A method of producing single cell protein from whey

Abstract

A method of producing single cell protein SCP or biomass comprises culturing microbial inoculum in a growth medium comprising dairy-derived whey. The method may comprise (i) inoculating growth medium

Description

INTRODUCTION [1] According to the Food and Agriculture Organization of the United Nations, the world’s population is projected to reach 9.7 billion by 2050. It is widely considered therefore that food sources would soon present a serious challenge. Malnutrition also continues to present a growing concern which needs urgent and concerted attention. These problems need immediate attention and effective long term solutions. [2] Food’s nutritional value, sometimes referred to as protein quality, relies on its amino acid content and the utilisation of specific amino acids. Concentrations and ratios of amino acids affect the quality of particular proteins and peptides, and the biological quality' or nutritional benefits is greater if the proportion of indispensable amino acids such as for instance histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine is greater. [3] Alternative proteins and peptides may be developed as a replacement for conventional proteins. These alternatives may be specifically designed to require less technologically intensive and thus cheaper production methodologies. Examples of such alternative proteins include for example microbial proteins, microbial peptides, microbial biomass, insect-based proteins, cell-based meat, plant-based meat substitutes and also of course dairy alternatives. Microbial proteins are colloquially referred to as single-cell protein (SCP) which can be are derived from different microorganisms such as microalgae, fungi, yeast, or bacteria. [4] The term ■’single-cell protein” was coined in 1966 by Professor Carroll Wilson, Massachusetts Institute of Technology (MIT). SCP represents microorganism biomass or protein extract that can be used in animal and human nutrition alike. Besides proteins and peptides, single cell protein products may contain free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Single-cell proteins are utilised mainly as protein-rich or enhanced food supplements or ingredients for human and animal nutrition alike. Production of single-cell proteins for animal feed is intimately connected to animal fanning, husbandry and agriculture which allows the exploitation of previously inedible materials such as waste materials and increases resource efficiency. 15 ] Milk products and particularly cheese is one of the most valuable food “sources” for humans (Fox et al., 2017). In recent years the dairy industry' has seen an ever increasing production of greater quantities and different types of cheese from dairy and dairy alternative courses which is inevitably accompanied by large amount of waste such as whey as a by- or co-product material. The disposal of dairy industry waste such as whey has led to considerable environmental problems due to its high organic matter content (Zandona et al., 2021). [6] Therefore, there exists a real need to develop not only an appropriate process for long term production of SCP but also the need to impactfully reduce the environmentally negative impact of waste products released by the dairy industry'. [7] The above problems are at least in part solved by the present invention as described below. BRIEF SUMMARY OF THE INVENTION [8] In the most broadest sense, the present invention is generally concerned with the field of microbial fermentation of dairy or dairy alterative industry waste material and microbial protein production such as microbial biomass or single cell protein (SCP). The present invention is further concerned with a method of identifying a strain of microorganism or mixture of strains of microorganisms, capable of effectively utilising dairy-derived waste such as whey, as a food source to produce microbial biomass as a source of SCP. 19] The present invention is therefore generally directed to a method of producing microbial biomass or single cell protein (SCP) which method comprises culturing microbial inoculum in a growth medium comprising dairy-derived waste material. The present invention is directed to a method of producing microbial biomass or single cell protein (SCP) which method comprises culturing microbial inoculum in a growth medium comprising dairy-derived whey. The present invention is also therefore directed to a method of identifying a strain of microorganism or mixture of strains of symbiotic microorganisms, capable of effectively utilising dairy-derived waste such as whey, as a food source to produce microbial biomass as a nutritional source of SCP for humans and animals.

[10] According to one aspect, the present invention provides a method of producing single cell protein (SCP) which method comprises culturing microbial inoculum in a growth medium comprising daily--derived whey.

[11] According to some embodiments, the method of the present invention further comprises: i) inoculating growth medium comprising dairy-derived whey with a microbial inoculum to form an inoculated microbial mixture; ii) incubating the inoculated microbial mixture at a first incubation temperature (ITc) to generate a propagated starter culture containing between about 103 - 5x10" cells / ml; iii) adding the propagated starter culture to a large scale growth medium comprising dairy-derived whey to form a fermentation mixture and incubating the fermentation mixture; and optionally iv) treating the fermentation mixture to produce a SCP.

