the fibre or the biologically derived product; wherein the conditions comprise wherein the conditions comprise: exposing the microorganism-inoculated growth medium to an electric field, that is essentially free of a magnetic component, generated by applying a voltage across at least a first terminal and a second terminal, wherein the first terminal and the second terminal are spaced apart and not in contact with each other, and wherein the first terminal and second terminal have opposite polarities, and iteratively reversing a polarity of the voltage at one or more frequency in the range of about 210 Hz to about 99 kHz.

[0314] The method may be performed as described in the thirteenth aspect of the disclosure, or as described elsewhere in this disclosure, unless indicated otherwise by context.

[0315] Device

[0316] In a twenty-seventh aspect, there is provided an electric field-generating device of the disclosure. The device comprises a power supply, a differential oscillator connected to the power supply, the oscillator comprising first and second outputs, at least a first voltage terminal connected to the first output, at least a second voltage terminal connected to the second output, wherein the first voltage terminal is spaced apart from the second voltage terminal, and the first voltage terminal is not in contact with the second voltage terminal, wherein the power supply supplies a voltage to the oscillator and wherein the oscillator supplies the voltage to the first and second voltage terminals, such that the first terminal and second voltage terminals have opposite polarities, and the oscillator reverses a polarity of the voltage at a frequency in the range of about 210 Hz to about 99 kHz to generate an electric field through the first and second voltage terminals that is essentially free of a magnetic component. Embodiments of the device are shown in Figures 21 to 22, and 24 to 27.

[0317] In some embodiments of this aspect, the device electric field-generating device to improve a stress resistance, a longevity, or both, of microorganisms, the device comprising: a controller to generate an electric field; wherein the generated electric field is oscillating at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is substantially free of a magnetic field; andwherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

[0318] The device may be used to treat, e.g., a fermentation, microorganism or enzyme. This may be performed by treating or exposing the fermentation, microorganism or enzyme to the electric field. If the fermentation, microorganism or enzyme is in a container, the device may be placed in or adjacent to the container to treat or expose the fermentation, microorganism or enzyme to the electric field.

[0319] In certain embodiments, the electric field has a strength as described elsewhere in the disclosure. The electric field, and the absence of a magnetic field, may be measured using an EMF meter or oscilloscope. In certain embodiments, the frequency is as described elsewhere in the disclosure. In certain embodiments, the voltage is as described elsewhere in the disclosure.

[0320] As would be appreciated by the person skilled in the art, there are multiple ways to generate the oscillating electric field. As such, the differential oscillator comprises multiple approaches to generating a differential oscillation. In certain embodiments, the differential oscillator comprises a phase-locked loop (PLL) circuit, where a voltage -controlled oscillator (VCO) produces oscillations stabilised by feedback through a phase detector and a loop filter. In other embodiments, the differential oscillator comprises a cross-coupled LC tank circuit, employing matched transistors or other active devices to drive a resonant LC network for differential output. In certain embodiments, the differential oscillator comprises a ring topology with an odd number of inverting stages, each producing a phase-shifted signal, resulting in stable differential oscillation. Additionally, some embodiments comprise a digitally controlled oscillator (DCO) driven by a microcontroller or field-programmable gate array (FPGA) to generate complementary pulse signals that are subsequently filtered for clean differential waveforms. For enhanced stability and frequency precision, a temperature -compensated crystal oscillator (TCXO) or surface acoustic wave (SAW) resonator may serve as a frequency reference. Collectively, the multiple approaches may be termed a differential oscillator or microcontroller.

[0321] In embodiments with a controller, the controller is configured to generate a waveform and supply a modulated voltage to at least a first and a second voltage terminals or antennae, wherein at least the first and second voltage terminals or antennae are controlled by an one or more output channels configured to supply at least a first and a second signal that are of the same phase as each other or are phase-shifted relative to each other; or to control the at least a first and a second voltage terminals to increase field exposure and uniformity of exposure to microbial cells; or both.

[0322] In certain embodiments, the modulated voltage supplied to at least the first voltage terminal or antenna is in a range of 1 V RMS to 40 kV RMS, and the controller or at least the first voltage terminal or antenna, or both, comprise a means to prevent formation of a magnetic component of the generated electric field. In some embodiments, the modulated voltage is relatively low, for example in a range of 1V RMS to 10 V RMS, such as 2 V RMS to 8 V RMS. Such lower-voltage embodiments can be advantageous in small-scale bench reactors, laboratory screening systems, or micro-bioreactors where the volume of the composition is small and only modest field strengths are required to provide surmounting energy to functional proteins. In other embodiments, the modulated voltage is in a range of 10 V RMS to 100 V RMS, such as 20 V RMS to 80 V RMS or 30 V RMS to 60 V RMS. Voltages in this range can be useful for pilot-scale fermenters and process -development systems, where the characteristic dimensions between the electrically -insulated emitter and the composition are moderate, and where an electric field of sufficient strength can be generated without resorting to kilovolt amplitudes. In further embodiments, the modulated voltage is in a range of 100 V RMS to 1 kV RMS, such as 150 V RMS to 800 V RMS. In yet further embodiments, the modulated voltage is in a range of 1 kV RMS to 10 kV RMS, such as 2 kV RMS to 8 kV RMS or 3 kV RMS to 7 kV RMS. These embodiments can be suitable, for example, for medium- to large-scale bioreactors where the spacing between emitter and composition is larger, and higher potentials are used to maintain an electric field of effective magnitude across the working volume. In still further embodiments, the modulated voltage is in a range of 10 kV RMS to 40 kV RMS, such as15 kV RMS to 30 kV RMS or 20 kV RMS to 35 kV RMS. Higher-voltage embodiments can be used where the emitter is placed outside the primary container wall, or where the dielectric between the emitter and the composition is relatively thick or of relatively low permittivity, such that a higher applied voltage is useful to generate the desired near electric field within the composition. In some embodiments, the modulated voltage is at least 1 V RMS, at least 5 V RMS, at least 10 V RMS, at least 50 V RMS, at least 100 V RMS, at least 500 V RMS, at least 1 kV RMS, or at least 5 kV RMS. In alternative or additional embodiments, the modulated voltage is at most 40 kV RMS, at most 30 kV RMS, at most 20 kV RMS, at most 10 kV RMS, at most 5 kV RMS, at most 1 kV RMS, at most 500 V RMS, or at most 100 V RMS. Any of these lower-bound and upper-bound values can be combined to define a closed sub-range within 1V RMS to 40 kV RMS.

