A method of producing non-alcoholic fermented drink

Abstract

The present invention is directed to a method of producing a non-alcoholic fermented drink such as a non-alcoholic drink. In particular, the present invention relates to a method of producing non-alcoholic fermented drink comprising propagating a microbial culture in a large-scale setting under suitable conditions to produce a non-alcoholic fermented drink with desired physicochemical and organoleptic properties, where the microbial culture comprises both yeast and bacterial species. The present invention also relates to a propagated microbial culture and use of the culture in fermentation. The present invention also relates to a method of producing sparkling non-alcoholic fermented drink. The present invention further provides a non-alcoholic fermented drink or a sparkling non-alcoholic fermented drink.

Description

[0001] Title: A METHOD OF PRODUCING NON-ALCOHOLIC FERMENTED DRINK

[0002] [1] The present invention is directed to a method of producing a non-alcoholic fermented drink such as a nonalcoholic drink. In particular, the present invention relates to a method of producing non-alcoholic fermented drink comprising propagating a microbial culture in a large scale setting under suitable conditions to produce a non-alcoholic fermented drink with desired physicochemical and organoleptic properties, where the microbial culture comprises both yeast and bacterial species. The present invention also relates to a propagated microbial culture and use of the culture in fermentation. The present invention also relates to a method of producing sparkling non-alcoholic fermented drink. The present invention further provides a non-alcoholic fermented drink such as a sparkling non-alcoholic fermented drink.

[0003] INTRODUCTION

[0004] [2] There are currently recognised some 69 genera of yeast with over 500 “true” strains, of which there are hundreds of individual variants. There are also 3,500 identified genera of bacteria with over 20,000 species. In nature, many hundreds of these microorganisms exist in complex competing or symbiotic relationships, creating an environment in which they inhibit one another or thrive together in concert.

[0005] [3] We know that alcoholic fermentations in wine (e.g. champagne), beer and spirits have been studied in great depth over centuries in order to understand not only the overall metabolism but also develop finely tuned processes for refined flavour development, fermentation process, microbial bioreactor relationships and commercial product consistency. The management of the artificial ecosystems which are routinely formed in aerobic and / or anaerobic bioreactors, require dynamic intervention to ensure certain essential physico-chemical conditions and parameters are maintained within strict and well understood parameters, such as for instance pH, temperature, oxidoreduction potential and nutritional needs.

[0006] [4] The understanding of non-alcoholic fermentation however is a nascent discipline having only really begun in the last decade. Consequently, the creation and management of artificial ecosystems of non-alcoholic bioreactor fermentations is in its infancy with little understanding or familiarity of the specific physico-chemical conditions, parameters and microbiota that may influence, for instance, microbial bioreactor relationships, fermentation process, flavour development and product consistency. Moreover, absent is a scientifically meaningful knowledge let alone profound appreciation of the conditions which could lead to a finely tuned non-alcoholic bioreactor fermentation process for refined flavour development and commercial product consistency.

[0007] [5] We understand that yeast is the primary contributor of flavour in both alcoholic and non-alcoholic fermentations. During reproduction and respiration (i.e. fermentation), yeast metabolise sugars, oxygen and other compounds to produce alcohol, carbon dioxide and a wide range of other compounds, many of which are detectable by the human palate. The challenge has been to identify yeasts that produce interesting and appropriate flavour compounds in the presence of substrate and produce the appropriate nutrient precursors for bacteria to thrive in but without the bacteria adversely affecting the flavour or spoiling the product.

[0008] [6] It is also known that certain bacteria can consume alcohol and also produce different acids. In a co-culture with yeast, bacteria will metabolize simple sugars produced by yeast invertase, alcohols, and other intermediate metabolites to produce for example acetic acid, gluconic acid, acetic acid and other acids. A formidable challenge therefore exists to determine if it is at all possible to create a bioreactor fermentation, using different yeast and bacteria that may provide a complex in flavour non-alcoholic fermented product that can be considered an adequate or even complete substitute for alcoholic drinks such as wine (e.g. champagne). A further challenge exists in selecting bacteria and conditions of incubation that allow bacteria to work well in conjunction with the flavour producing yeasts, to be able to produce an appropriate balance of metabolites such as acids.

[0009] [7] In recent years, consumers’ interest and taste in non-alcoholic fermented beverages has increased dramatically. The demand for more refined non-alcoholic fermented beverages to replace alcoholic drinks is also rising. [8] Therefore, there exists a real need to develop not only an appropriate process for producing a non-alcoholic fermented drink product on a commercial scale but also suitable microbial cultures which can be safely and effectively employed in non-alcohol fermentation industry.

[0010] BRIEF SUMMARY OF THE INVENTION

[0011] [9] In the broadest sense, the present invention relates to a method of producing non-alcoholic fermented beverage by relying on microbial cultures which under controlled conditions are able to facilitate, on an industrial scale and in a reproducible manner, the production of non-alcoholic beverages, without compromising on taste or flavour.

[0012]

[0010] The present invention relates to a method of producing non-alcoholic fermented beverage by incubating certain microbial cultures in the presence of suitable substrate and under controlled conditions which enable the production of a non-alcoholic beverage with refined taste and flavour. In some embodiments, the present invention relates to a method of producing non-alcoholic fermented beverage by incubating certain microbial cultures in the presence of suitable substrate and under controlled conditions which enable the production of a non-alcoholic beverage with refined taste and flavour.

[0013]

[0011] According to one aspect, the method of the present invention comprises the steps of: a. supplying an aqueous solution with a plant-based nitrogen source, combined with a plantbased carbon source to form a growth medium; b. inoculating the growth medium with a microbial inoculum to form an inoculated mixture; c. incubating the inoculated mixture at a first incubation temperature (ITc) to generate a propagated starter culture containing between about 103- 5xlO10viable cells / ml; d. to the propagated starter culture adding a large scale growth medium consisting of a aqueous solution of a plant-based nitrogen source, combined with a plant-based carbon source to form a fermentation mixture; e. incubating the fermentation mixture at a second incubation temperature (2Tc) for between about 1 and about 5 weeks; and f. treating the fermenting mixture to produce a non-alcoholic fermented drink.

[0014]

[0012] In some embodiments of the present invention, the microbial inoculum and the propagated starter culture are obtained from a microbiota. In some embodiments of the present invention, the microbial inoculum and the propagated starter culture are obtained by the isolation of specific microbes from kombucha. It is also contemplated that the microbial inoculum can be obtained from any suitable commercial microbial source or an official microorganism strain repository institution such as for instance the American Type Culture Collection (ATCC). Furthermore, different species of the microbial inoculum can be obtained from isolated viable microbial stocks or as viable predetermined mixed species microbial cultures.

[0015]

[0013] In some embodiments of the present invention, the propagated starter culture comprises a symbiotic mixture of yeast and bacteria. The present method is perfectly suited for producing a propagated starter culture comprising a symbiotic mixture of yeast and bacteria that contains different organic compounds, alcohol or metabolites such as physico-chemical parameters, which unless stated otherwise, can be readily measured and quantified with standard laboratory techniques.

[0016]

[0014] In some embodiments of the present invention, the yeast is selected from the group of genera consisting of Pichia, Saccharomyces , Zygosaccharomyces, Hanseniaspora, Mycotorula, Hanseniaspora, Zygosaccharomyces, Lachancea, Candida, Kazachstania, Kloeckera, Metschnikcnvi, Medusomyces, Brettanomyces, Saccharomy codes, Torulopsis, Torulaspora, Schizosaccharomyces and Kluyveromyces. In some embodiments, the yeast can be selected from the yeast genera listed in Table 4.

[0017]

[0015] In some embodiments of the present invention, the bacteria is selected from the group of general group consisting of Leuconostoc, Lactococcus, Acetobacter, Allobacullum, Bifidobacterium, Leuconostoc, Propionobacterium, Ruminococcus, Gluconob acter, Gluconacetobacter, Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Abiotrophia, Aerococcus, Aerosphaera, Agitococcus, Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes, Bavariicoccus, Carnobacterium, Carnococcus, Catellicoccus, Chungangia, Convivina, Desemzia, Dolosicoccus, Dolosigranulum, Enterococcus, Eremococcus, Facklamia, Floricoccus, Fructobacillus, Globicatella, Granulicatella, Ignavigranum, Isobaculum, Jeotgalibaca, Lacticigenium, Lactovum, Lachancea, Marinilactibacillus, Melissococcus, Metschnikowia, Oenococcus, Okadaella, Pilibacter, Pisciglobus, Sharpea, Komagataeib acter, Brevibacterium, Pediococcus, Nguyenibacter, Sporolactobacillus, Tetragenococcus, Torulaspora, Trichococcus, Thermus, Streptococcus, Staphylococcus, Vagococcus and Weissella. In some embodiments, the bacteria can be selected from the genera of bacteria listed in Table 5.

[0018]

[0016] In some embodiments of the present invention, the plant-based nitrogen source and plant-based carbon source are combined and used to prepare a growth medium. In some embodiments of the present invention, the aqueous solution is a growth medium.

[0019]

[0017] In some embodiments of the present invention, the plant-based nitrogen source and plant-based carbon source material are combined and used to prepare a growth medium. In some embodiments of the present invention, the growth medium is prepared by steeping the plant-based nitrogen source and plant-based carbon source material in cold, hot or lukewarm aqueous solution.

[0020]

[0018] In addition, the present invention can be carried out using only food grade materials such as plant-based materials. Furthermore, the present invention can be carried out using only food grade materials such as plant-based materials to prepare a growth medium.

[0021]

[0019] In some embodiments of the present invention, the plant-based nitrogen source comprises biomass from at least one selected from the group consisting of camellia sinensis (tea), berries, fruits, herbs, flowers, nuts, seeds, spices, vegetables and others.

[0022]

[0020] In some embodiments of the present invention, the plant-based carbon source comprises biomass from at least one selected from the group consisting of sugars, berries, fruits, herbs, flowers, grains, nuts, seeds, spices, vegetables and others.

[0023]

[0021] In some embodiments of the present invention, the carbon source comprises between about 1% v / v and about 15% of the growth medium. In some embodiments of the present invention, at least one organic compound is sucrose.

[0024]

[0022] In some embodiments of the present invention, the first incubation temperature (ITc) is in the range of between about 20°C to 37°C, preferably between about 22°C to 35°C, preferably between about 24°C to 32°C, preferably between about 26°C to 30°C. In some embodiments of the present invention, the second incubation temperature (2Tc) is in the range of between about 20°C to 37°C, preferably between about 22°C to 35°C, preferably between about 24°C to 32°C, preferably between about 26°C to 30°C.

[0025]

[0023] It would be appreciated that when generating a propagated starter culture, ITc incubation temperature may vary depending on for example the type of growth medium, the microbial mixture, duration of incubation, the concentration of nitrogen source, the concentration of carbon course and others. By way of non-limiting example, where the growth medium contains relatively high level of carbon source such as sucrose of about 25 g / L the ITc can be reduced and the duration of incubation can be increased. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 26°C to 29°C for about 14 weeks. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 26°C to 28°C for about 12 weeks. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 26°C to 28°C for about 10 weeks.

[0026]

[0024] In some embodiments of the present invention, the method further comprises the step of passing sterile air continually over the fermentation mixture. In some embodiments of the present invention, the method further comprises the step of passing sterile air continually over the fermentation mixture at a rate of between 8 to 20 L / minute.

[0025] In some embodiments of the present invention, the method further comprises the step of micro-oxygenation of the fermentation mixture.

[0027]

[0026] In some embodiments of the present invention, the method further comprises measuring the level of at least one test parameter selected from pH, sucrose, tannin, esters, polyphenols, minerals, acetic acid, glucuronic acid, amino acids and alcohol (ABV).

[0028]

[0027] In some embodiments of the present invention, the fermenting mixture is treated to stop fermentation when the level of sugars is between about 15% and 0.5%, the level of lactic acid is between about 1% and 0.2%, the level of acetic acid is between about 1% and 0.2%, the level of gluconic acid is between 0.5% and 0.1%, and the level of glucuronic acid is between about 0.05% and 0.01%.

[0029]

[0028] In some embodiments of the present invention, the fermenting mixture is treated to produce a non-alcoholic fermented drink.

[0030]

[0029] In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation.

[0031]

[0030] In some embodiments of the present invention, treating the fermented mixture comprises rapidly cooling the fermented mixture to between about 2°C to 6°C to stop fermentation.

[0032]

[0031] In some embodiments of the present invention, treating the fermented mixture comprises subjecting the mixture to filtration. In some embodiments of the present invention, filtration leads to the removal or elimination of the microbial culture. In some embodiments of the present invention, the non-alcoholic fermented drink is subjected to filtration. Preferably, treatment of the fermented mixture or non-alcoholic fermented drink does not adversely affect or modify the physico-chemical properties or test parameters of the fermented drink. Preferably, treatment of the fermented mixture or non-alcoholic fermented drink does not adversely affect or modify the taste or flavour of the fermented drink.

[0033]

[0032] In one aspect the present invention provides a non-alcoholic fermented drink produced according to the method described herein. In some embodiments of the present invention a non-alcoholic fermented drink is produced according to the method described herein.

[0034]

[0033] Although the inventors do not wish to be bound by theory, it is believed that the present invention may benefit from the quomm sensing capabilities of the microorganisms such as a symbiotic mixture of at least one type of yeast and at least one type of bacteria.

[0035] BRIEF DESCRIPTION OF THE DRAWINGS

[0036]

[0034] FIG. 1. Microbial growth (cells x 104per ml) over time (Days) such as viable cell numbers of yeast Saccharomyces spp. and bacteria Acetobacter spp. during fermentation.

[0037]

[0035] FIG. 2. TA Development (grams / litre / day) over time (Days) such as TA development (grams / volume / day) over duration of fermentation - for microbial cells e.g. symbiotic microbial mixture comprising yeast Saccharomyces spp. and bacteria Acetobacter spp.

[0038]

[0036] FIG. 3. Flavour intensity (1-10) over time (Days) such as development of flavour intensity over time for symbiotic microbial mixture yeast Saccharomyces spp. and bacteria Acetobacter spp.

[0039]

[0037] FIG. 4. Microbial growth (cells x 104per ml) over time (Days) such as viable cell numbers of yeast Saccharomyces spp. and Zygosaccharomyces spp. and bacterial constituent Acetobacter spp. during fermentation.

[0040]

[0038] FIG. 5. Represents TA development (grams / volume / day) over duration of fermentation step for microbial cells e.g. symbiotic microbial mixture comprising Saccharomyces spp. and Zygosaccharomyces spp. and bacterial constituent Acetobacter spp.