[12] In some embodiments, the method of the present invention comprises incubating the fermentation mixture at a second incubation temperature (2Tc). In some embodiments, the method of the present invention comprises incubating the fermentation mixture at 2Tc for at least 24 hrs, at least 36 hrs, at least 48 hrs, at least 72 hrs or more.

[13] 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 from dairy industry microorganisms. 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), the Bulgarian National Collection of Microorganisms and Cell Cultures, and LB Bulgaricum EAD. Furthermore, different species of the microbial inoculum can be obtained from isolated viable microbial stocks or as viable predetermined mixed species microbial cultures.

[14] In some embodiments of the present invention, the propagated starter culture comprises at least one of the strains in the microbial inoculum selected from microalgae, a yeast, a fungi or bacteria

[15] In some embodiments of the present invention, the propagated starter culture comprises a symbiotic mixture of at least one microalgae, at least one fungi, at least one yeast and at least one bacteria or a combination thereof. In some embodiments of the present invention, the propagated starter culture comprises a symbiotic mixture of at least two strains of microalgae, at least two strains of fungi, at least two strains of yeast and at least two strains of bacteria. In some embodiments of the present invention, the propagated starter culture comprises a symbiotic mixture of at least three strains of microalgae, at least three strains of fungi, at least three strains of yeast and at least three strains of bacteria.

[16] The present method is perfectly suited for producing a propagated starter culture comprising a symbiotic mixture of microalgae, a yeast, a fungi or bacteria that contains different organic compounds or metabolites such as physico-chemical parameters, which unless states otherwise, can be readily measured and quantified with standard laboratory techniques.

[17] In some embodiments of the present invention, the microalgae is selected from the group of genera comprising Anabaena, Bortycossus, Chaetoceros, Chlorella, Dunaliella, Haematocissus, Isocgrysis, Nannochloropsis, Nostoc, Nodontella, Oscillatoria. Chamudomonas, Parachlorella, Arthrospira. Porphyridium, Rhodomonas, Phaeodactylum, Scenedesmus and Tetraselmis.

[18] In some embodiments of the present invention, the fungi is selected from the group of genera comprising Fusarium, Paecilomyces, Rhizobium, Aspergillus, Cladosporidium, Monascus. Penicillium, Trichoderma and Altemaria.

[19] In some embodiments of the present invention, the yeast is selected from the group of genera consisting of 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, Yarrowia and Kluyveromyces.

[20] In some embodiments of the present invention, the yeast is selected from the group of yeast species comprising 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.

[21] In some embodiments of the present invention, the bacteria is selected from the group of general group consisting of Lactococcus, Leuconostoc. Allobacullum, Bifidobacterium, Propionobacterium, Ruminococcus, Ghiconobacter, Gluconacetobacter, Lactobacillus, Pediococcus, Lactococcus, Streptococcus. Abiotrophia, Aerococcus, Aerosphaera, Agitococcus, Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes. Bavariicoccus, Camobacterium, Camococcus, 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, Sharped, Komagataeibacter. Brevibacterium, Pediococcus, Nguyenibacter, Sporolactobacillus, Tetragenococcus, Torulaspora, Trichococcus, Thermus, Streptococcus, Staphylococcus, Vagococcus and Weissella.

[22] In some embodiments of the present invention, the symbiotic mixture comprises at least one of the strains of Bortycossus braumi, Pichia pastoris, Aspergillus ochraceus or Lactobacillus delbruckii.

[23] In some embodiments of the present invention, the symbiotic mixture comprises Bortycossus braunii, Pichia pastoris, Aspergillus ochraceus and Lactobacillus delbruckii.

[24] In addition, the present invention can be carried out using food grade materials such as dairy materials. Preferably, the present invention is carried out using waste food grade materials such as waste dairy materials. In addition, the present invention can be carried out using food grade materials such as dairy alternative material. Preferably, the present invention can be carried out using waste food grade materials such as waste dairy alternative material. In addition, the present invention can be carried out using food grade materials such as plant-based materials. Preferably, the present invention can be carried out using waste plant-based materials. In addition, the present invention can be carried out using a combination of waste dairy materials, waste dairy alternative material and waste plant-based materials.