[0323] In each of these embodiments, the controller, the first voltage terminal or antenna, or both, comprise a means to prevent formation of a significant magnetic component of the generated electric field, for example by configuring the emitter circuit so that substantially no net current flows through the composition, by providing capacitive or inductive blocking elements in series with the emitter, by avoiding closed current loops, or by any combination of these measures, such that the composition is predominantly exposed to an electric near field that is substantially free of a magnetic component. The means to prevent formation of a significant magnetic component of the generated electric field may also be as described elsewhere in this disclosure.

[0324] Turning Figure 21, there is illustrated a first embodiment of the device 100. The device is designed to produce an electrical field without significant magnetic components. The illustrated device consists of several key components including a power supply 110, a differential oscillator 120, first and second voltage terminals 130, 132, and two output signals (first output 122 and second output 124).

[0325] The power supply 110 is responsible for providing the necessary voltages to operate all components within the device. The differential oscillator 120 acts as a central component in the circuit, producing an output signal based on the input voltage from the power supply 110.

[0326] The first voltage terminal 130 is connected to the first output 122 and the second voltage terminal 132 connected to the second output 124. The first voltage terminal 130 is spaced apart from the second voltage terminal 132. These terminals receive the output signals from the oscillator and distribute them across a gap to create an electric field without significant magnetic components. The spacing between these two terminals ensures that they maintain opposite polarities at all times, in order to generate the desired electrical field.

[0327] As the person skilled in the art would appreciate, if only a voltage terminal is attached to the first output 122 or if only a voltage terminal is attached second output 124, then no electric field is created. This may be detected using an oscilloscope or an EMF meter set to electric mode. The person skilled in the art would appreciate that, in this illustrated embodiment, at least two terminals of opposite sign, i.e. one deriving from output 122 and one from output 124, may be required to create the electric field.

[0328] A further embodiment of the device is illustrated in Figure 22, which shows an embodiment of the functional features of the bioreactor control unit 160 and its power supply unit 110. The power supply unit (110) takes the AC input from a mains power supply and converts it to DC power of about 1 V to about 450 V or multiple DC voltages. The bioreactor control unit (160) in turn takes the DC input from the power supply unit (110) and uses that DC input to generate a DC oscillating voltage. Specifically, the sign of the voltage supplied to any output node (140, 142, 144, 146) oscillates between being positive or negative and does so at a frequency which is within the frequency range at which the device operates (e.g. range of about 210 Hz to about 99 kHz). To generate the signal of oscillating voltage, the bioreactor control unit contains a means such as a microcontroller circuit (also described as differential oscillator 120) which may generate a curved or so called “harmonic oscillation” waveform, or may generate a linear or so called “relaxation oscillation” waveform. In cases where the microcontroller circuit generates a relaxation oscillation, the microcontroller circuit is termed a relaxation oscillator. In cases where the microcontroller circuit generates a harmonic oscillation, the microcontroller circuit is termed a harmonic oscillator. Throughout other parts of the specification, these may be generically referred to as a differential oscillator. In the illustrated embodiment, the output signals (122 and 124) of the microcontroller is split (at node 185 and node 195) into two identical signals, so providing four output signals via the output nodes (140, 142, 144 and 146). At a given point in time, the output signal 122 is negative in voltage sign, while at that same point in time the output signal 124 is positive in voltage sign, unless they are both zero at that point in time. Signals from the output nodes 140, 142, 144 and 146 feed voltage terminals 130, 132, 134 136, embodiments of which are shown in Figures 27 to 31.

[0329] The device may comprise a means to prevent formation of a significant magnetic component to the electric field. In certain embodiments, that means is one or more capacitor. This is illustrated at nodes 185 and 195. In certain embodiments, a capacitor is located at node 185, at node 195 or at both nodes 185 and 195. In other embodiments, the means to prevent formation of any significant magnetic component to the electric field is one or more gyrator circuit, switched-capacitor circuit, inductor configured within a resonant circuit, capacitor or an integrated circuit comprising resistors and active components. In other embodiments, the means to prevent formation of a significant magnetic component to the electric field is as described elsewhere in the disclosure.

[0330] In embodiments in which a capacitor is emplaced at node 185 or node 195 or both, the degree of capacitance of the capacitor or capacitors may be selected so as not to reduce the oscillating voltage signal nor distort the oscillating voltage signal that is measured on an oscilloscope. As would be understood by the person skilled in the art, the signal may be derived from attachment of the input channels of the oscilloscope to either node 140 or 142 in combination with either node 144 or 146, such as by two test probes wired to the input channels of the oscilloscope and likewise to the nodes. If the signal on the oscilloscope shows attenuation or distortion, then, e.g., the next higher standard capacitor value may be selected.

[0331] By a capacitive means firmly emplaced at node 185 or node 195 or both, a desired wave shape and frequency of signal is made to feed into the voltage terminals of the bioreactor module 200, so that an essentially pure electric field is created and maintained between the voltage terminals, and a preventative block is placed on the formation of any significant magnetic component of such field. In certain embodiments, the electric field strength measured at the surface of the bioreactor module 200 is around 450 v / cm. In certain embodiments the electromagnetic field strength measured at the surface of the bioreactor module 200 is around 0. 1 milligauss to 4 milligauss, effectively background levels of electromagnetism, and not discemibly due to operation of the bioreactor module. Liquid in a fermenter vessel or incubation vessel affects measurements of electric fields and electromagnetic fields, and so these fields may rather be measured while the bioreactor module is operating in air, such as in an unfilled fermenter vessel or an unfilled incubation vessel.