[0039] FIG. 6. Represents development of flavour intensity over time for symbiotic microbial mixture comprising Saccharomyces spp. and Zygosaccharomyces spp. with bacterial constituent Acetobacter spp.

[0041]

[0040] FIG. 7. Microbial growth (cells x 104per ml) over time (Days) such as viable cell numbers of Saccharomyces spp. and Zygosaccharomyces spp and those of Acetobacter spp. and Gluconobacter spp. during fermentation.

[0042]

[0041] FIG. 8. TA Development (grams / litre / day) over time (Days) such as TA development (grams / volume / day) over duration of fermentation step for microbial cells e.g. symbiotic microbial mixture comprising Saccharomyces spp. and Zygosaccharomyces spp and those oi Acetobacter spp. and Gluconobacter spp. during fermentation.

[0043]

[0042] FIG. 9. Flavour intensity (1-10) over time (Days) such as development of flavour intensity over time for microbial cells e.g. symbiotic microbial mixture comprising Saccharomyces spp. and Zygosaccharomyces spp and those of Acetobacter spp. and Gluconobacter spp. during fermentation.

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045]

[0043] The disclosure illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms “comprising”, “including,” containing”, etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed.

[0046]

[0044] The present invention relates to a method of producing non-alcoholic fermented beverage relying on microbial cultures which under controlled conditions are able to facilitate, on an industrial scale and in a reproducible manner the production of non-alcoholic beverages, without compromising on taste or flavour.

[0047]

[0045] The present invention also relates to a method of producing non-alcoholic fermented beverage relying on microbial cultures which under controlled conditions are capable, on an industrial scale and in a reproducible manner, to produce a non-alcoholic drink, with taste or flavour comparable to a similar alcoholic drink. Specifically, the present invention relates to a method of producing non-alcoholic fermented beverage by incubating certain microbial cultures in the presence of suitable substrate and under controlled conditions which enable the production of a non-alcoholic beverage with refined taste and flavour. Furthermore, the present invention relates to a method of producing nonalcoholic fermented beverage by incubating certain microbial cultures in the presence of suitable substrate and under controlled conditions which enable the production of a non-alcoholic beverage with refined taste and flavour which can be used as a realistic or a suitable substitute for alcoholic drinks such as wine. The present invention relates to a method of producing non-alcoholic fermented beverage by incubating certain microbial cultures in the presence of suitable substrate and under controlled conditions which enable the production of a non-alcoholic beverage.

[0048]

[0046] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identifying citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference into the present disclosure to more fully describe the state of the art to which this disclosure pertains.

[0049]

[0047] As used herein, certain terms may have the following defined meanings.

[0050]

[0048] As used in the specification and claims, the singular form “a,” “an” and “the” include singular and plural references unless the context clearly dictates otherwise. For example, the term “yeast” or “bacterium” includes a single or plurality of yeasts or bacteria. By way of a further example, the term “test parameter” refers to one or more chemical or physico-chemical compounds, molecular or substance are capable of being identified, measured, or quantified.

[0051]

[0049] As used herein, the term “alteration” may be used interchangeably with the terms, “alter” or “modify” such as increase or decrease in the level of a metabolite such as a chemical or a physico-chemical parameter detected and / or analysed and / or monitored, as part of the present invention. In some embodiments, the alteration is at least 0.001%, 0.005%, 0.01%, 0.05%, 0.08%, 0.1%, 0.15%, 0.2%, 0.4%, 1%, 2%, 2.5%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% or greater compared to control or base level. In some embodiments the alteration may be at least 0.1, 0.25, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10-fold or greater compared to control or base level.

[0052]

[0050] In some embodiments the alteration of a test parameter is statistically significant. In some embodiments the alteration is determined qualitatively. In some embodiments the alteration is determined quantitatively. In some embodiments, alteration is assessed by a qualitative step and / or a quantifying step. In further embodiments, the qualitative step and / or a quantifying step is performed on a sample.

[0053]

[0051] In the present disclosure, the terms “beverage” and “drink” are used interchangeably and are intended to have the same meaning such as a non-alcoholic fermented aqueous liquid product which is non-toxic and is suitable for consumption for example by a subject. The non-alcoholic fermented liquid product can be non-alcoholic fermented drink or sparkling non-alcoholic fermented beverage. Therefore, according to some embodiments of the present invention, there is provided a method for producing a non-alcoholic fermented drink.

[0054]

[0052] As used herein, the term “organoleptic” refers to a perception, impact, quality, impression or an affect produced by a test parameter on a sensory organ such as touch, taste, flavour or smell, on a subject as a whole. Preferably, the organoleptic characteristics can be modified and controlled using the systems and apparatus contemplated herein. Therefore, according to some embodiments of the present invention, there is provided a method for producing a non-alcoholic fermented drink with finely tuned, such as desirable by the subject or preferred, organoleptic qualities beverage.

[0055]

[0053] In the present disclosure, the terms “flavour” and “taste” are used interchangeably. The terms “flavour” and “taste” refer generally to certain aspects of a subject’s sensory experience when coming into contact with a nonalcoholic fermented drink such as a for example sparkling non-alcoholic fermented drink according to embodiments of the present invention by the subject. The term “taste” refers to the basic sensations detected by taste buds, such as sweet, sour, salty, bitter, and umami. The term “flavour” is a much more complex perception that involves not only taste, but also for instance parameters such as smell, texture, feel, physical appearance, colour, fizziness, and even temperature. Preferably, flavour is also the overall sensory experience that a subject perceives during or after consumption including the combination of taste, aroma, and mouthfeel. While taste is generally limited to the basic sensations, flavour is considered a multi-dimensional experience that adds depth and complexity. It is contemplated that different parameters such as esters can affect the taste of the fermented beverage such as the fermented beverage as described herein.

[0056]

[0054] There has been progress since the turn of the century of the identification of buccal cell receptors or receptor candidates for all five basic tastes - bitter, sweet, umami, sour, and salty. The receptors for bitter, sweet, and umami appear to belong to the same superfamily of G-protein-coupled receptors (GPCRs), whereas the receptor for salty is an ion channel. The receptor function for sour is the least understood although it has been proposed that it may involve proton sensing. Taste and flavour as contemplated in the present disclosure can be determined or assessed by measuring certain parameters such as test parameters. Taste and flavour as contemplated in the present disclosure can be modified such as effectively modified or controlled by using the results of the test parameters determinations and the methods described herein.

[0057]

[0055] Preferably, the parameters can be measured and quantified. Preferably, the parameters can be measured with great accuracy and be qualitatively quantified. Preferably, the parameters can be measured and quantified continuously. Preferably, the parameters can be measured and quantified during microbial incubation or microbial propagation. Preferably, the parameters can be measured and quantified during microbial fermentation. Preferably, the parameters are physico-chemical parameters. Preferably, the parameters are chemical parameters. Preferably the parameters are sensory parameters. Preferably, the parameters are physico-chemical parameters of the non-alcoholic fermented drink such as a sparkling non-alcoholic fermented drink or certain chemical parameters of the non-alcoholic fermented drink such as a sparkling non-alcoholic fermented drink e.g. physico-chemical test parameters of the nonalcoholic fermented drink such as a sparkling non-alcoholic fermented drink or chemical test parameters of the non- alcoholic fermented drink such as a sparkling non-alcoholic fermented drink. Different assays and tests for assessing or measuring, e.g. continuously, concomitantly, separately or in real-time, test parameters e.g. physico-chemical and / or chemical parameters would be familiar to the skilled person, bay way of non-limiting example such assays and tests include for instance HPLC, Maldi Tof (MS), energy dispersive x-ray spectroscopy) EDX analysis and SEM (scanning electron microscopy) studies using FESEM (field emission scanning electron microscope), odour description tests, olfactory description tests etc.

[0058]

[0056] In Table 1 below, there is provided an exemplary list of sensory, tactile and taste / flavour parameters, attributes or factors, the Proposed Descriptor and non-exhaustive list of molecules or chemical involved.

[0059]

[0057] Table 1. Non-exhaustive sensory, tactile and taste / flavour parameters, attributes or factors contemplated in the present invention.

[0060]

[0058] As used herein, the term “subject” means any animal, such as a vertebrate, preferably a mammal such as human, who consumes or drinks a non-alcoholic fermented drink such as a sparkling non-alcoholic fermented drink according to embodiments of the present invention.

[0061]

[0059] According to one aspect, there is provided a method for producing a non-alcoholic fermented drink, the method comprising the steps of: a. supplying an aqueous solution with a plant-based nitrogen source, combined with a plantbased carbon source material to form a growth medium; b. inoculating the growth medium with a microbial inoculum to form an inoculated mixture; c. incubating the inoculated mixture at a first incubation temperature (ITc) to generate a propagated starter culture containing between about 103 - 5x1010 viable cells / ml; d. to the propagated starter culture adding a large scale growth medium consisting of a aqueous solution of a plant-based nitrogen source, combined with a plant-based carbon source to form a fermentation mixture; e. incubating the fermentation mixture at a second incubation temperature (2Tc) for between about 1 and about 5 weeks; and f. treating the fermenting mixture to produce a non-alcoholic fermented drink.

[0062]

[0060] As used herein, the term "comprising" means including, made up of, composed of, encompass, consist of, constitute and incorporate.

[0063]

[0061] All numbers or numerals as used herein that indicate amounts, ratios of materials, chemical properties, physical properties physico-chemical properties of materials or parameters such as test parameters, and / or use are to be understood as modified or qualified by the term "about," except as otherwise explicitly indicated.

[0064]

[0062] As used herein, the term "about" includes the recited number or number and + / - 10% from the recited numeral or number. By way of example only, the term "about ten (10)" would encompass nine (9) to eleven (11) or 9-11.

[0065] Plant-based material and growth medium

[0066]

[0063] The term "aqueous solution" as used herein refers to an aqueous growth medium or aqueous culture medium that supports the growth of microorganisms. In some embodiments, the aqueous growth medium comprises a combination of one or more plant-based nitrogen sources and one or more plant-based carbon sources at different ratios.

[0067]

[0064] As used herein, the term “plant-based material” refers to a biomass, extract, concentrate, juice or additive obtained or derived from a plant such as a non-toxic plant. The additive can be an artificial or synthetic flavour enhancing or masking compound. In some embodiments, the plant-based material is at least one selected from the group consisting of teas, sugars, berries, fruits, herbs, flowers, grains, nuts, seeds, spices, vegetables and others.

[0068]

[0065] It is within the scope of the present invention that the aqueous solution used in the present methods can be supplemented with different materials such as plant-based material. In some embodiments, the aqueous solution is supplemented with plant-based material. In some embodiments, the plant-based material comprises biomass, extract, concentrate, juice or additive obtained or derived from a plant such as a non-toxic plant. In some embodiments, the additive can be an artificial or synthetic flavour enhancing or flavour masking compound.

[0069]

[0066] In some embodiments, the plant-based material is at least one selected from the group consisting of teas, sugars, berries, fruits, herbs, flowers, grains, nuts, seeds, spices, vegetables and others. In some embodiments, the medium has to not be too acidic in order to accommodate plant-based material characterised with high acid content. In some embodiments, the plant-based material such as apple and grape juice which have high acidity such as around pH 3.0, would require the addition of buffer solution. In some embodiments, dried plant-based material is used.

[0070]

[0067] In some embodiments the aqueous solution contains a nitrogen source. In some embodiments, the nitrogen source comprises at least one organic compound such as one or more selected from the group of teas, berries, fruits, herbs, flowers, grains, nuts, seeds, spices, vegetables or combinations thereof. In some embodiments the aqueous solution contains a carbon source. In some embodiments, the carbon source comprises at least one organic compound such as one or more selected from the group of sugars, berries, fruits, grains, nuts, seeds, vegetables or combinations thereof. In some embodiments the aqueous solution is a tea solution. In some embodiments the aqueous solution is a brew solution. In some embodiments the aqueous solution is a fermentation solution.

[0071]

[0068] In some embodiments, the nitrogen source can be a juice or extract derived from berries, fruits or vegetables such as acai, acerola, bilberry, black cherry, black currant, blackberry, blueberry, boysenberry, cloudberry, cranberry, elderberry, goji berry, gooseberry, hawthorn, honeyberry, huckleberry, josta berry, juniper berries, logan berry, magellan barberry, marionberry, marlon berry, mulberry, pineberry, raspberry, red currant, salal berry, salmonberry, sea buckthorn, silvan berry, sloe, sour cherry, strawberry, suriname cherry, tamarillo, tayberry, thimbleberry, white currant, young berry, ackee, akebi, ancho chilli, apricot , aratiles, araza, avocado, banana, bergamot, birds eye chilli, blood orange fruit infusion, breadfruit, buddha's hand, canistel, cantaloupe, catmon, cempedak, ceyan pepper, cherimoya, chico fmit, chipotle chilli, clementine, crab apple, damson, dates, dragon fmit, durian, feijoa, figs, finger lime, gac, grape seed, grape skin, grapefmit, green apple, guava, habanero, hala fruit, homed melon, jabuticaba, jack fmit, japanese plum, jujube, kashmiri chilli, kiwi, kumquat, lanzones, lemon, lemon peel, lime, longan, loquat, lulo, lychee, macopa, mamey apple, mamey sapote, mamoncillo, mandarine, mango, mangosteen, mayapple, medlar, minneola, miracle fmit, momordica fmit, monstera deliciosa, nance, nectarine, orange, orange peel, papaya, paprika, passionfruit, pawpaw, peach, pear, persimmon, pineapple, pitaya, plantain, plum, plumcot, pomegranate, pommelo, prickly pear, pulasan, quince, rambutan, red apple, red berry blend, red grape, rhubarb, rose apple, salak, santol, sapodilla, sapote, sarguelas, satsuma, smoked guava, smoked paprika, soursop, star fmit, tamarind, tangelo, tangerine, ugli fmit, watermellon, white grape, white sapote, ximenia, yuzu, breadfruit, lotus, plantain, snake gourd, indian fig, beet, chicory, com, cucumber, ginger, ginseng, horseradish, hot paprika, inulin, wasabi, artichoke, broccoli, cauliflower, squash, broadbean, chayote, chickpea, common bean, com, cowpea, durian, aubergine, gerkin, tomato, lentil, lima bean, loofah, okra, olive, pea, pumpkin, soybean, tomatillo, tomato, water chestnut, wax gourd, courgette, asparagus, bamboo, beet, bok choy, bmssels sprouts, cabbage, celery, chard, kale, kohlrabi, lambs lettuce, leek, lettuce, spinach, watercress

[0072]

[0069] In some embodiments, the plant-based nitrogen source comprises 10% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 20% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 30% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 40% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 50% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 60% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 70% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 80% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 90% of the aqueous growth medium. In some embodiments, the plant-based nitrogen source comprises 100% of the aqueous growth medium.