[25] Furthermore, the present invention can be carried out using whey as a by- or co-product material of the dairy industry'. In some embodiments of the present invention, dairy derived whey is growth medium. In some embodiments of the present invention, dairy alternative material is growth medium. In some embodiments of the present invention, plant-based material is comprised in the growth medium.

[26] In some embodiments of the present invention, the solids content of die growth medium comprises at least 30% by weight of lactose, preferably at least 40% by weight of lactose, preferably at least 50% by weight of lactose or more.

[27] In some embodiments of the present invention, the solids content of the whey waste comprises at least 30% by weight of lactose, preferably at least 40% by weight of lactose, preferably at least 50% by weight of lactose or more.

[28] In some embodiments of the present invention, microbial inoculum is added to the growth medium.

[29] In some embodiments, the addition of the microbial mixture to the growth medium forms an inoculated microbial mixture.

[30] In some embodiments of the present invention, microbial substrate is added to the growth medium. In some embodiments of the present invention, microbial substrate is added to the inoculated microbial mixture.

[31] In some embodiments of the present invention, the microbial substrate comprises at least one organic compound such as at least one organic compound selected from the group of lactose, glucose, sucrose, fructose, galactose, maltose, raffinose or combinations thereof. In some embodiments of the present invention, the growth medium is an aqueous solution.

[32] In some embodiments of the present invention, the microbial substrate comprises at least one organic compound such as at least one organic compound selected from the group of lactose, glucose, sucrose, fructose, galactose, lactose, maltose, raffinose or combinations thereof. In some embodiments of the present invention, at least one organic compound is selected from the group of lactose, glucose, sucrose, fructose, galactose, maltose, raffinose, 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.

[33] In some embodiments of the present invention, the first incubation temperature (1 Tc) is in the range of is in the range ofbetween about20°C to 38°C, preferably between about 22°C to 37°C, preferably between about 24°C to 36°C, preferably between about 26°C to 35°C.

[34] In some embodiments of the present invention, the method further comprises measuring the level of at least one test parameter selected from pH, lactose, sucrose, esters, polyphenols, minerals, minerals, lactic acid, citric acid, ascorbic acid, acetic acid, glucuronic acid and amino acids.

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

[36] In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation when predetermined criteria are reached. In some embodiments of the present invention, the predetermined criteria comprise a level of biomass yield. In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation when the predetermined criteria reach a level of biomass yield greater than 20% w / w, preferably greater than 30% w / w, preferably greater than 40% w / w, preferably greater than 50% w / w, preferably greater than 60% w / w, preferably greater than 70% w / w, preferably greater than 80% w / w or more.

[37] In some embodiments of the present invention, the fermenting mixture is treated to stop fermentation when the level of lactose is between about 3% and 1%.

[38] In some embodiments of the present invention, the fermentation mixture is lyophilised.

[39] In some embodiments of the present invention, the fermentation mixture is subjected to further treatment. In some embodiments of the present invention, treating the fermented mixture comprises 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. In some embodiments of the present invention, treating the fermented mixture comprises rapidly cooling the fermented mixture to stop fermentation.

[40] In some embodiments the fermentation mixture comprises the microbiota, microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals.

[41] 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. Preferably, treatment of the fermented mixture does not adversely affect or modify the physico-chemical properties or test parameters of the composition. Preferably, treatment of the fermented mixture does not adversely affect or modify the organoleptic properties of the composition. Preferably, treatment of the fennented mixture does not adversely affect or modify the microbial biomass, SPC, free single ammo acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals such as calcium of the composition.

[42] Preferably, treatment of the fennented mixture enhances the organoleptic properties of the composition. Preferably, treatment of the fermented mixture enhances the microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals such as calcium of the composition.

[43] In one aspect of the present invention, there is provided a propagated starter culture. In some embodiments of the present invention, the propagated starter culture is lyophilised. In some embodiments of the present invention, the propagated starter culture is used in fermentation. In some embodiments of the present invention, the propagated starter culture is used in producing a food product or SCP. In some embodiments of the present invention, the propagated starter culture is used in producing a function food.

[44] In one aspect of the present invention, there is provided a use of dairy-derived whey waste as a food source for a microorganism which microorganism is a source of single cell protein.