[0332] In the art, pure electric fields are known to exist, such as between the plates of a capacitor. The force acting on a charged particle located within an electric field, such as between the plates of a capacitor, is given by the formula F=qE where F is the force acting on the particle, q is the charge on the particle (the charge being of either sign, positive or negative) and E is the strength of the electric field, measured in Volts per meter or Volts per cm. By means of the device provided, an electric field is similarly set up between voltage terminals, just as it can be set up between the plates of a capacitor. Inclusion of a capacitor is optional in the methods, device and system provided, and is one means of generating an essentially pure electric field.

[0333] Other means of generating a purely electric field could be envisaged and are explained elsewhere in this disclosure. For example, a microcontroller 120 may itself contain means of removing the magnetic component of a signal. In embodiments, the feature of deliberately not electrically connecting a voltage terminal from node 140 or node 142 to a voltage terminal from node 144 or 146, is a feature that prevents the formation of any significant magnetic component in the electric field that is setup between voltage terminal 130 or 134 and voltage terminal 132 or 136. It is by not connecting together voltage terminals via contact (e.g., not connecting voltage terminals 13 and 132 from nodes 140 and 144, for instance, being voltage terminals that connect to both nodes 185 and 195), that the formation of a closed circuit is prevented. A closed circuit would conduct current and generate a magnetic field around such connected voltage terminals.

[0334] Formation of a magnetic field occurs by electron flow in a conductor such as a wire. Current flow in a wire requires electrons entering and leaving the wire. Copper wires are an example of a material that contains an essentially fixed number of electrons to which few electrons can be added or subtracted, although addition or subtraction of enough electrons can occur to generate a charge in the copper. The number of electrons addable or subtractable is limited by the mass of the piece of copper. By contrast, electrons can freely flow through the copper wire if they are entering and leaving at the same rate, and such free flow generates a more considerable magnetic field than merely charging a copper wire or plate does. Circuits are also conductors, around which electrons flow, although yet resisted, impeded and transisted by many components. As such, electronic circuits can inadvertently produce a magnetic field at some level in excess of the ambient magnetic field. In certain embodiments, the device uses insulated copper wire of from gauge 12 to gauge 16, to carry a signal from the differential oscillator or microcontroller 120 to the voltage terminals 130, 132, 134, 136. In certain embodiments the material of voltage terminals is itself insulated copper wire of gauge 12 to gauge 16.

[0335] In embodiments in which the differential oscillator is a microcontroller 120, there may be provided a signal generator and a signal amplifier, in that order. In the case of the signal generator, very little current would be expected to circulate in that circuit, and thus very little magnetic field would be generated. In the amplifier, a greater amount of current could reasonably be expected to circulate. As such some minimal measurable electromagnetic field may be generated in the microcontroller circuit as a whole. The microcontroller circuit may be located away from the bioreactor module 200, so as to not impart an electromagnetic field to the incubator or fermenter vessel. In certain embodiments, when a voltage terminal is attached to a node such as node 185 or node 195 that delivers the output of the amplifier circuit of the microcontroller, little or no current is expected to flow up the wire from node 185 or node 195 to the voltage terminal nor along the conducting material of the voltage terminal, owing to the fact that neither voltage terminal 130 or 132 contacts voltage terminal 134 or 136. Because significant current does not flow from node 185 to a voltage terminal, nor from node 195 to a voltage terminal,neither the connecting wires from node to voltage terminal, nor the voltage terminals themselves, carry significant current.

[0336] In the art, the phenomenon called voltage is described purely as the potential for a charged particle to release or obtain kinetic energy by moving between two points. The presence of a voltage does not imply that a current is moving between two points. Similarly, the presence of a voltage does not imply that a charged particle is moving between two points. In certain embodiments, in the methods, device and system of the disclosure, such voltage is set up between two voltage terminals.

[0337] In certain embodiments, the differential oscillator or microcontroller 120 sets up a voltage between node 185 and node 195, and thus between voltage terminals 130-134 and 132-136. To inhibit the formation of a magnetic field component creatable between voltage terminals, that may be due to creation of a voltage difference by the microcontroller, the microcontroller may employ means to minimise current flow within the circuit of either the signal generator or the amplifier (or both) that together comprise the microcontroller . Alternatively, other types of differential oscillator, such as a relaxation oscillator or harmonic oscillator that do not employ a microcontroller of the given description, may achieve these same ends. Other alternatives to a microcontroller, relaxation oscillator or harmonic oscillator as provided elsewhere in this disclosure. As explained with regard to certain embodiments of the methods, device and system provided herein, a differential oscillator generates an essentially pure electric field between one or more pairs of voltage terminals, and the directionality of that electric field, also called polarity of that electric field, is a characteristic that oscillates. The voltage itself may also oscillate periodically if the shape of the field is not of a square wave shape. The oscillation of polarity of the electric field, describes a periodic reversal of polarity, such as occurring at a frequency of 210 Hz, or a frequency of about 99 kHz, or at some frequency between those two bounds, or at frequencies within the range of those two bounds, in series. In certain embodiments, the methods, device or system employs both a square wave produced by a relaxation oscillator, and a series of frequencies freely selected in the range 1 kHz to 5 kHz.

[0338] Figure 23A is a graph depicting voltage over time, of a signal received from the microcontroller at a single output node, being any one node of the pair 140 or 142. That signal at that node at a given moment in time may be positive or negative in voltage, or may be zero in voltage. In the graph shown, the signal oscillates between being positive and negative in voltage. The oscillation, when graphed, takes a so-called wave shape. The shapes that a relaxation-oscillator produces are generally either square-wave shaped, or sawtooth-wave shaped. The shapes that a harmonic-oscillator produces are either sine-wave shaped or in some way curve shaped. In certain embodiments, the oscillation comprises a sine-wave shape, a square-wave shape, or sawtooth-wave shape. In certain embodiments, a relaxation oscillator is preferable. An advantage of a square wave or sawtooth wave over a sine wave is that the transition toopposite sign is sudden, so giving a sudden impulse to any charged moieties that are present in the liquid or medium exposed to the electric field.