[0073]

[0070] In some embodiments, the plant-based nitrogen source is dried plant-based material such as the plant-based material comprises less than about 50% moisture content (MC), preferably less than 40% moisture content (MC), preferably less than 30% moisture content (MC), preferably less than 20% moisture content (MC), preferably less than about 18% MC, preferably less than about 16% MC, preferably less than about 14% MC, preferably less than about 12% MC, preferably less than about 10% MC, preferably less than about 8% MC, preferably less than about 6% MC, preferably less than about 5% MC or less. In some preferred of the present invention, the plant-based material comprises about 5% MC.

[0074]

[0071] In some embodiments, the plant-based nitrogen source is dried plant-based material. In some embodiments, the dried plant-based nitrogen source is dried fmits and vegetables such as dried grape skins, dried grape seed, dried apple, dried sugarbeet, dried strawberry, dried blackberry, dried raspberry, dried melon, dried watermelon, dried grapefmit, dried grape, dried orange, dried plum, dried prune, dried pomegranate, dried pineapple, dried kiwifruit, dried quince, dried cranberries, dried pear, dried carrot, dried passion fmit, dried fig, tea and others. In some embodiments, the dried plant-based nitrogen source is herbs, flowers, nuts, seeds and spices, such as basil, bay leaf, borage, bumet, camomile flowers, camelia sinensis, capsicum, catnip, chervil, chives, cicely, cilantro, coriander, cornflowers, costmary, dill, elderflower, fennel, fenugreek, green rooibos, grewia asiatica, hibiscus, hibiscus special cut, holy basil, honeybush, hops, horehound, hyssop, jasmine, kaffir lime, lavender, lemon balm, lemon grass, lemon verbena, lovage, maijoram, mint, moroccan mint, oregano, parsley, peppermint leaf, peppermint special cut, rose, rose petals whole, rosehip, rosemarry, rue, safflowers, sage, satemwa hibiscus, satemwa peppermint, savory, sorrel, tarragon, thyme, verbena, yerba mate, caper, amaranth, arugula, borage, burdock, cardoon, chicory, chive, endive, fennel, lambs quarters, stinging nettle, tossa jute, chocolate, cocoa, cocoa de mer, coconut, poppy seed, sesame, peanut, all spice, angelica root, asafoetida, black pepper, caraway, cardomom, cassia, celery seed, chai, chilli powder, cinnamon, cloves, coffee, cumin, curry, file, grains of paradise, liquorice , mace, mustard, nutmeg, saffron, star anis, turmeric, vanilla, white pepper, collard and others.

[0075] [1] In some embodiments the plant-based nitrogen source comprises 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100% of the aqueous growth medium. In some embodiments, the nitrogen source comprises 2% of the aqueous growth medium. In some embodiments, the nitrogen source comprises 3% of the aqueous growth medium. In some embodiments, the nitrogen source comprises 5% of the aqueous growth medium. In some embodiments, the nitrogen source comprises 10% of the aqueous growth medium. In some embodiments, the nitrogen source comprises 20% of the aqueous growth medium. In some embodiments, the nitrogen source comprises 30% of the aqueous growth medium.

[0076]

[0072] In some embodiments of the present invention, the plant-based nitrogen source comprises tea plant biomass. In some embodiments, the tea plant biomass is used to form a brew solution. In some embodiments, the brew solution is prepared by steeping tea plant biomass in cold, hot or lukewarm aqueous solution. In some embodiments, the teaplant medium comprises 0.5 % of the aqueous growth medium. In some embodiments, the tea-plant medium comprises 1% of the aqueous growth medium. In some embodiments, the tea-plant medium comprises 2.5% of the aqueous growth medium. In some embodiments, the tea-plant medium comprises 5% of the aqueous growth medium. In some embodiments, the tea-plant medium comprises 10% of the aqueous growth medium. In some embodiments, the teaplant medium comprises 20% of the aqueous growth medium. In some embodiments, the tea-plant medium comprises 30% of the aqueous growth medium.

[0077]

[0073] In some embodiments, the tea plant biomass comprises at least one selected from the group of imperial pluck bud + 1 leaf, fine pluck bud + 2 leaves and course pluck bud + 3 leaves or more leaves.

[0078]

[0074] In some embodiments, the tea plant biomass is derived from at least one selected from the group of Camellia sinensis, Lamiaceae species (including peppermint and spearmint), Verbascum species, Cinnamomum species, Elettatia cardamomum, plants of the Aquilaria genus, yarrow flower, Salvia species, Angelica species, catnip plants, lavender (Lavandula angustifolia), Taraxacum species, linden tree flowers, lemon verbena (Aloysia citrodora), gotu kola (Centella asiatica), Leonotis species, blue lotus (Nymphaea caerulea), kratom, herbal non-tobacco, mugwort, coltsfoot leaf, Heimia salicifolia, St. John's wort, Melissa officinalis, skullcap, yerba lenna yesca, Capillaris herba, Calea zacatechichi, Leonurus sibericus, damiana, Withania somnifera, Mucuna pruriens, Celastrus paniculatus, Emblica officinalis, Terminalia chebula, Terminalia bellerica, Solanum xanthocarpum, Glycyrrhiza glabra, Ocimum sanctum, Zingiberaceae family and Zingiber officinale, Commiphora mukul, Boswellia serrata, Vitex negundo, Bacopa monnieri, Cyperus rotundus, Myristica fragrans, Valeriana wallichii, Nardostachys jatamansi, Mimosa hostilis, Avena sativa, kanna, Lactuca virosa, Comus, pipsissewa (Chimaphila umbellata), pyrola, kinnikinnik, camomile, manzanita, Arctostaphylos species, madrone, Arbutus menziesii, Rubus species, scotch broom, calamus, Crocus sativus, Santalum album, Acacia confusa, Passifioraceae species, Echinacea purpurea and milk thistle (Silybum marianum).

[0079]

[0075] In some embodiments of the present invention, the tea plant biomass is derived from at least one selected from the group of camelia sinensis tea styles including African Orthodox, African Orthodox Organic, Apricot Flavoured Black Tea, Assam, Assam Black Orthodox, Assam Breakfast, Assam Black CTC, Assam 2nd Flush CTC, Assam CTC Bold Broken, Assam CTC Bold Broken QE Project, Assam GFBOPS, Assam Finest 2nd Flush, Assam Good 2nd Flush, Assam BOP Organic, Assam TGFOP1 Organic, Assam-Kenya Breakfast Blend , Bi Luo Chun Green Silvery Snail Green Tea, Black Cherry Flavoured Tea , Black Tea Chai , Black Tea and Lemon , Black Tea Peach , Blood Orange Fruit Infusion , Blue Lady Black Tea Grapefruit Scented with flowers, Breakfast Blend Vacuum-Packed, Christmas Blend , Colombian Black OP1 , Colombian Green Tea , Ceylon Low Grown OP1, Ceylon High Grown OP1, Ceylon Western BOP, China Black OP , Chun Mee , Daijeeling First Flush - Fiori tendency, Daijeeling First Flush Angrove March Crop, Daijeeling First Flush FTGFOP1 Finest, Daijeeling First Flush FTGFOP1, Daijeeling First Flush Moonshine, Daijeeling First Flush Flowery CIO, Daijeeling 2nd Flush FTGFOP1, Daijeeling 2nd Flush TGBOP1, Daijeeling Green FTGFOP, Decaffeinated Earl Grey , Dong Ding Oolong , Earl Grey , Earl Grey Finest , Earl Grey Finest with Flowers , Extra Strong Blend , Floral Blooming Tea , Fu Tea Brick Dark Tea , Genmaicha toasted rice & Sencha, Gin and Tonic Tea , Good Night Blend , Green Tea Exotic green tea, pineapple, flavours, safflower, cornflower, Green Tea Ginger Orange , Green Tea Nilgiri Organic, Gunpowder Green Tea , Gyukuro , High Grown STGFOP Bespoke Blend , Honeybush , Jade Oolong , Japanese Hojicha , Japanese Macha Organic , Japanese Sencha , Jasmine Leaf Scented Tea , Jasmine Pearls Scented Tea , Keemun Black Tea , Kenyan Decaf PF1 , KTDA East-of- Rift Bold Broken, KTDA East-of-Rift PF1, KTDA East-of-Rift Orthodox Pekoe, KTDA East-of-Rift OP1, Kukicha, Lapsang Souchong Tea, Livingstone's Twist & Dry Oolong, Lung Ching Green Tea Dragonwell, Malawi First Flush, Malawi PF1, Mao Jian, Matcha, Matcha Green Tea , Milky Oolong , Mozambique BPI Organic , Mulled Wine Blend , Nandi Purple Tea , Nilgiri Frost , Nilgiri Bamboo, Oolong Standard, Oriental Beauty Oolong , Orthodox FBOP Decaffeinated, Orthodox Peko Decaffeinated , Orthodox Peko EA Method , Pai Mu Tan White Tea , Peppermint Flavoured Black Tea , Pu Erh Leaf Dark Tea , Red Berry Blend , Rose Congou Scented Tea , Ruby 18 , Russian Caravan Blend , Rwandan BPI , Satemwa Antlers , Satemwa Hibiscus , Satemwa Peppermint , Sea Buckthorn , Se Chung Oolong , Sencha Green Tea , Silver Needle White Tea , Sunon Yellow Tea , Thyolo Oolong , Thyolo Peony , Tieguanyin Oolong, Tinderet Green Tea, Tippy Kenyan , Tumoi Nandi Gold Turmeric Chai , Turmeric Chai , Vanilla Flavoured Black Tea, Whiskey Tea , White Downy White Tea, White Tea and Mango, Yerba Mate, Yunnan, Yunnan Ying Ming, Yunnan Black FOP, Yunnan Gold, Yunnan Gold, Yunnan Golden Tips, Yunnan Green FOP, Yunnan Noir.

[0080]

[0076] In some embodiments, the brew solution is prepared by steeping at least 0.5 g / L tea biomass in aqueous solution, at least 1 g / L tea biomass in aqueous solution, preferably at least 2 g / L tea biomass in aqueous solution, preferably at least 3 g / L tea biomass in aqueous solution, preferably at least 5 g / L tea biomass in aqueous solution, preferably at least 6 g / L tea biomass in aqueous solution, preferably at least 7 g / L tea biomass in aqueous solution, preferably at least 8 g / L tea biomass in aqueous solution, preferably at least 9 g / L tea biomass in aqueous solution, preferably at least 10 g / L tea biomass in aqueous solution, preferably at least 15 g / L tea biomass in aqueous solution, preferably at least 20 g / L tea biomass in aqueous solution, preferably at least 40 g / L tea biomass in aqueous solution or more to form the brew solution.

[0081]

[0077] In some embodiments, the brew solution is prepared by steeping the tea plant biomass in an aqueous solution for at least 1 min, for at least 2 min, for at least 5 mins, for at least 10 min, for at least 15 min, for at least 20 min, for at least 30 min, for at least 40 min, for at least 50 min, for at least 60 min, for at least 70 min, for at least 80 min, for at least 90 min, for at least 100 min, for at least 120 min, for at least 130 min, for at least 150 min, for at least 180 min, for at least 4 hrs, for at least 5 hrs, for at least 6 hrs, for at least 7 hrs, for at least 8 hrs, for at least 10 hrs, for at least 12 hrs, for at least 16 hrs or more, to form the brew solution.

[0082]

[0078] In some embodiments, the tea plant biomass comprises about 0.1% v / v of the growth medium, preferably about 0.2% of the growth medium, preferably about 0.4% of the growth medium, preferably about 0.8% of the growth medium, preferably about 1% of the growth medium, preferably about 1.2% of the growth medium, preferably about 1.4% of the growth medium, preferably about 1.8% of the growth medium, preferably about 2% of the growth medium, preferably about 3% of the growth medium, preferably about 4% of the growth medium, preferably about 5% of the growth medium, preferably about 6% of the growth medium, preferably about 7% of the growth medium, preferably about 8% of the growth medium, preferably about 9% of the growth medium, preferably about 10% of the growth medium, preferably about 11% of the growth medium, preferably about 12% of the growth medium, preferably about 13% of the growth medium, preferably about 14% of the growth medium, preferably about 15% of the growth medium or more of the tea plant biomass.

[0083]

[0079] The skilled person would be familiar with different methods of preparing a tea brew solution. It would also be familiar to the skilled person that different tea plant biomass materials can lead to the preparation of different types of brew solutions. Indeed, the same type of tea plant biomass can be used where the resulting brew solution can be different with a minor modification of for example, the steeping temperature, duration of steeping, humidity of the plant biomass, growth stage of the tea plant or tea plant constituents. The sensory, tactile and flavour attributes or characteristics of the brew solution can be modified, to a different extent, by the skilled person to meet the individual requirements or the preferred characteristics.

[0084]

[0080] In some embodiments of the present invention, the growth medium described herein is supplemented with a plant-based carbon source comprising at least one organic compound such as one or more selected from the group of sugars, berries, fruits, grains, nuts, seeds, vegetables or combinations thereof. In some embodiments of the present invention, at least one organic compound is selected from the group of sugars, berries, fruits, grains, nuts, seeds, vegetables or combinations thereof to deliver between 0.1% v / v and about 25% sugar content.