[45] In one aspect the present invention, there is provided a composition comprising single cell protein (SCP) obtained from microorganisms that have been cultured in a medium comprising dairy-derived whey w aste. In some embodiments, there is provided a product or supplement comprising the composition of the present invention.

[46] In one aspect the present invention, there is provided a method of identifying a strain of microorganism, or mixture of strains of microorganisms, capable of efficiently using dairy-derived whey waste as a food source to produce biomass as a source of single cell protein, which method comprises cultivating one or more microorganisms in a cell culture medium comprising dairy-derived whey waste and selecting a microorganism, or mixture thereof, based on one or more predetermined criteria that indicate efficient use of the whey waste to produce said biomass, such as growth rate, biomass yield, overall nutritional content, protein content and / or resistance to contaminants. In some embodiments, the predetermined criteria include biomass yield. In some embodiments, the predetermined criteria include biomass yield greater than 20% w / w, preferably greater than 30% w / w, preferably greater than 40% w / w, preferably greater than 50% w / w, preferably greater than 60% w / w, preferably greater than 70% w / w, preferably greater than 80% w / w or more. In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation when the predetermined criteria reach a level of biomass yield greater than 50% w / w.

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

[48] 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, as a result of the controlled conditions and substrate, can produce enhanced quantities of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. 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, as a result of the controlled conditions, substrate and growth medium, can produce an increased quality of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals with particular physico-chemical parameters and / or characteristics. The present inventors also surprisingly and unexpectedly observed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, as a result of the controlled conditions, substrate and growth medium, can produce microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, 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, as a result of the controlled conditions, substrate and growth medium, can provide a long term supply of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, 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, as a result of the controlled conditions, substrate and growth medium, can provide a suitable functional food supplement of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, the present inventors surprisingly and unexpectedly observed that the produced by the methods of the present invention, SCP has further beneficial properties in that it offers functional protein properties characterised with greater gain in muscle mass in the subjects following consumption. BRIEF DESCRIPTION OF THE DRAWINGS

[49] FIG. 1. Schematic of the methods described here showing culture medium (such as whey), mixing rotor, CO2 source, photobioreactor, light source, fermentation in the reactor which incorporates culture medium inoculated with microbial inoculum comprising one or more of microalgae, fungi, yeasts or bacteria and separation of single-cell protein. DETAILED DESCRIPTION OF THE INVENTION

[50] 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 recognised that various modifications are possible within the scope of the disclosure claimed.

[51] The present invention relates generally to the field of microbial fermentation of dairy or dairy alterative industry waste material and microbial protein production such as microbial biomass or single cell protein (SCP). The present invention is further concerned with a method of identifying a strain of microorganism or mixture of strains of microorganisms, capable of effectively utilising dairy-derived waste such as whey, as a food source to produce microbial biomass as a source of SCP. The present invention is therefore generally directed to a method of producing microbial biomass or single cell protein (SCP) which method comprises culturing microbial inoculum in a growth medium comprising dairy-derived waste material. The present invention is directed to a method of producing microbial biomass or single cell protein (SCP) which method comprises culturing microbial inoculum in a growth medium comprising dairy-derived whey. The present invention is also therefore directed to a method of identifying a strain of microorganism or mixture of strains of symbiotic microorganisms, capable of effectively utilising dairy-derived waste such as whey, as a food source to produce microbial biomass as a nutritional source of SCP for humans and animals.

[52] According to one aspect, the present invention provides a method of producing single cell protein (SCP) which method comprises culturing microbial inoculum in a growth medium comprising dairy-derived whey.

[53] 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.

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

[55] 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 “microalgae”, “fungi”, “yeast” or “bacterium” includes a single or plurality of microalgae, fungi, 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 by methods which are known to those of skill in the art.

[56] As used herein, the tenn “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 0.1%, 0.5%, 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 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.

[57] 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.

[58] In this disclosure, the term “waste”, “by-“, “co-product” or “whey” are used interchangeably and are intended to have the same meaning such as an aqueous liquid or material which is obtained or harvested after milk has been curdled or coagulated and separated. By way of example as used herein, the term whey refers to an aqueous liquid or serum obtained from manufacturing of cheese or casein. Without wishing to be bound by theory, cheese derived whey, is a liquid left over such as a by-product of the dairy industry containing between about 66-77% lactose, 8-15% proteins (B-lactoglobulin and a-lactalbulmin), 7-15% minerals (calcium and phosphorus), and vitamins (vitamins A, D, and B5). Lactose is the main component of whey and therefore results in a high chemical oxygen demand (COD) of40-80 g / 1 and biochemical oxygen demand (BOD) of 30-50 g / 1.