[0339] Figure 23B is a graph depicting voltage over time, of a signal received from the microcontroller at a single output node, being any one node of the pair 144 or 146 depicted in concurrent time with the signal received from the microcontroller at nodes 140 or 142 (panel A). Time proceeds from left to right. As an example of the relationship between signals that control voltage terminals, time D is the same moment of time in panels A and B. At time D the square wave signal in panel B is transitioning almost instantaneously from negative 100V to positive 100V, meanwhile at that same time D, the square wave signal in panel C is transitioning almost instantaneously from positive 100V to negative 100V. Thus, by time E (an equivalent moment in time in panels A and B) the square wave signal in panel B is at 100 V and stable, while the square wave signal in panel C is at negative 100V and stable. As would be appreciated by the person skilled in the art, the device output (e.g. field strength) may be measured with an oscilloscope which shows the wave shape and the voltage between peaks, taken together with the distance between voltage terminal and target.

[0340] Figures 24 to 27 show different embodiments of the voltage terminals. In these embodiments, the device consists of a power supply (110), a microcontroller or differential oscillator (120), first and second voltage terminals (130 and 132), two output signals (122 and 124) from the oscillator, and optionally non-conducting material (190).

[0341] In one embodiment, as shown in Figure 24, the device includes a first voltage terminal 130 and second voltage terminal 132 that are parallel plates. The power supply 110 provides a voltage to the oscillator 120, which produces two output signals (first output 122 and second output 124). These signals are then distributed across the gap between the first and second voltage terminals 130, 132, creating an electric field without significant magnetic components.

[0342] In another embodiment, as shown in Figure 25, the device includes a first voltage terminal 130 and second voltage terminal 132 that are parallel plates separated by non-conducting material 190. The power supply 110 provides a voltage to the oscillator 120, which produces two output signals (first output 122 and second output 124). These signals are then distributed across the gap between the first and second voltage terminals 130, 132 through non-conducting material 190. This ensures that an electric field with minimal or no magnetic component is generated across the gap. The parallel plates may be contacting the non-conducting material, or attached via, e.g., a fastener, cognate surfaces or by an adhesive. The illustrated non-conducting material 190 is a cuboid.

[0343] In yet another embodiment, as shown in Figure 26, the device includes a first voltage terminal 130 and second voltage terminal 132 that are helically coiled wire, wrapped around non-conducting material 190. The power supply 110 provides a voltage to the oscillator 120, which produces two outputsignals (first output 122 and second output 124). These signals are then distributed across the gap between the first and second voltage terminals 130, 132 through non-conducting material 190. This ensures that an electric field with minimal or no magnetic component is generated across the gap. The illustrated nonconducting material 190 is a rod.

[0344] While the illustrated voltage terminals take the form of plates or helical coils, the voltage terminals are not so limited. In some embodiments, various emitter configurations, including parallel plates, helically coiled wires, or other linear, folded, grid, lattice, spiral, or conical arrangements of conductive materials (e.g., copper, aluminium), may be employed. These terminals are insulated, and the insulation can be of any non-conducting material, further preventing current flow and ensuring the integrity of the electric field. These emitter configurations are used to generate electric fields as described herein which, in use, provide surmounting energy to facilitate protein breathing in the cells’ functional proteins and thereby support stress resistance and longevity. In alternative embodiments, the first and second voltage terminals may take the form of various configurations for generating an electric field, including but not limited to linear arrangements, folded designs, closed-loop structures, and helical patterns. The first and second voltage terminals may also be arranged in planar configurations, such as grid or lattice structures, or in volumetric patterns, such as spiral or conical setups. Additional configurations may include fractal geometries to enhance field distribution and compactness, or meandered paths to optimise spatial efficiency. In embodiments, the first and second voltage terminals may be planar, elongate or take the form of wires. These first and second voltage terminals may be composed of conductive materials, such as copper (or any other noble metal), aluminium, or conductive composites. As would be appreciated by the person skilled in the art, the first and second voltage terminals may be arranged in predetermined patterns to achieve specific field characteristics and operational performance.

[0345] In certain embodiments, when in an uninsulated wire configuration, the first and second voltage terminals may take the form of any of the abovementioned configurations in which adjacent winds of wire do not contact each other. The insulation around the wires can be of any non-conducting material.

[0346] In other embodiments, when in an insulated wire configuration, the first and second voltage terminals may take the form of any of the abovementioned configurations and any other form in which adjacent winds of wire contact each other. In certain embodiments, the first and second voltage terminals comprise insulated wires that are coiled, twisted or wrapped together. The insulation around the wires can be of any non-conducting material.

[0347] In some embodiments, a novel passive reradiation system is incorporated into the device to further enhance the electric field's interaction with the probiotic culture. Reradiation components may for instance consist of coils of conductive material (e.g., insulated copper wire, gauge 12-16) placed in proximity to the primary electric field emitters but not electrically connected to the main circuit. Instead,the re-radiators are designed to capacitively couple with the oscillating electric field, absorbing and then re-emitting the electric field energy as a combined semi-electric field that may contain an electric component combined with a minor magnetic component. This passive re-emission effectively increases the spatial reach and uniformity of the electric field within the growth medium, ensuring that a larger volume of the probiotic culture is subjected to the beneficial field, thereby maximising the treatment's efficiency. The reradiation coils can take various forms, such as co-helical windings around the nonconducting material alongside the primary voltage terminals, or as separate windings or planar grids or other reradiating conductors strategically positioned within the container holding the probiotic culture. The enhanced field distribution provided by reradiation is advantageous for ensuring consistent probiotic growth and activity throughout the medium, high overall productivity, and effective treatment of large culture volumes.

[0348] Typically, a probiotic culture container or culture vessel is composed of stainless steel impervious to the ingress of electric fields and electromagnetic fields which may be present in the space that surrounds the exterior of the culture vessel. The electric field modulator device, method and system specified in this disclosure benefits from exclusion of external fields, but may also operate in culture vessels that are not protected from external fields, owing to the specific biological activity of electric field frequencies modulated in the specific frequency range from about 210 Hz to about 99 kHz.

[0349] Pre-existing coils that may be present in or near to an existing culture vessel such as motor windings, may act as reradiation coils for the specific frequencies that the disclosure covers, from about 210 Hz to about 99 kHz which enhance bioprocessing. Reradiation from such coils may comprise a combined-semi -electric field that has an electric component combined with a minor magnetic field component. The magnetic field component from a reradiation coil, not being caused by a powered electric current, and not being driven by the controller provided, is not anticipated to interfere with increase in cell viability, or increase in longevity or stress resistance of microorganisms.