[0085]

[0081] In some embodiments of the present invention, the plant-based carbon source comprising at least one organic compound such as one or more selected from the group whitebeam, acai, acerola, amelanchier intermedia, barberry, billberry, blackcurrant, blue lilly pilly , bunchberry, calligonum junceum, changunga, cherry, chokeberry, conkerberry, crowberry, curry berry, didgen berry, elderberry, emblic, engkala, gooseberry, guavaberry, hackberry, hawthorn, jomboloan, juneberry, lemonadeberry, lilly pilly, lingonberry, lotus, lovi-lovi, maqui berry, mortino, muntrie, musho, nannyberry, native currant, partridge berry, raspberry, red huckleberry, redcurrant, riberry, rowan, rukam, salal, saskatoon, scarlet firethom, serviceberry, shoebutton ardisia, silver buffaloberry, silverberry, sloe, staghorn sumac, sumac, vilmorin's rowan, water berry, wild dilly, wolfberry, aguasiqui, ambarella, apple, apricot, arazo, atoto, avocado, babacoa, bambangan, banana, bignay, bilimbi, binjai, bola verde, bolowarra, bombona, borojo, burahol, bush butter fruit, butia, cainito, calabur fruit, cambuci, camu camu, canistel, caqui, cardon fruit, casimiroa, Chinese date, coolamon, corozo palm fruit, crabapple, curuba, damson, date, desert quandong, dhung, dragon fruit, dutchamns laudanum, feioa, fibrous satinash fmit, fuchsia, gandaria, gomortega, granadilla, grape, greene's mountain-ash fmit, greengage, guarana, guava , gubinge, hairless rambutan, jackal jujube, jagua, jocote, jujube, kaong, khimi, kiwi, korlan, kubal, langsat, lime, lipote, longan, loquat, lucuma, lychee, mains sieversii, mains x zumi, mammee, mandacaru, mangaba, mango, mangosteen, manilikara, maracuja , maracuja bravo, mardarrugu, manila, mayapple, maypop, medlar, melon, millaa millaa, miracle fruit, mistol, mombin, musa ingens, muscadine, nance, narajilla, nectarine, neem, nokaido, oil palm fruit, olive, olosapo, ooray, orange, papaya, passionfruit, peach, peanut butter fruit, pear, pequi, persimon, phalsa, pitanga, pitomba, pitomba-da-bahia, plantain, plum, pomegranate, poro poro, prickly pear fruit, prunus , pmnus alaica, pulasan, pumi, pyrus, quince, rambutan, ramontchi, saguaro fruit, sapodilla, satsuma, saw palmetto fruit, sea apple, sea coconut, sea grape, shipova, soh-sang, sorb, sourplum, star fruit, strawberry, swamp maire, swartabas, syzygium , syzygium acuminatissimum, tamarind, tangerine, tapia, toyon, watermellon, wongi, yangmei, ziziphus budhensis, ziziphus incurva, zwetschge, barley , wheat, rice , rye, millet, sorghum , blue guarri, buffalo-thorn, bushveld bluebush, passiflora , passiflora alata, almond, coconut, coffee, pili, agave, barbados, barley malt, beet sugar, brown sugar, buttered syrup, cane sugar, caramel, carob syrup, castor sugar, coconut palm, coconut sugar, confectioners, com sweetner, com symp, date sugar, dehydrated cane juice, demerara sugar, dextrin, dextrose, free-flowing brown sugar, fmctose, glucose, golden sugar, golden symp, grape sugar, high-fructose com symp, honey, icing sugar, invert sugar, malt symp, maltodextrin, maltol, maltose, mannose, maple symp, molasses, muscovado sugar, palm sugar, panocha, powdered sugar, raw sugar, refiner's symp, rice symp, saccharose, sorghum symp, sucrose, granulated sugar, treacle, turbinado sugar, yellow sugar, beet , carrot, cassava, celeriac, water chestnut, garlic, ginger, horseradish, jemsalem artichoke, jicama, onion, parsnip, pepino, potato, radish, rutabaga, salsify, sansapote, shallot, sweet potato, taro, turnip, yam and others.

[0086]

[0082] In a preferred embodiment, at least one organic compound comprises sucrose. In some embodiments, sucrose comprises about 25 % v / v of the growth medium, preferably sucrose comprises about 20 % v / v of the growth medium, preferably sucrose comprises about 15 % v / v of the growth medium, preferably sucrose comprises about 12 % v / v of the growth medium, preferably sucrose comprises about 10 %v / v of the growth medium, preferably sucrose comprises about 9 % v / v of the growth medium, preferably sucrose comprises about 8 % v / v of the growth medium, preferably sucrose comprises about 7 % v / v of the growth medium, preferably sucrose comprises about 6 % v / v of the growth medium, preferably sucrose comprises about 5 % v / v of the growth medium, preferably about 4.5 % v / v of the growth medium, preferably about 4 % v / v of the growth medium, preferably about 3.5 % v / v of the growth medium, preferably about 3 % v / v of the growth medium, preferably about 2.5 % v / v of the growth medium, preferably about 2 % v / v of the growth medium or less.

[0087]

[0083] In some embodiments, the aqueous medium employed in the present method typically contains at least 70 wt.% water. More preferably, the aqueous culture medium contains at least 80 wt.%, most preferably 90 wt.% water. Besides water, the aqueous culture medium contains a carbon and nitrogen source and optionally any other ingredients needed by the organisms to grow, such as salts providing essential elements such as magnesium, phosphorus and sulphur.

[0088]

[0084] It is believed that the addition of plant-based material in the aqueous growth medium contributed to the flavour of the growth medium. It is believed that the addition of plant-based material in the aqueous growth medium can enhance the release of yeast metabolites which contribute to the sensory, tactile and flavour parameters or attribute of the fermented drink.

[0089]

[0085] In some embodiments the growth medium is a large-scale growth medium such as commercial scale growth medium. As used herein the term “large scale” is synonymous with “commercial scale” and means about 5 to 500L or more of total microbial culture or total microbial growth culture. In some embodiments, large scale is about 5 to WOOL or more of total microbial culture or total microbial growth culture. In some embodiments, large scale is about 5 to 2000L or more of total microbial culture or total microbial growth culture. In some embodiments, large scale is about 5 to 5000L or more of total microbial culture or total microbial growth culture.

[0090] Microbial Composition

[0091]

[0086] In some embodiments the aqueous medium is inoculated with microorganisms such as one or more yeast and / or one or more bacteria. The term “inoculated” as used herein refers to introducing or adding microorganisms such as yeast and / or bacteria or combinations thereof, into the aqueous solution.

[0092]

[0087] As used herein, the term “inoculum” means a microbial material or cell culture which is added to some other material or substance, such as aqueous growth medium. In some embodiments the inoculum comprises live microorganism cells. In some embodiment, the inoculum employed in the present method comprises at least one microorganism. The microorganism that can be employed can include prokaryote or eukaryote. The microorganism can be selected from fungi, such as yeast, or bacteria.

[0093]

[0088] As used herein, the term “incubating inoculated microbial mixture” means a method of multiplying microorganisms such as yeast and / or bacteria by letting them reproduce or proliferate in predetermined culture solution such as growth medium, under controlled conditions.

[0094]

[0089] As used herein, the term “fermentation mixture” means a symbiotic microbial culture comprising yeast and bacterial species. Preferably, yeast metabolises a nitrogen source, a carbon source, oxygen and other chemicals to produce carbon dioxide, alcohol and taste or flavour compounds whereas bacteria metabolise the yeast produced alcohol and remaining sugars and produce acids. Preferably, yeast metabolises a nitrogen source, a carbon source, oxygen and other chemicals to produce carbon dioxide, alcohol and taste or flavour compounds whereas bacteria metabolise the yeast produced alcohol and produce acids without adversely affecting or modifying the taste or flavour of the fermented drink.

[0095]

[0090] As used herein, the term “fermenting” means yeast producing alcohol and taste or flavour compounds in a growth medium and where alcohol is broken down by bacteria without adversely affecting the taste or flavour of the fermented drink such as by an increase in alcohol, carbon dioxide and different acids. As used herein, the term “fermented drink” means a product, such as a beverage produced by the methods described herein.

[0096]

[0091] Preferably the microorganisms are selected from yeast or bacteria. Preferably the microorganisms comprise both yeast and bacteria species.

[0097]

[0092] The bacteria kingdom has over 70,000 species and each carry very different characteristics. The microorganisms that are propagated using the present method can be sampled from, for instance, complex cultures for food or feed fermentation, mixed cultures for bioprotection, complex probiotics, from microbiota or from kombucha microbial culture.

[0098]

[0093] In some embodiments, the inoculum is incubated to generate between about 102-5xl06viable cells / ml or more. According to some embodiments the inoculum can be produced or propagated using microfluidic systems.

[0099]

[0094] In some embodiments, the inoculated microbial mixture is incubated to generate a propagated starter culture.

[0100]

[0095] In some embodiments, the propagated starter culture contains between about 103- 5xlO10viable cells / ml. It is contemplated within the context of the present invention that when generating a propagated starter culture, ITc incubation temperature may vary depending on for example the type of growth medium, the microbial mixture, duration of incubation, the concentration of carbon course and others. By way of non-limiting example, where the growth medium contains relatively high level of carbon source such as sucrose of about 25 g / L the ITc can be reduced and the duration of incubation can be increased. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 25°C to 30°C for about 14 weeks. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 25°C to 30°C for about 12 weeks. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 25°C to 30°C for about 10 weeks.

[0101]

[0096] In some embodiments, the propagated starter culture comprises a symbiotic mixture of at least one type of yeast and at least one type of bacteria. In some embodiments the propagated starter culture comprises a mixture of at least two types of yeast and at least two types of bacteria. In some embodiments the propagated starter culture comprises a mixture of at least three types of yeast and at least three types of bacteria or more.

[0102]

[0097] Most yeast or bacterial species can be exploited in the context of the present methods. Usually, yeast which produce low alcohol levels are well suited in the present methods.

[0103]

[0098] Preferably, the yeast can be selected from the group of genera consisting of Pichia, Saccharomyces, Zygosaccharomyces, Hanseniaspora, Mycotorula, Hanseniaspora, Zygosaccharomyces, Lachancea, Candida, Kazachstania, Kloeckera, Metschnikcnvi, Medusomyces, Brettanomyces, Saccharomycodes, Torulopsis, Torulaspora, Schizosaccharomyces and Kluyveromyces. In some embodiments, the yeast is selected from the group of genera comprising Pichia, Saccharomyces, Zygosaccharomyces, Hanseniaspora, Mycotorula, Hanseniaspora, Zygosaccharomyces, Lachancea, Candida, Kazachstania, Kloeckera, Metschnikowi, Medusomyces, Brettanomyces, Saccharomycodes, Torulopsis, Torulaspora, Schizosaccharomyces and Kluyveromyces. In some embodiments, the yeast can be selected from the yeast genera listed in Table 4.

[0104] Table 4. List of example yeast microorganisms which can be employed in the present invention.

[0105]

[0099] In some embodiments, the yeast is selected from the group consisting of Saccharomyces bayanus, Saccharomyces cerevisiae, Saccharomyces boulardii, Saccharomyces fructicola, Saccharomyces pastorianus, Saccharomyces bruxellensis, Saccharomyces carlsbergensis, Saccharomyces japonicus, Mycotorula intermedia, Mycotorula humilis, Brettanomyces nanus, Brettanomyces naardenensis, Brettanomyces custerisianus, Brettanomyces anomalus, Brettanomyces bruxellensis, Zygosaccharomyces bailii, Zygosaccharomyces rouxii, Zygosaccharomyces pseudorouxii, Zygosaccharomyces mellis, Zygosaccharomyces bisporus, Zygosaccharomyces lentus, Hanseniaspora valbyensis, Hanseniaspora osmophila, Candida lactis-condensi, Candida stellata, Lachancea thermotolerans, Metschnikowia pulcherrima Saccharomycodes ludwigii, Torulaspora delbrueckii, Zygosaccharomyces bailii, Schizosaccharomyces pombe, Saccharomyces ludwigii, Zygosaccharomyces rouxii, Torulaspora delbrueckii, Brettanomyces bruxellensis, Brettanomyces lambicus, Brettanomyces custerii, Pichia membranaefaciens and Kloeckera apiculate.

[0106]

[0100] In some embodiments, the bacteria can be selected from the group of genera consisting of Leuconostoc, Lactococcus, Acetobacter, Allobacullum, Bifidobacterium, Leuconostoc, Propionobacterium, Ruminococcus, Gluconob ac ter, Gluconacetobacter, Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Abiotrophia, Aerococcus, Aerosphaera, Agitococcus, Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes, Bavariicoccus, Carnobacterium, Carnococcus, Catellicoccus, Chungangia, Convivina, Desemzia, Dolosicoccus, Dolosigranulum, Enterococcus, Eremococcus, Facklamia, Floricoccus, Fructobacillus, Globicatella, Granulicatella, Ignavigranum, Isobaculum, Jeotgalibaca, Lacticigenium, Lactovum, Lachancea, Marinilactibacillus, Melissococcus, Metschnikowia, Oenococcus, Okadaella, Pilibacter, Pisciglobus, Sharpea, Komagataeibacter, Brevibacterium, Pediococcus, Nguyenibacter, Sporolactobacillus, Tetragenococcus, Torulaspora, Trichococcus, Thermus, Streptococcus, Staphylococcus, Vagococcus and Weissella. In some embodiments, the bacteria can be selected from the genera of bacteria listed in Table 5.

[0107]

[0101] Table 5. List of example bacterial microorganisms which can be employed in the present invention.

[0108]

[0102] In some embodiments, the bacteria can be selected from the group of species consisting of Lactobacillus nagelii, Lactobacillus rhamnosus, Lactobacillus sanfranciscansis, Lactobacillus fermentum, Limosilactobacillus fermentum, Companilactobacillus paralimentarius, Lactiplantibacillus plantarum, Fructilactobacillus sanfranciscensis, Acetobacter pasteurianus, Acetobacter obeodiens, Acetobacter pomorum, Acetobacter intermedius, Acetobacter malorum, Acetobacter aceti, Acetobacter nitrogenifigens, Acetobacter oeni, Acetobacter cerevisiae, Komagataeibacter xylinus, Gluconobacter oxydance, Gluconacetobacter europaeus, Gluconacetobacter entanii, Gluconacetobacterxylinus, Gluconacetobacter ketogenus, Gluconacetobacter hensenii and Gluconacetobacter sacchari.

[0103] In some embodiments, symbiotic mixture comprises Saccharomyces bayanus, Saccharomyces cerevisiae, Lactiplantibacillus plantarum and Gluconacetobacter europaeus. In some embodiments, symbiotic mixture comprises Saccharomyces bayanus, Zygosaccharomyces bisporus and Gluconacetobacter europaeus. In some embodiments, symbiotic mixture comprises Saccharomyces bayanus, Zygosaccharomyces bisporus, Gluconacetobacter europaeus and Acetobacter cerevisiae. In some embodiments of the present invention, the symbiotic mixture comprises Saccharomyces bayanus, Zygosaccharomyces bisporus, Gluconacetobacter europaeus and / or Acetobacter pasteurianus . In some embodiments of the present invention, the symbiotic mixture comprises Saccharomyces luchvigii, Zygosaccharomyces kombuchaensis, Gluconacetobacter ketogenus and / or Acetobacter zylinoides. In some embodiments of the present invention, the symbiotic mixture comprises Saccharomyces apiculatus, Zygosaccharomyces lentus, Komagataeibacter xylinus and / or Acetobacter xylinum. In some embodiments of the present invention, the symbiotic mixture comprises Saccharomyces cerevisiae, Zygosaccharomyces bisporus, Lactobacillus nagelii and / or Gluconacetobacter ketogenus.