[0350] A combined-semi-electric field is one in which the electric component does the work on affecting protein breathing by providing surmounting energy to facilitate increased, enhanced or improved protein function. A combined-semi-electric field may be produced by a powered emitter or by an unpowered emitter.

[0351] A near electric field may be used in conjunction with a combined-semi-electric field such as a combined-semi -electric field emitted from another type of field emission technology, such as an uninsulated current carrying loop, or an insulated current carrying loop, to achieve an industrial benefit for stress resistance and viability of a microorganism without ohmic heating, where the improvement in stress resistance and viability is specifically by means of the electric component of the field influencing protein breathing in a microorganism without involvement of ohmic heating.

[0352] Similarly, a near electric field may be used in conjunction with a combined-semi-electric field such as emitted from another type of field emission technology such as an electrode or electrodes, or an uninsulated current carrying loop or an insulated current carrying loop, to achieve an industrial benefit for stress resistance and viability of a microorganism without electrochemical damage, where the improvement in stress resistance and viability is specifically by means of the electric component of the near electric field influencing protein breathing in a microorganism despite the presence of potentially damaging field emission technology.

[0353] In the embodiments illustrated in Figures 25 and 26, the non-conducting material 190 is a cuboid or rod. In other embodiments, the non-conductive material may be configured into various shapes, including but not limited to rods, cuboids, rectangular prisms, or other geometric forms tailored to the application. A rod configuration may be used in systems requiring linear separation, providing an elongated barrier that ensures sufficient distance between terminals to prevent completion of the circuit. A cuboid or rectangular prism design may be employed in cases where broader or flatter surfaces are necessary to support terminals securely while maintaining structural stability. Additional configurations may include cylindrical shapes for uniform separation in rotational or symmetrical systems, spherical forms for compact applications requiring multidirectional insulation, and custom contoured geometries designed to fit irregular or specific spatial constraints. Honeycomb structures may be incorporated for weight reduction while maintaining high mechanical strength, and laminated sheets may be layered to enhance dielectric properties. In embodiments comprising laminated sheets, the first and second voltage terminals may be positioned in the same layer or separated by non-conducting material into a different or adjacent layer.

[0354] The first and second voltage terminals may be separated by any suitable distance to generate an electric field. The distance in Figures 21, 22 and 24 to 26 is not explicitly defined, as the distance can be adjusted based on the specific application and design requirements, taking into account factors such as voltage, voltage terminal configuration and desired electric field strength. In certain embodiments, the first and second voltage terminals are arranged in close proximity without direct contact, minimising a separation distance to enhance a strength of the electric field between them.

[0355] In certain embodiments, the distance is greater than 1mm. In certain embodiments, the distance is between about 1mm and about 5 m, about 1mm and about 500mm, such as between about 1mm and about 50mm, about 20mm and 50mm, about 1mm and about 20mm, about 1mm and 5mm or about 3mm and about 20mm.

[0356] The voltage terminals may be arranged to maximise the electric field while preventing dielectric breakdown. A person skilled in the art would appreciate that the electric field ( / ■.') between two terminals is governed by the relationship E=V / d, where V represents the voltage difference and d the distance between the terminals. To achieve a high electric field, the distance d should be minimised. However, theminimum distance is constrained by the dielectric strength of the intervening material, which must be sufficient to prevent electrical breakdown or arcing. For example, in air, the dielectric strength is approximately 3 x 106V / m under standard conditions. The minimum safe distance (dmin) for a given voltage can thus be calculated as dmm=V / Emax, where Emaxdenotes the dielectric strength of the medium. To further enhance field uniformity, the terminal geometry may be optimised, such as by using large, flat, and parallel surfaces for uniform fields, or by minimizing sharp edges and protrusions that could induce localised field concentrations and lead to premature breakdown.

[0357] The device may efficiently produce the electric field using different configurations of the voltage terminals. In certain embodiments, the first and second voltage terminals are formed into helical shapes positioned relative to each other in a manner that concentrates the electric field in specific regions. In certain embodiments, the first and second voltage terminals comprise conductive material helically wound around a non-conducting material. In certain embodiments, the first and second voltage terminals comprise insulated conductive material helically wound around a non-conducting material, the nonconducting material comprises an elongate member having a longitudinal axis, the first voltage terminal emplaced at a first axial position along a length of the elongate member, and the second voltage terminal emplaced at a second axial position along the length of the elongate member.

[0358] The strength of the electric field for a given configuration could readily be determined by the person skilled in the art. For example, in an emitter configuration where first and second voltage terminals — each consisting of 1 -meter coils of wire wrapped around a 50 mm diameter plastic rod and separated by 20 mm — are connected to an oscillator supplying a 100-volt alternating voltage at 1000 Hz, the electric field at a distance of 20 mm from the emitter is calculated as follows. Between the coils, the electric field E is determined by dividing the voltage V (100 V) by the separation distance d (0.02 m), resulting in E=V / d=100 V / 0.02 m=5000 V, which is equivalent to 50 V / cm. Outside one of the coils, at a point 20 mm from the coil surface (total radial distance of 45 mm from the coil axis), the electric field is calculated using the formula for a long charged cylinder: E(r)=[V / r] x [l / ln(r / R)], where His 100 V, r is 0.045 m, and R is the coil radius of 0.025 m. Substituting the values givesE(r)=[100 V / 0.045 m]x[l / ln(0.045 m / 0.025 m). Calculating the natural logarithm, ln(0.045 m / 0.025 m)=ln(1.8)«0.5878, the expression becomes E(r)=|2222.22 V / m | / | l / 0.5878|=3780 V. which is approximately 37.8 V / cm. The electric field oscillates at 1000 Hz due to the oscillator's polarity reversal, causing the field to change direction 1000 times per second. The person skilled in the art could readily calculate the electric field for any other configurations.

[0359] The embodiments illustrated in Figures 21, 24, 25 and 26 only show a first and a second voltage terminal. However, the device may comprise four, six, eight or more voltage terminals. In other embodiments, the device comprises an odd number of voltage terminals. As such, in certain embodiments, the device further comprises a third voltage terminal connected to the first output and afourth voltage terminal connected to the second output, wherein the third voltage terminal is spaced apart from the fourth voltage terminal, and the third and fourth voltage terminals are spaced apart from the first and second terminals. In embodiments that have a non-conducting material, the non-conducting material may comprise an elongate member having a longitudinal axis. In such embodiments, there may be the first voltage terminal at a first axial position along a length of the elongate member, the second voltage terminal at a second axial position along the length of the elongate member, the third voltage terminal at a third axial position along the length of the elongate member, the fourth voltage terminal at a fourth axial position along the length of the elongate member.

[0360] An embodiment of the device with four voltage terminals is illustrated in Figure 27. The device (100) is illustrated with a housing 220. The device has a bioreactor control unit (120), connected by a cord of a suitable length (240) to an emitter (210). The bioreactor control unit (120) is powered by a power supply unit PSU (110) which may be mains-supplied or otherwise supplied, e.g., by a generator or via renewable energy.

[0361] The bioreactor module (200) consists of the emitter (210) inserted within a bioreactor housing (220) via the entry (255) of the housing. The emitter (210) comprises four voltage terminals (also called filaments) (130, 132, 134 and 136) emplaced on or connected to a non-conductive material (190). In this embodiment, the voltage terminals are conductive material helically wound around the non-conductive material (190). Insulated wires connect each voltage terminal to the bioreactor control cord and by that means also connect to the bioreactor control unit (120). All bioreactor voltage terminals (130, 132, 134 and 136) are insulated and are also insulation-end-capped, to prevent any closure of any circuit. The connections between the bioreactor control unit (120) and voltage terminals (130, 132, 134 and 136) may be as described or illustrated with reference to Figures 21 to 26.

[0362] In this embodiment, the bioreactor control unit (120) generates an oscillating voltage signal in the range 1 kHz to 5 kHz, and at an amplitude of 100 V. In other embodiments, the oscillating voltage signal is in the range of the 210 Hz to 99 kHz, and the amplitude is 20V to 450V or 20V to 240V. The oscillating voltage signal from the bioreactor control unit (120) powers the voltage terminals of the emitter (210) that are affixed to or near to the non-conductive material (190).

[0363] The entry (255) of the bioreactor housing (220) in this embodiment consists of an attached ferrule (260) which is machined flat on both sides to facilitate attachment of the ferrule to a fermenter vessel or incubator vessel. The ferrule may be integrally formed, permanently attached or removably attached.

[0364] In this embodiment, the emitter (210) may be removably fixed within the bioreactor housing (220). The bioreactor module head (270) may protrude from the bioreactor housing (22) after the emitter (210) is inserted into the bioreactor housing via the bioreactor housing ferrule (260) or the bioreactormodule head (270) may be hidden within the bioreactor module housing. In certain embodiments, a seal is positioned between the bioreactor module head (270) or the emitter (210) and the bioreactor housing (220). In certain embodiments, the bioreactor module head (270) or the emitter (210) fits in the housing to create a seal whereby splashes into the bioreactor module are prevented. In certain embodiments, the bioreactor housing is inserted or emplaced into a fermenter or container.

[0365] The housing (220) is constructed of any suitable sturdy material, such as a food-grade polymer. The person skilled in the art could readily select a suitable material that is permeable to the electric field, so that the electric field can also penetrate into the liquid or medium in a fermenter vessel, without the bioreactor emitter contacting the liquid or medium. For example, the material of the housing may be made from any one or more of polyethylene (PE), polypropylene (PP), Teflon (PTFE), Kynar (polyvinylidene fluoride, PVDF), fluorinated ethylene propylene (FEP), perfluoroalkoxy (PFA), silicone, or glass. These materials are electrically non-conductive and exhibit high dielectric strength, making them permeable to electric fields. Alternatively, if the material of the housing (220) is copper, then the electric field can pervade the sturdy material to affect the liquid in the fermenter vessel, without the bioreactor emitter contacting the liquid.

[0366] The non-conductive material (190) is preferably composed of non-metallic material or combination of non-metallic materials, but may be alternately composed of any capacitive material. As such, the non-conductive material (190) may be made from one or more of: polymers such as polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), polyvinylidene fluoride (PVDF), fluorinated ethylene propylene (FEP), and perfluoroalkoxy alkane (PFA); ceramics such as barium titanate (BaTiO3), lead zirconate titanate (PZT), and aluminum oxide (A12O3); glass materials including borosilicate and soda-lime glass; silicone-based compounds; and advanced composites like epoxy resins embedded with high-dielectric fillers. The non-conductive material (190) may be partly hollow but, if so, it does not carry a flowing liquid. The capacitive material (1909) may be porous or non-porous, and may be a compound material or a pure material.

[0367] While the housing (220) is illustrated as a blind and hollow cylinder, in other embodiments, the housing (220) may be any suitable shape to contain the emitter (190), depending on the specific application and operational requirements. Suitable shapes comprise a blind hollow cylinder, a fully enclosed cylinder, a rectangular or square prism, a conical structure, a spherical or hemispherical enclosure, an elliptical or oval cross-section housing, a polygonal prism (e.g., triangular, hexagonal, or octagonal), a toroidal structure, a tapered or stepped design, or a custom asymmetrical geometry designed to conform to the spatial constraints of the surrounding system. These shapes may include features such as internal partitions, baffles, or supports to position or secure the emitter, as well as openings, flanges, or threaded connections for integration with other components or for ease of assembly and maintenance.