[0109]

[0104] In some embodiments, the propagated starter culture contains between about 102-5xl010viable cells / ml. In some embodiments, the propagated starter culture contains between about 102-5xl09viable cells / ml. In some embodiments, the propagated starter culture contains between about 102-5xl08viable cells / ml. In some embodiments, the propagated starter culture contains between about 102-5xl07viable cells / ml. In some embodiments, the propagated starter culture contains between about 102-5xl06viable cells / ml. In some embodiments, the propagated starter culture contains between about 102-5xl05viable cells / ml.

[0110]

[0105] In some embodiments, the propagated starter culture contains about IO10viable cells / ml. In some embodiments, the propagated starter culture contains about 109viable cells / ml. In some embodiments, the propagated starter culture contains about 108viable cells / ml. In some embodiments, the propagated starter culture contains about 107viable cells / ml. In some embodiments, the propagated starter culture about 106viable cells / ml. In some embodiments, the propagated starter culture contains about 105viable cells / ml.

[0111]

[0106] The skilled person would know that viability of microbial cells can be assessed or estimated using colonyforming units per millilitre (CFU / mL) in case of a liquid being tested or grams (CFU / g) if a solid material is tested.

[0112]

[0107] If the propagated starter culture is a liquid, the concentration of the viable cells in the culture can be estimated via colony-forming units per millilitre (CFU / mL or growth medium). In some embodiments, the concentration of the viable cells in the starter culture is from 0.5 million to 1 billion CFU / mL, 0.5 million to 500 million CFU / mL, 0.5 million to 400 million CFU / mL, 0.5 million to 300 million CFU / mL, 0.5 million to 200 million CFU / mL, 0.5 million to 150 million CFU / mL, 0.5 million to 125 million CFU / mL, 0.5 million to 100 million CFU / mL, 0.5 million to 75 million CFU / mL, 0.5 million to 50 million CFU / mL, 200 million to 300 million CFU / mL, 300 million to 1 billion CFU / mL, 300 million to 500 million CFU / mL, 300 million to 400 million CFU / mL, 400 million to 1 billion CFU / mL, 400 million to 500 million CFU / mL, or 500 million to 1 billion CFU / mL or more such as 10 billion CFU / ml.

[0113] Incubation, apparatus and systems

[0114]

[0108] In order to try and address some of the limitations of the current systems, for example speed of growth of the microorganism and appropriately controlled flavour and taste development, the methods described herein can employ suitably adapted incubation apparatus and techniques which utilise different types and forms of physical forces. Examples of the different types and forms of physical forces include for instance pseudo forces. The different types and forms of physical forces, such as for example pseudo forces can be generated for example as a result of a rotating platform or structure within an apparatus (e.g. a fermentation vat or vessel), resulting in increased advection and chaotic mixing of microorganism, e.g. yeast and bacteria, comprising aqueous growth medium and carbon source.

[0115]

[0109] Without wishing to be bound by theory, in a rotating incubation platform system, Euler pseudo force (which is perpendicular to centrifugal pseudo force), may be used to generate vortical flow and provide uniform mixing within for example a microfluidic chamber of a microfluidic system. Euler pseudo forces are inertial forces that are produced when a microfluidic system experiences cycles of unidirectional acceleration-and-deceleration rotation. Thus, mixing is dependent on chamber geometry, acceleration / deceleration rate, and angular spin.

[0116] [HO] For liquid microbial cultures, such as yeast and bacterial cultures as contemplated herein, rapid and healthy growth may depend on factors or parameters including but not limited to:

[0117] (1) aeration, so that the symbiotic microbes in the mixture have access to for example where aerobic, fresh oxygen for growth;

[0118] (2) substrate and nutrient availability, where samples are thoroughly mixed to provide nutrients homogenously throughout the culture to facilitate optimal flavour development;

[0119] (3) minimisation of biofilms and clumping, where shaking and agitation prevents microbial culture from settling to the bottom of a fermentation chamber and forming biofilms or clumps that hinder reproduction;

[0120] (4) optimal interaction of symbiotic microbes in the mixture with metabolites during fermentation which leads to alcohol production by yeast and assimilation of the alcohol by the co-cultured bacteria in the mixture, without adversely affecting the flavour or taste of the non-alcohol fermented drink;

[0121] (5) minimisation or complete elimination of the production of by-product such as acids in the non-alcohol fermented drink;

[0122] (6) Temperature and / or duration of incubation of the symbiotic microbes; and

[0123] (7) optimal level of production of parameters such as physico-chemical or chemical parameters selected from and not limited to pH, sucrose, tannin, esters, polyphenols, minerals, acids such as lactic acid, citric acid, ascorbic acid, acetic acid, glucuronic acid, amino acids and alcohol (ABV).

[0124] [Hl] In some embodiments, the liquid microbial culture is a fermentation microbial culture. In some embodiments, the liquid microbial culture is a fermentation mixture.

[0125]

[0112] It is contemplated herein that any detection, monitoring, sampling or measurements of the above exemplary factors or parameters can be continuous or intermittent. Furthermore, it is contemplated that any detection, monitoring, sampling or measurements of the above exemplary factors or parameters is in real time. In some embodiments, detection, monitoring, sampling or measurements are qualitative or quantitative. In order to detect, monitor, sample or measure different factors and parameters as contemplated herein, for example during microbial culture e.g. during the process of microbial fermentation, an apparatus or system can be used which comprises one or more detection, monitoring, sampling or measurement devices. Exemplary devices or techniques include detecting, monitoring, sampling or measurement devices to continuously assess, evaluate and control factors or parameters during an ongoing microbial fermentation. In some embodiments, the devices or techniques are in communication with the growth medium. In some embodiments, the devices or techniques are in communication with the fermenting mixture.

[0126]

[0113] The devices or techniques include and are not limited to visual imaging, infrared (IR) / thermal imaging, laser techniques, oxygen levels, acid level, nitrogen source, carbon source,, alcohol levels and other physico-chemical and / or chemical parameters which can deploy devices or techniques such as HPLC, MALDI TOF (MS), Energy Dispersive X-ray Spectroscopy, EDX analysis and SEM (Scanning Electron Microscopy) studies using FESEM (Field Emission Scanning Electron Microscope), odour description tests, olfactory description tests and techniques listed in Table 6 below.

[0127]

[0114] Furthermore, the method of the present invention provides a mechanism for correction such as real time correction of factors or parameters which have been determined to be at variance relative to control or desirable values or parameters as determined from examples during the process of microbial fermentation. In order to facilitate accomplishment of this, one or more correction devices or techniques are contemplated which can lead to cooling, heating, aeration, micro-oxygenation, addition of further nitrogen or carbon source, or propagated starter culture, in order to produce a non-alcoholic fermented drink such as sparkling non-alcoholic fermented drink with desired flavour or taste.

[0128]

[0115] It is contemplated that one of the main compounds which is associated with imparting different flavour or taste to the drink is the development during fermentation is the different esters in the drink such as the non-alcoholic fermented drink. Some factors affecting the formation of esters are for example the selection of the yeast strain and fermentation temperature. Different yeast strains yield different quantities of esters. Yeast strains that produce lower quantities are called neutral yeasts. Those that give higher quantities are usually referred to as aromatic yeasts; they’re typically used to craft different fermented drinks such as wine which are fruity in style and intended to be drunk early while being young i.e. before the esters degrade. Fermentation temperature is another factor which may impact the quantity and type of esters produced, as well as the degree to which they are retained in the fermented drink. In some embodiments, a high-ester content will usually use an aromatic yeast which ferments the plant-based medium at as cold a temperature as the yeast can tolerate. According to the present inventors, cooler temperatures usually lead to the production of fewer esters than warmer fermentations, however, more esters seem to be retained during and after fermentation at cooler temperatures according to the present invention.

[0129]

[0116] It is anticipated that aeration of the fermentation mixture can improve the overall health of microorganisms, such as yeast and bacteria during fermentation and also the quality of the mixture. In some embodiments of the present invention, the method further comprises the step of aeration. In some embodiments of the present invention, aeration comprises the step of passing sterile air continually over the fermentation mixture. It is contemplated that aeration can influence for example the sensory, tactile and taste / flavour characteristics of the fermenting mixture. In some embodiments of the present invention, the method comprises the step of passing sterile air continually over the fermentation mixture at a rate of between 8 to 20 L / minute.

[0130]

[0117] Further it is anticipated that micro-oxygenation may improve the overall health of microorganisms, such as yeast and bacteria during fermentation. It is further suggested that micro-oxygenation can lead to improved colour intensity and stability while also helping in the development of more complex organoleptic characteristics, a reduction in off-odours such as sulphur. Moreover, it is anticipated that micro-oxygenation can enhance the ability to mimic the reactions that occur during ageing of the fermenting drink such as for example oak-aging.

[0131]

[0118] In some embodiments of the present invention, the method further comprises the step of micro-oxygenation of the fermenting mixture. As used herein, the term “micro-oxygenation” means introducing an amount of oxygen such as a measured amount of oxygen into the fermented non-alcoholic drink. In some embodiments, method of the present invention further comprising the step of micro-oxygenation of the fermenting mixture. In some embodiments, micro-oxygenation can influence the sensory, tactile and taste / flavour parameters of the fermented non-alcoholic drink. It would be appreciated that temperature, total and free SO2, taste and malolactic fermentation must be controlled. The measurement of free and total SO2has been found to be an effective tool for monitoring microoxygenation of fermented alcoholic drinks such as wine.

[0132]

[0119] Example micro-oxygenation systems and equipment include but are not limited to Stavin Ox Box.

[0133]

[0120] In some embodiments the methods of the present invention rely on propagating microbial cultures in a commercial or large-scale manufacturing facilities. In some embodiments, the methods of the present invention rely can rely on fermentation systems consisting of large fermentation tanks, bioreactors, large-scale cooling and large- scale purification systems.

[0134]

[0121] In some embodiments of the present invention, the fermenting mixture is treated to stop fermentation when the level of sugars is between about 15% and 0.5%, the level of lactic acid is between about 1% and 0.2%, the level of acetic acid is between about 1% and 0.2%, the level of gluconic acid is between 0.5% and 0.1%, and the level of glucuronic acid is between about 0.05% and 0.01%.

[0135]

[0122] It has been observed by the present inventors that ongoing fermentation occurs in the fermenting mixture which can lead to the production of increased levels of for example flavour or organoleptic properties spoiling compounds and substances such as alcohol, carbon dioxide and acids such as acetic acid. Therefore, ongoing fermentation must be stopped after, such as, soon or immediately after, the preferred flavour characteristics and organoleptic properties of the fermentation mixture have been reached. In some embodiments, treating the fermenting mixture leads to stabilisation of the fermented drink. In some embodiment, treating the fermenting mixture substantially eliminates or completely prevents the production of spoiling compounds and molecules such as alcohol, carbon dioxide and acids such as acetic acid, enzyme activity, after completion of the fermentation and the preferred flavour characteristics and organoleptic properties of the fermentation mixture have been reached.

[0136]

[0123] It is anticipated that treating the fermenting mixture such as for example to remove the microbial culture and other unwanted particles, sediment and unwanted molecules plays an important enhancing clarity, stability, and overall sensory attributes of the resulting non-alcoholic fermented drink.

[0137]

[0124] In some embodiment, treating the fermenting mixture removes the microbial culture such as the symbiotic mixture of at least one type of yeast and at least one type of bacteria. In some embodiment, treating the fermenting mixture removes unwanted particles, sediment and other unwanted molecules.

[0138]

[0125] Different equipment and techniques for removing microbial culture from a liquid would be familiar to the skilled person such as centrifugal separation, filtration techniques such as depth filtration, plate filtration, cross-flow filtration, diatomaceous filtration, membrane filtration and combinations thereof.

[0139]

[0126] In some embodiments, the fermenting mixture is treated to stop fermentation when the fermenting mixture contains < 1.2% alcohol (ABV). In some embodiments, the fermenting mixture is treated to stop fermentation when the fermenting mixture contains < 0.5% alcohol (ABV). In some embodiments, the fermenting mixture is treated to stop fermentation when the fermenting mixture contains < 0.25% alcohol (ABV). In some embodiments, the fermenting mixture is treated to stop fermentation when the fermenting mixture contains < 0.1% alcohol (ABV). In some embodiments, the fermenting mixture is treated to stop fermentation when the fermenting mixture contains < 0.0% alcohol (ABV).

[0140]

[0127] In some embodiments, treating the fermented mixture comprises rapidly cooling the fermented mixture to between about 2°C to 7°C, preferably between about 2.5°C to 6.5°C, preferably between about 3°C to 6°C, preferably between about 3.5°C to 5.5°C, preferably between about 4°C, to stop or cease fermentation.

[0141]

[0128] In some embodiments, the treated non-alcoholic fermented drink is subjected to fdtration such as large-scale filtration.

[0142] Methods of incubating a Microorganism culture

[0143]

[0129] In some embodiments, the microbial mixture is conducted at temperature such as first incubation temperature (ITc) and / or second incubation temperature (2Tc) in the range of 20 °C to 40 °C.

[0144]

[0130] In some embodiments, the first incubation temperature (ITc) can be the same as the second incubation temperature (2Tc).

[0145]

[0131] In some embodiments of the present invention, the first incubation temperature (ITc) is in the range of between about 20°C to 37°C, preferably between about 22°C to 35°C, preferably between about 24°C to 32°C, preferably between about 26°C to 30°C. In some embodiments of the present invention, the second incubation temperature (2Tc) is in the range of between about 20°C to 37°C, preferably between about 22°C to 35°C, preferably between about 24°C to 32°C, preferably between about 26°C to 30°C.

[0146]

[0132] It would be appreciated that when generating a propagated starter culture, ITc incubation temperature may vary depending on for example the type of growth medium, the microbial mixture, duration of incubation, the concentration of carbon course and others. By way of non-limiting example, where the growth medium contains relatively high level of carbon source such as sucrose of about 25 g / L the ITc can be reduced and the duration of incubation can be increased. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 26°C to 29°C for about 14 weeks. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 26°C to 28°C for about 12 weeks. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) of around 26°C to 28°C for about 10 weeks.

[0147]

[0133] In some embodiments, the microbial inoculum is incubated at the first incubation temperature (ITc). In some embodiments, the inoculated microbial mixture is incubated at the first incubation temperature (ITc). In some embodiments, the propagated starter culture is incubated at the first incubation temperature (ITc). In some embodiments, the propagated starter culture comprises a symbiotic mixture of at least one type of yeast and at least one type of bacteria. In some embodiments, the propagated starter culture comprises a symbiotic mixture of at least two types of yeast and at least two types of bacteria.

[0148]

[0134] In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 2 weeks and about 5 weeks. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 1 to about 5 weeks or more. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least 2 weeks. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 3 weeks. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 3.5 weeks. In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for about 4 weeks or more. In some embodiments, the fermentation mixture is incubated at a second incubation temperature (2Tc). In some embodiments, the fermentation mixture is incubated at a second incubation temperature (2Tc) for between about 1 and about 6 weeks or more. In some embodiments, the fermentation mixture is incubated at a second incubation temperature (2Tc) for between about 2 and about 5 weeks.

[0149]

[0135] One of the steps of the method described herein involves treating such as cooling of a microorganism culture. One of the steps of the method described herein involves treating such as cooling of the fermenting mixture. In some embodiments the cooling is carried out on a microbial culture such as the fermenting mixture incubated under second incubation temperature (2Tc). In some preferred embodiments the cooling is carried on a microbial culture such as the fermenting mixture, incubated under the second incubation temperature (2Tc) as per step f. of the method.

[0150]

[0136] According to the method described herein, a step of cooling of a microorganism culture, such as the fermenting mixture, during a second incubation temperature (2Tc) leads to cessation of growth of the microorganism culture. In some embodiments, the step of cooling of a microorganism culture, such as the fermenting mixture, during a second incubation temperature (2Tc) leads to cessation of growth of the microorganism culture, such as the fermenting mixture, and fermentation itself.

[0151]

[0137] It is contemplated that cessation of growth of the microorganism culture leads to cessation of fermentation and concomitant preservation of the desired sensory, tactile and taste / flavour parameters, attribute and factors of the fermented drink. In some embodiments of the present invention, cessation of growth of the microorganism culture leads to cessation of fermentation and concomitant preservation of the desired taste and flavour parameters of the nonalcoholic fermented drink. In some embodiments of the present invention, cessation of growth of the microorganism culture leads to cessation of fermentation and concomitant preservation of the desired taste and flavour parameters of the non-alcoholic fermented drink.

[0152] Quorum sensing

[0153]

[0138] Without wishing to be bound by theory, quorum sensing is generally considered to represent a response to fluctuations in cell-population density. Quorum sensing microorganisms for instance yeast and bacteria, produce and release chemical signal molecules generally called autoinducers that increase in concentration as a function of cell density. The detection of a minimal threshold stimulatory concentration of an autoinducer leads to an alteration in gene expression. Microorganism such as yeast (e.g. Saccharomyces cerevisiae and Candida albicans) and bacteria such as Gram-positive and Gram-negative bacteria use quorum sensing communication circuits to regulate a diverse array of physiological and physicochemical metabolic activities and processes. These processes include for example symbiosis, virulence, switch between yeast to hyphal form, competence, conjugation, antibiotic production, motility, sporulation, metabolite utilisation, acid production and biofilm formation.

[0154]

[0139] In general terms and without wishing in any way to be bound by theory, in yeast such as Saccharomyces cerevisiae it is suggested that 2-phenylethanol, tyrosol, and tryptophol are the main quorum sensing molecules while Candida albicans are known to produce farnesol, famesoic acid and tyrosol. In bacteria on the other hand, such as Gram-negative bacteria use acylated homoserine lactones as autoinducers while Gram-positive bacteria use for example processed oligo-peptides to communicate. Recent advances in the field indicate that cell-cell communication via autoinducers occurs both within and between different yeast and bacterial species.

[0155]

[0140] It is believed that when the microbial cultures of the present invention are propagated according to the method of the present invention, the propagated microorganisms, particularly yeast and bacteria such as a symbiotic mixture of at least one type of yeast and at least one type of bacteria, due to the continuous parameter monitoring aiming to maintain certain physico-chemical parameters at a desired level, the metabolic processes of the microorganisms can be controlled in such a manner so as to effectively control the microorganisms to release defined levels of favourable or even the desirable microbial metabolites. In particular, when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, particularly yeast and bacteria such as a symbiotic mixture of at least one type of yeast and at least one type of bacteria, due to the continuous parameter monitoring aiming to maintain certain physico-chemical parameters at a desired level, the metabolic processes of the microorganisms can be controlled to influence certain sensory, tactile and taste / flavour parameters, attributes or factors of the fermented drink.

[0156]

[0141] By exposing the yeast and bacteria microorganism cultures such as the symbiotic mixture of at least one type of yeast and at least one type of bacteria, due to the continuous parameter monitoring aiming to maintain certain physico-chemical parameters at a desired level, the metabolic processes of the microorganisms can be controlled to lead to maximisation of the desired sensory, tactile and taste / flavour parameters, attributes or factors of the fermented drink.

[0157] Propagated starter culture and uses thereof

[0158]

[0142] In general, the present invention provides a method for producing propagated microbial starter culture which can be used in fermentation. In some embodiments, the propagated starter culture which can be used in fermentation. In some embodiments, the propagated starter culture which can be used in fermentation in the production of nonalcoholic fermented drink. In some embodiments, the propagated starter culture comprises viable yeast and bacteria such as a symbiotic mixture of at least one type of yeast and at least one type of bacteria. In some embodiments, the propagated starter culture comprises a symbiotic mixture of at least two types of yeast and at least two types of bacteria. In some embodiments, the propagated starter culture can be used in the production of a food product. In some embodiments, the propagated starter culture can be used in the production of a fermented food product.

[0159]

[0143] In some embodiments, the propagated starter culture can be added directly into an ongoing fermentation process. In some embodiments, the propagated starter culture can be added directly into an ongoing fermentation process in the preparation of non-alcoholic fermented drink. In some embodiments, the propagated starter culture can be added directly into an ongoing fermentation process in the preparation of non-alcoholic fermented drink. In some embodiments, the non-alcoholic fermented drink is a tea based non-alcoholic fermented drink.

[0160]

[0144] In some embodiments, the propagated starter culture is lyophilised. As used herein the term “lyophilised” means preserving the propagated antimicrobial culture by freezing it very quickly and then subjecting it to a vacuum or sublimation to remove the ice. In some embodiments, the lyophilised the propagated starter culture is preserved longterm. In some embodiment the lyophilised propagated starter culture comprises viable microbial cells. In some embodiment the lyophilised propagated starter culture comprises viable yeast and bacteria such as a symbiotic mixture of at least one type of yeast and at least one type of bacteria. In some embodiment, lyophilisation can be used to prepare a dosage form that is to be reconstituted for direct addition into an ongoing fermentation process. In some embodiment, the reconstituted dosage form can be used for direct addition into an ongoing fermentation process in the preparation of non-alcoholic fermented drink. In some embodiment, the reconstituted dosage form can be used for direct addition into an ongoing fermentation process in the preparation of non-alcoholic fermented drink.

[0161] Statistical Analysis

[0162]

[0145] Analysis of different chemical and physico-chemical parameters as described herein, can be carried out in situ e.g. on the fermentation vessel, or remotely using systems and software programs known in the art such as for example IBM SPSS version 25. Variables in measurement and data with skewed distributions can be log-transformed to ensure normality. Comparisons can be performed with t-test, Wilcoxon-Mann- Whitney, and one-way ANOVA as appropriate. Significance was defined as p<0.05. Non-parametric tests were used for comparing ordinal or non-normal variables. Data can be presented as mean standard deviation (mSD).

[0163] Reports and Data Transmission

[0164]

[0146] In some embodiments, the methods and systems disclosed herein further comprise generating one or more reports such as measurement reports of different sensory, tactile and taste / flavour parameters, attributes or factors. In some embodiments, the methods disclosed herein further comprise storing one or more reports. In some embodiments, the methods disclosed herein further comprise transmitting one or more reports. In some embodiments, the report includes information on the capability of a microorganism to propagate and generate a propagated starter culture.

[0165]

[0147] In some embodiments, the report includes information on the capability of a microorganism to ferment suitable substrate for the generation of desirable sensory, tactile and taste / flavour parameters, attributes or factors profile of a non-alcoholic fermented drink. In some embodiments, the report provides recommendations on the selection of suitable symbiotic mixture of at least one type of yeast and at least one type of bacteria, growth medium and in the production of a particular taste or flavour profile of a non-alcoholic fermented drink.

[0166]

[0148] In some embodiments, the report provides recommendations on the timing of treatment of the fermentation mixture. In further embodiments, the test parameters of the present disclosure such as those that are characterised with or associated with certain such as desirable organoleptic, sensory, tactile and taste / flavour parameters, attributes or factors may also be implemented in the form of a recording medium including instructions executable by a computer, such as a program module executed by the computer. Computer-readable media may be any available media that may be accessed by a computer and include both volatile and nonvolatile media and removable and non-removable media. In addition, the computer-readable media may include all computer storage media. The computer storage media includes both volatile and nonvolatile media and removable and non-removable media implemented by any method or technology of storing information, such as a computer readable instruction, a data structure, a program module, and other data. The storage may be in the iCloud.

[0167]

[0149] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non-limiting examples.

[0168] MATERIALS & METHODS and EXPERIMENTAL EXAMPLES

[0169]

[0150] Test parameters / factors considered and monitored when carrying out the methods of the present invention.

[0170]

[0151] 1. Oxygen (Oi) and Carbon dioxide (CO2)

[0171]

[0152] 1.1. Oxygen (O2)

[0172]

[0153] In brewing - Oppb when boiled;

[0173]

[0154] During fermentation - consumed by microbial culture such as symbiotic microbial culture - starts between 2500ppb-3500ppb; drops to 1200ppb after for example 48 hours and end in lOOppb;

[0174]

[0155] Filtration - filtration increases to 200ppb-300ppb; and optionally

[0156] In packaging - can increase up to 1000 ppb.

[0175]

[0157] In one particular example of the present invention, the level of oxygen during brewing is maintained usually at between 30-70ppb,

[0176]

[0158] filtration usually 100-250ppb

[0177]

[0159] post carbonation maybe 150

[0178]

[0160] Post fermentation, reduction of oxygen is desirable.

[0179]

[0161] 1.2. Carbon dioxide (COi)

[0180]

[0162] In brewing - Oppb when boiled - boiled out = low pH;

[0181]

[0163] During fermentation - produced by microbial culture such as yeast constituent of symbiotic microbial culture - starts between 2500ppb-3500ppb; drops to 1200ppb after for example 48 hours and end in lOOppb; and optionally.

[0182]

[0164] In packaging this parameter causes carbonation and product shelf life stability e.g. the non-alcoholic fermented drink such as for example a sparkling non-alcoholic fermented tea drink.

[0183]

[0165] In some examples of the present invention, no carbonation of the product is performed after fermentation.

[0184]

[0166] 2. Factors affecting ester formation

[0185]

[0167] Fermentation temperature, free amino nitrogen, yeast, pitch rate (i.e. incorporation of starter culture), oxygenation, sulphur, sugar dosage, starting pH, fermentation vessel and volume.

[0186]

[0168] 3. Non-exhaustive list of desirable organic acids

[0187]

[0169] Acetic acid (pungent and sharp), gluconic acid, glucuronic acid, citric acid, L-lacric acid, malic acid, tartaric acid, malonic acid, oxalic acid, succinic acid, pyruvic acid, usinic acid, lacti acid (soft and long).

[0188]

[0170] 3.1. Non-exhaustive list of undesirable organic acid intermediate metabolites

[0189]

[0171] Butyric acid (vomit), Capric acid (barnyard), Caproic acid (cheesy), Caprylic acid (rancid), Heptanoic acid (sweaty), Isovaleric (parmesan cheese), Lauric (soapy), Nanoic acid (rancid), succinic acid (sour taste), undecanoic acid (coconut, vanilla).

[0190]

[0172] 4. Impact of temperature

[0191]

[0173] One of the parameters which is monitored during the methods of the present invention is temperature. This parameter is measured and monitored closely during for example incubation of the different microorganisms employed in the present invention. In addition to microbial growth, temperature also impacts for example the constitution of the microbial solution, mineral content and physico-chemical properties and chemical characteristics of different molecules and substances as summarised below in Table 2.

[0192]

[0174] Table 2.

[0193]

[0175] 12. Other factors such as test parameters considered in the methods of the present invention.

[0194]

[0176] In carrying out the methods of the present invention it was observed that the following additional factors could affect the performance of the methods of the present invention and where ever necessary when these characterised additional factors are effectively controlled, different such as desirable non-alcoholic fermented product such as a nonalcoholic fermented drink is obtained.

[0195]

[0177] Such additional factors include but are not limited to for example:

[0196]

[0178] Alcohol - needed to start fermentation and production of acids.

[0197]

[0179] Nitrogen - microbial health (such as symbiotic microbial culture) and growth.

[0198]

[0180] Free Amino Nitrogen - low = slow fermentation, high = off flavours & spoilage

[0199]

[0181] pH - in brewing - high brewing pH will extract tannins; while in fermentation - low pH will stall fermentation and affect yeast health; low pH affect the protein transport chain in yeast cells as one of the main functions of yeast is its ability to transport K+; transporting K+ depends on the electric membrane potential difference generated by proton pumping by the plasma membrane H+-ATPase

[0200]

[0182] sugar concentration

[0201]

[0183] light - e.g. during packaging and storage.

[0202]

[0184] Equipment such as fermentation vessel or vat - The material that the equipment is made of or can have an effect e.g. Copper, can impart ions which may or may not be beneficial. Stainless steel and Glass are known to be the most neutral and generally may not impart any flavour into the brew. Height of fermentation vessels can impact on pressures and can result in over carbonation of liquid and stalled fermentations. Size of fermentation vessels can impact on circulation of nutrients and metabolites affecting both ester formation, yeast health and bacterial conversion of for example ethanol to acids. Depending on the shape of the fermentation vessels, critical difficulties are encountered when volumes reachabout 10,000-12,000 hL. Larger fermentation vessels lead to poor yeast growth, poor diacetyl reduction and poor ester production. The impact of fermentation vessels design on flavour production is principally attributed to increase of carbon dioxide as a result of higher hydrostatic pressure in tall fermentation vessels. Excessive dissolved carbon dioxide usually leads to an inhibition of yeast growth and metabolism, presumably because of the inhibition of essential decarboxylation reactions. As decarboxylation reactions are also necessary for the formation of both fusel alcohols and acetyl-CoA, it is alleged that the effect of carbon dioxide on ester production is due to the inhibition of substrate formation. The inhibitory effect of carbon dioxide on ester production is found in most strains. Infusion vessel can affect the extraction of free amino nitrogen from tea particles. Method of heating and addition order can impact Maillard Reactions and impact both colour i.e. darker colour and caramelisation of flavour.