[0368] In certain embodiments, the device described can be termed a bioenergetic accelerator. A person skilled in the art would be aware that the application of the bioenergetic accelerator may encompass the acceleration of in vitro and / or in vivo functions chosen from among any of a broad range of bioenergetic phenomena, such as enzyme function, regulatory functions, membrane gradients, chemiosmotic gradient generation and use, proton gradients, sodium and potassium gradients, advantageous metabolic rate of a cell or of a biochemical pathway, electron transport, oxidative phosphorylation, organellar functions, biosynthesis of organelles, transport functions, signalling functions, homeostasis, efficiency of the function of multi-protein complexes, promoter binding, cell dynamics, cell division, cell division rate, gene transcription, gene transcription-complex function, protein translation-complex function, DNA replication, adjustment of microbial cell density, chromatin unpacking, cell program rejuvenation, purified enzyme function, enzymatic catalysis of a substrate or substrates to generate a product or products, catalysis involving cofactors, reactions within biochemical pathways, reactions requiring free energy, Gibbs dynamics of enzyme catalysis, Arrhenius dynamics of enzyme catalysis, Michaelis -Menten dynamics of protein-protein binding, Michaelis-Menten dynamics of enzyme-substrate binding, industrial enzyme function, orderly function of an enzyme or cell to manufacture therapeutics, orderly function of an enzyme or cell to manufacture foods or substances, orderly function of an enzyme or cell to manufacture a beverage, orderly function of an enzyme or cell to manufacture a biofuel or fibre, orderly function of an enzyme or cell to manufacture a pharmaceutical, efficient utilisation of a sugar, the bypassing of sugar utilisation, accelerating the diauxic shift, efficient integration between the catabolism and anabolism of glucose, efficient integration between the catabolism and anabolism of reduced carbon sources, accelerating an incubation, efficiency of protein-breathing, efficient resistance to stress, and the like. The term bioenergetic accelerator may be shortened, abbreviated, derivatised or made into an acronym, and still be understood as covering the general phenomenon of bioenergetic acceleration. For example, the bioenergetic accelerator may be termed, BioEncel.

[0369] System

[0370] In a twenty-eighth aspect, there is provided a system of the disclosure. The system comprises an emitter system for treating a composition, comprising: a container configured to contain a composition; an electric field-generating device comprising: a power supply a differential oscillator connected to the power supply, the oscillator comprising first and second outputs; an emitter comprising at least a first voltage t...

Claims

CLAIMS1. A method of improving a stress resistance, a longevity, or both, of microorganisms, comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

2. A method of improving a stress resistance, a longevity, or both, of microorganisms, during a fermentation process of the microorganisms to produce one or more of: dairy products, yoghurt, cheese, a probiotic, a feed, an encapsulated probiotic, probiotic manufacturing, and a probiotic packaged with excipients, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

3. A method of improving a stress resistance, a longevity, or both, of microorganisms, during a fermentation process of the microorganisms, and of improving survival of the microorganisms during storage or transport, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

4. A method of treating a composition to provide surmounting energy to improve protein breathing during a fermentation of microorganisms to increase production of one or more of: a functional protein, a therapeutic protein, a bacteriocin, an antibiotic protein, an insulin, a growth hormone, a hormone replacement, a functional part of an antibody, and other functional protein in the microorganisms, the method comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component;wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

5. A method of improving a stress resistance, a longevity, or both, of microorganisms, comprising: subjecting the microorganisms to a generated electric field substantially free of a magnetic component; wherein the generated electric field provides improved protein breathing and is generated by a controller to oscillate at one or more frequency in a range of about 210 Hz to 99 kHz.

6. The method of any one of claims 1 to 5, wherein the generated electric field is generated by an AC signal, or a pulsed DC signal with a peak-to-peak voltage and a duty cycle; and wherein the stress resistance, or the longevity, or both, of the microorganisms is used to reduce loss of viable cell count, or loss of stress resistance or longevity during one or more of: a fermentation, a freeze-drying, a packaging and a long-term storage.

7. The method of claim 6, wherein the AC signal, or the pulsed DC signal, with a waveform, is applied to at least one voltage terminal or antenna, the AC signal or the pulsed DC signal comprising a single frequency, a set of frequencies, a frequency sweep, or a stochastic or shuffled progression of frequencies in the range of about 210 Hz to 99 kHz; and the AC signal or pulsed DC signal operating at a peak-to-peak voltage between 1 V and 45 kV.

8. The method of claim 7, wherein an integrated monitor is embodied in or connected to the controller to monitor a presence of a signal in the antenna or antennae, comprising monitoring a waveform of the signal, an amplitude of the signal, or a frequency of the signal.

9. The method of claim 7 or 8, wherein the progression of frequencies is: a stepped quartic sweep defined by a formula F(t)=at4+bt+ct2+dt+e that expresses frequency as a function of time t, and with a, b, c, d, e being coefficients; or a curved sweep, or a linear sweep, or is a stepped non-quartic sweep progressing upward in frequency or downward in frequency, or is a non-stepped sweep progressing upward in frequency or downward in frequency, the sweep taking a fixed period time before rebeginning.

10. The method of claim 7 or 9, wherein the progression of frequencies is defined using a lookup table or subsets of a lookup table.

11. The method of any one of claims 1 to 10, wherein the microorganisms are cultivated in a medium optimised to maintain a selected dissolved oxygen concentration, with or without pH control by an intermittent compensatory dosing with alkali or acid.

12. The method of any one of claims 1 to 11, wherein the microorganisms are selected from one or more of: eubacteria, fungi, actinomycetes, probiotic yeast, probiotic bacteria, Lactobacilli, Lactobacillus species, Lactobacillus-\i e species, Bifidobacterium species, Bifidobacierium-\\ c species, Saccharomyces spp., and other probiotic species, strains, and genera, which together comprise the probiotic group, being a non-taxonomic grouping.

13. The method of claim any one of claims 1 to 12, wherein each of the at least one voltage terminal or antenna comprises one or more of (i) insulated conductive material helically wound around a nonconducting material; (ii) an insulated conductive terminus material of any shape such as an insulated conductive plate; or (iii) two or more insulated conductive plates separated by a non-conducting material or by the composition; to provide surmounting energy to facilitate increased protein breathing in the cells’ functional proteins.

14. The method of any one of claims 1 to 13, wherein the electric field sweeps through sequential frequencies in a range of about 1400 Hz to about 4540 Hz or sequential frequencies in the range of about 210 Hz to about 99 kHz.

15. An electric field-generating device to improve a stress resistance, a longevity, or both, of microorganisms, the device comprising: a controller to generate an electric field; wherein the generated electric field is oscillating at one or more frequency in a range of about 210 Hz to 99 kHz; wherein the generated electric field is substantially free of a magnetic field; and wherein the generated electric field is produced by supplying the signal to at least one electrically insulated emitter.