[0203]

[0185] 13. Chemical Composition

[0204]

[0186] One of the parameters which is monitored and closely monitored during the methods of the present invent is the chemical composition. In Table 3 below is provided a list of exemplary chemical compositions which we assessed and accurately determined and their concentration effectively changed to attain the desired concentrations. In different methods, we treated the fermentation mixtures of the present invention in order to for example stop fermentation when the values below are reached.

[0205]

[0187] Table 3.

[0206]

[0188] 14. Sugar, type of sugar, source of sugar such as plant source used as a carbon source and flavours.

[0207]

[0189] Cane - sweeter aftertaste, fruity aroma; Beet - earthy, oxidised aroma, burnt sugar aftertaste; Brown - caramel, richness; Molasses - Slow fermentation, deep dark colour, funk aromas and flavours; Muscovado - deep caramel flavours; Glucose - higher alcohol production; Fmctose - slower fermentation, maturity and perception of an aged fermented product such as cave age non-alcoholic fermented drink produced by the method of the present invention; Sucrose - sweetness of fermented product such as cave age non-alcoholic fermented drink produced by the method of the present invention; Honey - clean, high TA development, mixed saccharides and others.

[0208]

[0190] 15. Water (e.g. aqueous solution)

[0209]

[0191] Alkalinity - affects the amount of acid required to bring down pH; Calcium - higher ie hard water helps in yeast flocculation (settling); Chlorides - higher increases malty flavours, fullness of flavour and sweetness; Hardness - affects extraction of tea solution - 120< flat and little flavour 10> bitter catechins; produces film on top of tea - Temporary Hardness = calcium & magnesium ie Bicarbonate precipitate when boiled : Permanent Hardness = chlorides and sulphates; Iron - higher ie hard water helps in yeast flocculation (settling) which also helps ester formation; Sodium - enhances roundness; Sulphates - higher produces dryer outcomes and can enhance bitterness; TDS - helps micro metabolism and growth; and Zinc - helps micro metabolism and growth and helps ester formation.

[0210]

[0192] 16. Metabolites

[0211]

[0193] Other metabolites which are also routinely tested, measured and monitored during the fermentation methods of the present invention include for instance one or more of the following: 3,4-Dihydroxyphenylethyleneglycol 3-0- glucuronide; 3,4-Dihydroxymandelate 3-0-glucuronide; Isomer 1 of {E,E}-2,4-Hexadienedial; Isoferuloyl C 1- glucuronide; Isoferuloyl C 1 -glucuronide; 5-Aminopentanal; 1-Deoxymannojirimycin; Isomer 1 of 1-0-Vanilloyl- beta-D-Glucose; cyclic 6-Hydroxymelatonin; 2-Hydroxyisophthalic acid; 1-0,6-0-Digalloyl-beta-D-Glucose; Isomer 1 of 4-Hydroxy-5-(3',5'-dihydroxyphenyl)-valeric acid; Isomer 1 of Trimescic acid; 3-Hexenedioic acid; Salicylic Acid; Isomer 1 of N-Phenylacetylapartic acid; Malonic Acid; Isobutyric Acid / Butonic Acid; Dodecanedioic Acid; Isomer 1 of 9-Oxononamoic acid; Dimethylmalonic acid; Cinnamic Acid / Trans-Cinnamic Acid; 3-Oxodecanoic acid; Isomer 1 of Gallic Acid; beta-(2-Methoxyphenoxy)-lactic acid; 2-Methyl-3-oxopropanoic acid; Hydroxyoctanoic Acid; cis-2,3-Dihydroxy-2,3-dihydro-p-curric acid; Isomer 2 of (R)-2,3 -Dihydroxy-3 -methylpentanoic acid; Isomer 2 of 1- O-Vanilloyl-beta-D-glucose; 4-Hydroxy-5-(3',4'-dihyroxyphenyl)-valeric acid; Isomer 1 of Neuraminic acid; Pilosin; Isomer 1 of 4-0-beta-D-Glucosyl-Esculetin; 2-Dehydropantolactone; (R)-l,2-dimethyl-5,6-dihydroxy- tetrahydroisequinoline; 1 -(beta-D-Ribofuranosyl)-l ,4-dihydomicotinamide; 7,8-Dihydroxanthopterin; 4-Chioro-L- lysine; Isomer 2 of 2-Dechloroethylifosfamide; 4-Hydroxycyclohexylcarboxylic acid; N-(Carbethoxyacetyl)-4-chloro- L-tryptophan; Isomer 2 of (+)-Gallocatechin / (-)-Epigallocatechin; Isomer 1 of Ribonolactone / L-Arabinono-1,4- lactone; 2-Hydroxy-2-hydropyrone-4,6-dicarboxylic acid; Caffeoylmalic acid; isomer 1 of 2D-5-O-Methyl-2,3,5 / 4,6- Pentahydroxy cyclohexanone; (+ / -)-2-(5-Methyl-5-vinyltetrahydrofuran-2-y()propionaldehyde; l,5-Dihydroxy-3- methoxy-2-prenylcanthone; Isomer 2 of Prolyl-Valine; Guaiacol; Parapyruvic acid; L-Anticapsin; Valyl-Histidine; Bisdemethoxycurcumin; Isomer 1 of Deoxyrubroskyrin; and Isomer 1 of 2-Ethyl-3,4-dihydroxyfuran.

[0212]

[0194] 17. Detection methods, test equipment, assays and techniques

[0213]

[0195] Although different detection methods, test equipment, assays and techniques would be known to the skilled person in the art, in carrying out the methods of the present invention for the purposed of testing, measuring and monitoring different metabolites, physico-chemical parameters, chemical compounds and molecules such as organic acids, which can be assessed continuously, concomitantly or separately in real time, rely on some of the following assays and methodologies - Ultraviolet (UV) spectrophotometry, Nuclear magnetic resonance (NMR), capillary electrophoresis, thin-layer chromatography, gas chromatography (GC), and liquid chromatography (LC).

[0214]

[0196] Table 6. Below describes some of the methodologies, techniques and assays which were used in the present methods however others may also be known to the skilled person and these other methodologies, techniques and assays may also be used for the purposes of the present invention provided they recognised to provide comparable level of data confidence:

[0215]

[0197] Table 6. Example methodologies, techniques and assays.

[0216]

[0198] 18. Taste and chemical compound or mixtures.

[0199] Although different measurements and parameters would be known to the skilled person in the art in determining or characterising different organoleptic properties such as taste and flavour, in carrying out the methods of the present invention for the purposed of determining the different for example tastes the present inventors relied on testing, identifying and if necessary quantifying some of the following taste and the associated with that particular taste chemical compound(s).

[0217]

[0200] Some of these chemical compounds would have desirable taste and their concentration would preferably be increased while others would have undesirable taste and their concentration reduced or completely eliminated. Our chemical compounds with desirable taste and their concentration were selected from one or more of the compounds in the group comprising: aged honey - phenylacetaldehyde; alkaline - sodium bicarbonate; almond - Benzaldehyde, benzylaldehyde; anise - anethole; aniseed or apple - Ethyl Hexanoate; astringent - rutin hydrate; Banana - Isoamyl acetate; barnyard - 4-Methyl Phenol; basmati rice - 2-Acetyl Pyrazine; bitter - denatonium benzoate, sucrose acta- acetate; Blueberry - 2-Ethyl-5,5-dimethyl-l,3-Dioxane; boiled cabbage - methyl thioacetate; bready, fruity oily, grape - Ethyl caprate, Ethyl decanoate; Bromophenol - 2-Bromophenol; brown sugar - sotolon; Burnt Coffee - 2- Furfurylthiol; burnt sugar - furaneol; butyric - butyric acid; caprylic - otanoic acid; caraway seed - D-Carvone; cardamom - terpinyl acetate; catty - 4-Mercapto-4-Methylpentan-2-one, P-methane-8-Thiol-3-one; cedar - betacaryophyllene; cherry - benzyl acetate; chlorophenol - 2,6-Dichlorophenol; cinnamon - cinnamaldehyde; citrus, fruity, floral - Linalylacetate; citrusy, waxy - decanal; clove - eugenol; clove, chemical, plastic - Amyl octanoate; coconut - gamma-nonalactone, whisky lactone; cucumber - trans, CIS-2, 6-nonenal; damasconone - beta-damascenone; dark chocolate - 2,3,5-Trimethylpyrazine; diacetyl - 2,3-Butanedione; dill - d-carvone; DMS, sweat com - dimethyl sulphide; earthy -compost - 2-Methylisobomeol; earthy, damp soil - ethyl fenchol; earthy, green pepper - 2-Isobutyl-3- methoxypyrazine; earthy, potato skins - 2-Isopropyl-3 -Methoxypyrazine; exotic fmits - Ethyl 3 -methylthio propionic acid; fennel - estragole; Floral - 2-phenyl ethyl acetate; floral, citrus - beta-citronellol; floral, flowery - geraniol; floral, fruity - benzyl acetate; floral, fruity, berry - p-Ionone; floral, fruity, citrus - linalyl acetate; floral, sweet - Phenylethanol; floral, sweet, fruity - geranyl acetate; floral, violet - beta-ionone; formed from valine and isoleucine - vicinal diketones; freshly cut grass - CIS-3 -Hexenol; fruity - Isobutyl acetate; fruity, creamy - 6-undecalactone; fruity, creamy, gum - Ethyl lactate; fruity, floral(raspberry) - 4-(p-hydroxyphenyl)-ethyl 2-butanon; fruity, pear - ethyl cis-4-deca-dienoic acid; fruity, refreshing - acetaldehyde diethyl acetal; fruity, sweet, berry, artificial grape - Ethyl isovalerate; fruity, tutti- fuity - hexyl acetate; fruity, pineapple - 3 -methylthio pro-pionic acid tester; geosmin - geosmin; grainy - isobutyraldehyde, isovaleraldehyde; grapefruit - 3 -Mercaptohexanol; grapefruit, bitter, fruity - 2-Aminoacetophenone, Nootkatone; grassy - cis-3 -hexenol, trans-2-hexenal; green banana - CIS-3-Hexenyl acetate; green, earthy - 2-sec.butyl- 3-methoxy-pyrazine; green, floral - 2-pentyl-4,5-dimethyl-thiazole; green, fresh - trans-2-hexenal; green, grassy - cis- 3-hexenol; green, leafy - cis-3 -hexenal; green, piney - alpha-pinene, hexyl 2-methylbutyrate; hay - nonanal; hazelnut - 5-Methyl-2-Hepten-4-one; herbaceous, sweet-medicinal, warm, spicy, thyme - thymol; herbaceous, sweet, anise - trans-anethole; honey - phenylthyl acetate, ethyl phenylacetate; honey, butter, caramel - pentanedione; hop bitterness - iso-alpha-acids; leathery - 6-Isobutylquinoline as well as others.

[0218]

[0201] 19. Example microbial mixtures such as Symbiotic Microbial mixtures and methods.

[0219]

[0202] Example 1 - results are summarised in Figures 1, 2 and 3.

[0220]

[0203] Symbiotic Microbial Mixture 1 - comprising as the yeast constituent Saccharomyces spp. and the bacterial constituent is Acetobacter spp. The data presented in Figure 1 represents a viable cell numbers of yeast Saccharomyces spp. and bacteria Acetobacter spp. during fermentation. The data presented in Figure 2 represents TA development (grams / volume / day) over duration of fermentation step. In this particular example using Symbiotic Microbial Mixture 1 a value around 5 represented the desired physicochemical distribution of the test parameters. Figure 3 represents flavour intensity over time. In more detail, when Symbiotic Microbial Mixture 1 was employed perceived acidity, ester intensity and flavour of the solution such as tea flavour was measured and maintained at the desired levels in the nonalcoholic fermented drink such as for example sparkling non-alcoholic fermented tea drink.

[0221]

[0204] Example 2 - results are summarised in Figures 4, 5 and 6.

[0205] Symbiotic Microbial Mixture 2 - comprising as the yeast constituent two distinctly different yeast species selected from Saccharomyces spp. and Zygosaccharomyces spp while the bacterial constituent was Acetobacter spp. The data presented in Figure 4 represents viable cell numbers of yeast Saccharomyces spp. and Zygosaccharomyces spp while the bacterial constituent / as, Acetobacter spp. during fermentation. The data presented in Figure 5 represents TA development (grams / volume / day) over duration of fermentation step. In this particular example using Symbiotic Microbial Mixture 2 a value above 4 represented the desired physicochemical distribution of the test parameters. Figure 6 represents flavour intensity over time. In more detail, when Symbiotic Microbial Mixture 2 was employed perceived acidity, ester intensity and flavour of the solution such as tea flavour was measured and maintained at the desired levels in the non-alcoholic fermented drink such as for example sparkling non-alcoholic fermented tea drink.

[0222]

[0206] Example 3 - results are summarised in Figures 7, 8 and 9.

[0223]

[0207] Symbiotic Microbial mixture 3 - comprising as the yeast constituent two distinctly different yeast species selected from Saccharomyces spp. and Zygosaccharomyces spp while the bacterial constituent contained two distinctly different bacterial species selected from Acetobacter spp. and Gluconobacter spp. The data presented in Figure 7 represents a viable cell numbers of Saccharomyces spp. and Zygosaccharomyces spp and those of Acetobacter spp. and Gluconobacter spp. during fermentation. The data presented in Figure 8 represents TA development (grams / volume / day) over duration of fermentation step. In this particular example using Symbiotic Microbial Mixture 3 a value above 4 and below 6 represented the desired physicochemical distribution of the test parameters. Figure 9 represents flavour intensity over time. In more detail, when Symbiotic Microbial Mixture 3 was employed perceived acidity, ester intensity and flavour of the solution such as tea flavour was measured and maintained at the desired levels in the non-alcoholic fermented drink.

[0224]

[0208] CONCLUSION

[0225]

[0209] The present inventors surprisingly and unexpectedly observed that each combination of the plant-based nitrogen source, combined with the plant-based carbon source required a different blend of yeast and bacterial strains to result in a successful fermentation. In addition, the present inventors surprisingly and unexpectedly observed that each combination of the plant-based nitrogen source, with the plant-based carbon source required a different concentration of viable cells per ml for each microbial strain used in the fermentation mixture in order to create a symbiosis between the microbial strains in the fermentation mixture.