16. The electric field-generating device of claim 15, wherein the controller is configured to generate a waveform and supply a modulated voltage to at least a first and a second voltage terminals or antennae, wherein at least the first and second voltage terminals or antennae are controlled by an one or more output channels configured to supply at least a first and a second signal that are of the same phase as each other or are phase-shifted relative to each other; or to control the at least a first and a second voltage terminals to increase field exposure and uniformity of exposure to microbial cells; or both.

17. The electric field-generating device of claim 15 and 16, wherein the modulated voltage supplied to at least the first voltage terminal or antenna is in a range of 1 V RMS to 40 kV RMS, and wherein the controller or at least the first voltage terminal or antenna, or both, comprise a means to prevent formation of the magnetic component to the generated electric field.

18. An electric field-generating device configured to perform the method of any one of claims 1 to 14.

19. An electric field-generating device configured to perform the method of any one of claims 1 to 14 comprising one or more reradiation emitter or emitters, that is or are separate from the at least one electrically-insulated voltage terminal or antenna and separate from any other electrically-insulated voltage terminals or antennae.

20. An electric field-generating system for treating a composition, comprising: a controller configured to generate a waveform and supply a modulated voltage to at least a first voltage terminal or antenna of an emitter; wherein the modulated voltage is supplied to at least the first electrically -insulated voltage terminal at one frequency or more frequency in a range of about 210 Hz to 99 kHz, to generate an electric field substantially free of a magnetic component; and wherein the controller is configured to supply an AC signal or a pulsed DC signal; and a container configured to contain the composition, wherein at least the first electrically -insulated voltage terminal positioned within the container to optimise exposure of the composition to the generated electric field.

21. The system of claim 20, wherein the container is configured to perform a batch culture, a fed- batch, a continuous culture, or a repeated fed-batch or semi-continuous culture, a perfusion process or a continuous or non-continuous feeding process and, in use, supplies a nutrient at a selected time within a culturing process, to augment the nutritional status of microorganisms to increase or maximise obtainable viable cell density per gram of dry cell.

22. The system of claim 20, wherein the container is separate from a fermenter vessel but connected by liquid flow to a fermenter vessel by a conduit, and wherein the container or conduit facilitates recirculation of a composition past an electrically-insulated electric field emitter and then back to a fermenter vessel for continued fermentation, such that the composition is exposed to a near electric field.

23. An electric field-generating system configured to perform the method of any one of claims 1 to 14 or configured to employ the device of any one of claims 15 to 19.

24. An electric field of the device of any one of claims 15 to 19, the method of any one of claims 1 to 14, or the system of any one of claims 20 to 23, used in conjunction with a combined-semi-electric field such as the combined-semi-electric field emitted from a reradiation coil or a combined-semi-electric field emitted from another type of field emission technology, to achieve an industrial benefit for stressresistance and viability of a microorganism, specifically by means of the electric component of the field providing surmounting energy to influence and improve protein breathing in a microorganism.

25. An electric field of the device of any one of claims 15 to 19, the method of any one of claims 1 to 14, or the system of any one of claims 20 to 23, used in conjunction with a combined-semi-electric field such as emitted from another type of field emission technology, to achieve an industrial benefit for stress resistance and viability of a microorganism without ohmic heating, where the improvement in stress resistance and viability is specifically by means of the electric component of the field influencing protein breathing in a microorganism without involvement of ohmic heating.

26. An electric field of the device of any one of claims 15 to 19, the method of any one of claims 1 to 14, or the system of any one of claims 20 to 23, used in conjunction with a combined-semi-electric field such as emitted from another type of field emission technology such as an electrode or electrodes, or an uninsulated current carrying loop or an insulated current carrying loop, to achieve an industrial benefit for stress resistance and viability of a microorganism without electrochemical damage, where the improvement in stress resistance and viability is specifically by means of the electric component of the near electric field influencing protein breathing in a microorganism despite the presence of potentially damaging field emission technology.

27. A nutritional supplement, a food, a supplement supporting gut health, a supplement supporting digestive health, a supplement containing beneficial therapeutic proteins derived from a microorganism, or a supplement containing beneficial therapeutic metabolites derived from a microorganism, prepared by fermenting with the device of any one of claims 15 to 19, the method of any one of claims 1 to 14, or the system of any claim 20 to 23.

28. A skin health or beauty supplement, a bioenergy compound, a pharmaceutical molecule, an enzyme, a prebiotic, an oligosaccharide, a cofactor, a fibre or a biologically derived product prepared by fermenting with the device of any one of claims 15 to 19, the method of any one of claims 1 to 14, or the system of any one of claims 20 to 23, for the purpose of producing a microbial lysate having skin health or beauty properties.

29. The method of any of claims 1 to 14 or the device of any of claims 15 to 19, or the system of any of claims 20 to 23, for manufacturing a product from microorganisms in a fermenter, or for manufacturing a product being microorganisms themselves using a fermenter, wherein the controller is configured to execute a deterministic sequencing algorithm, and be continuously monitored or data- logged for internal auditing purposes or external auditing purposes, thereby providing a highly repeatable and verifiable exposure regime suitable for GMP (Good Manufacturing Practice) compliance.

30. A metabolite manufactured using the device of any claim 15 to 19, or the method of any claim 1 to 14, or the system of any claim 20 to 23, wherein the metabolite is of cellular origin, and is increased in amount or activity by the electric field providing surmounting energy to a functional protein or to a set of functional proteins that work together for increased production of the metabolite.

31. The method of any of claims 1 to 14 or the device of any of claims 15 to 19, or the system of any of claims 20 to 23, where the emitter is inserted within a bioreactor housing that is in turn inserted or emplaced into a fermenter or container.

32. The device of claim 16, the method of claim 7 or 8, or the system of any one of claims 20 to 22, wherein the rise and fall respectively of the waveform are defined using a quartic formula of the form V(t)=at4+bt+ct2+dt+e that expresses voltage V as a function of time t, and with a, b, c, d, e being coefficients that may differ in sign or quantity between the rise and fall equations.

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