[0226]

[0210] The present inventors surprisingly and unexpectedly observed that the use of specific yeast and bacterial strains to match the nutrient composition of the specific growth medium, fermented under specific controlled conditions was critical to the production of a non-alcoholic fermented drink with physico-chemical parameters and / or characteristics associated with particular flavour.

[0227]

[0211] The present inventors surprisingly and unexpectedly observed that each growth medium, inoculated with a unique microbial inoculum required a specific level and method of oxygen delivery in order to result in a successful fermentation. In addition, the present inventors surprisingly and unexpectedly observed that for each growth medium, inoculated with a unique microbial inoculum deviation from this oxygen delivery protocol could lead to an overwhelming growth of one or other microbe in the fermentation mixture which would lead to stalled fermentation, off-flavours, and / or lack of flavour development.

[0228]

[0212] The present inventors surprisingly and unexpectedly observed that the nutrient available to the specific yeast and bacterial strains in the fermentation mixture at the beginning of fermentation was critical to the successful outcome of fermentation. In addition, the present inventors surprisingly and unexpectedly observed that the nutrient available to the specific yeast and bacterial strains in the fermentation mixture at the beginning of fermentation was critical to the production of a non-alcoholic fermented drink with physico-chemical parameters and / or characteristics associated with particular flavour.

[0229]

[0213] The present inventors surprisingly and unexpectedly observed that certain combinations of esters produced by yeast fermentation, combined with certain acids produced by bacterial respiration, can produce a non-alcoholic fermented drink with physico-chemical parameters and / or characteristics with refined taste and flavour which can be used as a realistic or even a complete substitute for alcoholic drinks such as for instance wine (e.g. champagne) whereas other combinations of esters and acids do not or may be distasteful.

[0230]

[0214] The present inventors surprisingly and unexpectedly observed that when the specific microbial cultures were propagated as described herein, the propagated microorganisms, for instance the symbiotic culture of yeast and bacteria, as a result of the controlled conditions and substrate, can reduce the amount of alcohol in the fermented drink to a specific and controlled level. In particular, the present inventors surprisingly and unexpectedly observed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, for instance the symbiotic culture of yeast and bacteria for example symbiotic mixtures 1, 2 and 3, as a result of the controlled incubation conditions, substrate and growth medium, can produce a non-alcoholic fermented drink with desirable physico-chemical parameters and / or characteristics with a controlled amount of alcohol below 0.5%

[0231]

[0215] The present inventors surprisingly and unexpectedly observed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, for instance the symbiotic culture of yeast and bacteria, as a result of the controlled conditions, substrate and growth medium, can produce a non-alcoholic fermented drink with physico-chemical parameters and / or characteristics with refined taste and flavour (e.g. esters) which can be used as a realistic or even a complete substitute for alcoholic drinks such as for instance wine (e.g. champagne).

[0232] * * *

[0233]

[0216] Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure claimed. It will also be appreciated that the device(s), method(s), use(s), detector(s), sensor(s), physiological parameters(s) such as test parameters, may be subject to numerous rearrangements, modifications and substitutions without departing from the scope of the present disclosure as set forth and defined by the following claims.

[0234]

[0217] REFERENCES

[0235] Holm T. Aspects of the mechanism of the flame ionization detector. J. Chromatogr. A. 1999;842:221-227. doi: 10.1016 / 80021-9673(98)00706-7.

[0236] Huang M.Q., Wang L., Sun B.G. Analysis of Non-Volatile Organic Acids in Sweet Sauce. Food Sci. 2013;34: 123- 130.

[0237] Talamond P., Gallon G., Treche S. Rapid and sensitive liquid chromatographic method using a conductivity detector for the determination of phytic acid in food. J. Chromatogr. A. 1998;805: 143-147. doi: 10.1016 / S0021- 9673(98)00050-8.

[0238] Birks J.W., Kuge M.C. Chemiluminescent aerosol spray detector for liquid chromatography. Anal. Chem. 1980;52:897-901. doi: 10.1021 / ac50056a028.

[0239] Kotani A., Miyaguchi Y., Tomita E., Takamura K., Kusu F. Determination of organic acids by high-performance liquid chromatography with electrochemical detection during wine brewing. J. Agric. Food Chem. 2004;52:1440- 1444. doi: 10.1021 / jf0306486.

[0240] Zheng Y.J., Duan Y.T., Zhang Y.F., Pan Q.H., Li J.M., Huang W.D. Determination of Organic Acids in Red Wine and Must on Only One RP-LC-Column Directly After Sample Dilution and Filtration. Chromatographia. 2009;69: 1391-1395. doi: 10.1365 / sl0337-009-1085-0. Kuligowski J., Quintas G., Garrigues S., Lendl B., de la Guardia M. Recent advances in on-line liquid chromatography-infrared spectrometry (LC-IR) TrAC Trends Anal. Chem. 2010;29:544-552. doi: 10.1016 / j.trac.2010.03.004.

[0241] Valentao P., Andrade P.B., Areias F., Ferreres F., Seabra R.M. Analysis of vervain flavonoids by HPLC / diode array detector method. Its application to quality control. J. Agric. Food Chem. 1999;47:4579M582. doi: 10.1021 / jf990444i.

[0242] Pang R.L., Fang J.B., Guo L.L., Xie H.Z. Extraction Conditions Optimization of Mai n Organic Acids from Fruits. Sci. Agric. Sin. 2014;47:2625-2633.

[0243] Mato I., Suarez-Luque S., Huidobro J.F. Simple determination of main organic acids in grape juice and wine by using capillary zone electrophoresis with direct UV detection. Food Chem. 2007;102:104-112. doi: 10.1016 / j.foodchem.2006.05.002

[0244] Yang Y., Pan D., Sun Y., Wang Y., Xu F., Cao J. 1H NMR-based metabolomics profiling and taste of stewed porkhock in soy sauce. Food Res. Int. 2019;121:658-665. doi: 10.1016 / j.foodres.2018.12.035.

[0245] Vereda Alonso E., Garcia de Torres A., Rivero Molina A., Cano Pavon J.M. Determination of organic acids in wines. A review. Quim. Anal. Bellaterra. 1998;17:167-176.

[0246] Zhou J.D., Dong M.S., Huang K.H. Determination of water-soluble organic acids and vitamins in juicy peach using HPLC-DAD. J. Nanjing Agric. Univ. 2009;32:151-154.

[0247] Spizzirri U.G., Restuccia D., Curcio M., Parisi O.I., Iemma F., Picci N. Determination of biogenic amines in different cheese samples by LC with evaporative light scattering detector. J. Food Compos. Anal. 2013', 29:43-51. doi: 10.1016 / j.jfca.2012.09.005.

Claims

What is claimed is:

1. A method of producing a non-alcoholic fermented drink, the method comprising the steps of: a. supplying an aqueous solution with a plant-based nitrogen source, combined with a plant-based carbon source material to form a growth medium; b. inoculating the growth medium with a microbial inoculum to form an inoculated mixture; c. incubating the inoculated mixture at a first incubation temperature (ITc) to generate a propagated starter culture containing between about 103- 5xlO10viable cells / ml; d. to the propagated starter culture adding a large scale growth medium consisting of a aqueous solution of a plant-based nitrogen source, combined with a plant-based carbon source to form a fermentation mixture; e. incubating the fermentation mixture at a second incubation temperature (2Tc) for between about 1 and about 5 weeks; and f. treating the fermenting mixture to produce a non-alcoholic fermented drink.

2. A method of producing a non-alcoholic fermented drink according to claim 1, wherein the non-alcoholic fermented drink is a sparkling non-alcoholic fermented drink.

3. A method of producing a non-alcoholic fermented drink according to anyone of claims 1 or 2, wherein the microbial inoculum and the propagated starter culture are obtained from a microbiota or kombucha.

4. A method of producing a non-alcoholic fermented drink according to anyone of claims 1 to 3, wherein the propagated starter culture comprises a symbiotic mixture of at least one type of yeast and at least one type of bacteria.

5. A method of producing a non-alcoholic fermented drink according to anyone of claims 1 to 4, wherein the propagated starter culture comprises a symbiotic mixture of at least two types of yeast and at least two types of bacteria.

6. A method of producing a non-alcoholic fermented drink according to anyone of claims 4 or 5, wherein the yeast is selected from the group of genera comprising Pichia, Saccharomyces, Zygosaccharomyces, Hanseniaspora, Mycotorula, Hanseniaspora, Zygosaccharomyces, Lachancea, Candida, Kazachstania, Kloeckera, Metschnikmvi, Medusomyces, Brettanomyces, Saccharomycodes, Torulopsis, Torulaspora, Schizosaccharomyces and Kluyveromyces and the bacteria is selected from the genera consisting of Leuconostoc, Lactococcus, Acetobacter, Allobacullum, Bifidobacterium, Leuconostoc, Propionobacterium, Ruminococcus, Gluconob acter, Gluconacetobacter, Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Abiotrophia, Aerococcus, Aerosphaera, Agitococcus, Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes, Bavariicoccus, Carnobacterium, Carnococcus, Catellicoccus, Chungangia, Convivina, Desemzia, Dolosicoccus, Dolosigranulum, Enterococcus, Eremococcus, Facklamia, Floricoccus, Fructobacillus, Globicatella, Granulicatella, Ignavigranum, Isobaculum, Jeotgalibaca, Lacticigenium, Lactovum, Lachancea, Marinilactibacillus, Melissococcus, Metschnikowia, Oenococcus, Okadaella, Pilibacter, Pisciglobus, Sharpea, Komagataeib acter, Brevibacterium, Pediococcus, Nguyenib acter, Sporolactobacillus, Tetragenococcus, Torulaspora, Trichococcus, Thermus, Streptococcus, Staphylococcus, Vagococcus and Weissella.

7. A method of producing a non-alcoholic fermented drink according to anyone of claims 4 to 6, wherein the symbiotic mixture comprises Saccharomyces bayanus, Zygosaccharomyces bisporus, Gluconacetobacter europaeus and / or Acetobacter pasteurianus.

8. A method of producing a non-alcoholic fermented drink according to anyone of claims 4 to 6, wherein the symbiotic mixture comprises Saccharomyces luchvigii, Zygosaccharomyces kombuchaensis, Gluconacetobacter ketogenus and / or Acetobacter zylinoides.

9. A method of producing a non-alcoholic fermented drink according to anyone of claims 4 to 6, wherein the symbiotic mixture comprises Saccharomyces apiculatus, Zygosaccharomyces lentus, Komagataeibacter xylinus and / or Acetobacter xylinum.

10. A method of producing a non-alcoholic fermented drink according to anyone of claims 4 to 6, wherein the symbiotic mixture comprises Saccharomyces cerevisiae, Zygosaccharomyces bisporus, Lactobacillus nagelii and / or Gluconacetobacter ketogenus.

11. A method of producing a non-alcoholic fermented drink according to any one of the preceding claims, wherein the plant-based material comprises a combination of one or more plant-based nitrogen sources and one or more plant-based carbon sources.

12. A method of producing a non-alcoholic fermented drink according to claim 11, wherein the plant biomass is used to form a brew solution, and wherein the brew solution is prepared by steeping, dissolving or mixing plant biomass in cold, hot or lukewarm aqueous solution.

13. A method of producing a non-alcoholic fermented drink according to claim 12, wherein the brew solution is prepared by steeping at least 1 g / L tea biomass in aqueous solution, preferably at least 2 g / L tea biomass in aqueous solution, preferably at least 3 g / L tea biomass in aqueous solution, preferably at least 5 g / L tea biomass in aqueous solution, preferably at least 6 g / L tea biomass in aqueous solution, preferably at least 7 g / L tea biomass in aqueous solution, preferably at least 8 g / L tea biomass in aqueous solution, preferably at least 9 g / L tea biomass in aqueous solution, preferably at least 10 g / L tea biomass, preferably at least 20 g / L tea biomass, preferably at least 25 g / L tea biomass in aqueous solution or more.

14. A method of producing a non-alcoholic fermented drink according to claim 13, wherein the brew solution is prepared by steeping the tea plant biomass in an aqueous solution for at least 1 min, for at least 2 min, for at least 5 mins, for at least 10 min, for at least 15 min, for at least 20 min, for at least 30 min, for at least 40 min, for at least 50 min, for at least 60 min, for at least 70 min, for at least 80 min, for at least 90 min, for at least 100 min, for at least 120 min, for at least 130 min, for at least 150 min, for at least 180 min, for at least 4 hrs, for at least 5 hrs, for at least 6 hrs, for at least 7 hrs, for at least 8 hrs, for at least 10 hrs, for at least 12 hrs, for at least 16 hrs or more, to form the brew solution.

15. A method of producing a non-alcoholic tea-based fermented drink according to anyone of the preceding claims, wherein the at the plant-based carbon source delivers a sugar content of between about 1% v / v and about 15% of the growth medium.

16. A method of producing a non-alcoholic fermented drink according to anyone of the preceding claims, wherein the first incubation temperature (ITc) is in the range of between about 20°C to 37°C, preferably between about 22°C to 35°C, preferably between about 24°C to 32°C, preferably between about 26°C to 30°C.

17. A method of producing a non-alcoholic fermented drink according to anyone of the preceding claims, wherein the second incubation temperature (2Tc) is in the range of between about 20°C to 37°C, preferably between about 22°C to 35°C, preferably between about 24°C to 32°C, preferably between about 26°C to 30°C.

18. A method of producing a non-alcoholic fermented drink according to anyone of the preceding claims, the method further comprising the step of oxygenation of the fermenting mixture.

19. A method of producing a non-alcoholic fermented drink according to anyone of claims 1 to 18, the method further comprising measuring the level of at least one test parameter selected from sucrose, tannin, esters, polyphenols (nitrogenous compounds), minerals, alcohol content and acidity and alcohol (ABV).

20. A method of producing a non-alcoholic fermented drink according to anyone of the preceding claims, wherein treating the fermented mixture comprises rapidly cooling the fermented mixture to between about 2°C to 8°C, preferably between about 2.5°C to 7°C, preferably between about 3°C to 6.5°C, preferably between about 3.5 °C to 6°C, preferably about 7°C, to stop fermentation.

21. A method of producing a non-alcoholic fermented drink according to claim 20, wherein the treated nonalcoholic fermented drink is subjected to filtration.

22. A propagated starter culture obtained according to the method of any one of claims 1 to 21.

23. A propagated starter culture according to anyone of claims 22, wherein the propagated starter culture is lyophilised.

24. A propagated starter microbial culture according to anyone of claims 22 or 23, for use in fermentation.

25. A propagated microbial culture according to anyone of claims 22 to 24, for use in producing a food product or a non-alcoholic fermented drink.

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