[59] As used herein the term “milk” and “dairy” are used interchangeably and are intended to have the same meaning. By way of example the term covers milk and / or milk alternatives such as synthetic milk or plant-based milk.

[60] As used herein, the term “organoleptic” refers to a perception, impact, quality, impression or an affect produced by a test parameter on a scnson 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 SPC with finely tuned, such as desirable by the subject or preferred, organoleptic qualities fermentation mixture.

[61] 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 fermentation mixture such as a SCP according to embodiments of the present invention by 8 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 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 mixture as described herein.

[62] There has been significant progress in 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 tire 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.

[63] Preferably, the parameters can be measured and quantified. Preferably, the parameters can be measured with great accuracy and be qualitatively quantified. Preferably, tire 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 fermented mixture or certain chemical parameters of the fermented mixture e.g. physico-chemical test parameters or chemical test parameters. 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. By way of nonlimiting 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) etc.

[64] As used herein, the term “subject” means any animal, such as a vertebrate, preferably a mammal such as farmed animal or human who consume the fermentation mixture, composition or product according to embodiments of the present invention. In some embodiments of the present invention the fermentation mixture, composition or product comprise SCP or microbial biomass as obtained by the methods described herein.

[65] According to an embodiment of the present invention, there is provided a method comprising: i) inoculating growth medium comprising dairy -derived whey with a microbial inoculum to form an inoculated microbial mixture; ii) incubating the inoculated microbial mixture at a first incubation temperature (ITc) to generate a propagated starter culture containing between about 103 - 5xlO10 cells / ml; iii) adding the propagated starter culture to a large scale growth medium comprising dairy - derived whey to form a fermentation mixture and incubating the fermentation mixture; and optionally iv) treating the fermentation mixture to produce a SCP.

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

[67] 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.

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

[69] 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 solution comprises whey. In some embodiments the aqueous solution contains a microbial substrate. In some embodiments, the microbial substrate comprises at least one organic compound such as one or more selected from the group of lactose, glucose, sucrose, fructose, galactose, maltose, raffinose or combinations thereof. In some embodiments the aqueous solution is whey. In some embodiments the aqueous solution is synthetic milk whey. In some embodiments the aqueous solution is plant-based milk whey.

[70] In some embodiments, the aqueous medium employed in the present method typically contains at least 70wt.% 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 calcium, magnesium, phosphorus and sulphur. In some embodiments, the aqueous growth medium contains at least 30% lactose or more. In some embodiments, the aqueous growth medium contains at least 1% protein. In some embodiments, the aqueous growth medium contains at least 5% calcium. In some embodiments, the aqueous growth medium contains at least 5% phosphorus.

[71] In some embodiments the aqueous medium is substantially derived from animal or daily products or by-products. In some embodiments the aqueous medium contains artificial or synthetic flavour enhancing or masking compounds.

[72] In some embodiments the aqueous medium is substantially free of animal or dairy products or byproducts. In some embodiments the aqueous medium is substantially free of artificial or synthetic flavour enhancing or masking compounds.

[73] As used herein the term “animal products” means any material derived from the body of an animal. Animal products include milk, microalgae, fungi, eggs, fat, flesh, blood, fish, crustacean shellfish etc.

[74] As used herein, the term "substantially free" means that the content is sufficiently low or negligent that no appreciable danger to a subject such as animal or humans will result from contact with the cultures or products described herein.

[75] As used herein, the term “substantially” refers to up to 80% or more of an entirety. Recitation of ranges of values herein are not intended to be limiting, referring instead individually to any and all values falling within the range, unless otherwise indicated, and each separate value within such a range is incorporated into the specification as if it were individually recited herein.

[76] For the purposes of this disclosure, the term “above” generally means superjacent, substantially superjacent, or higher than another parameter although not directly overlying the parameter. Further, for purposes of this disclosure, the term “in communication” generally refers to components being m direct physical contact with each other or being in indirect physical contact with each other where changes of one component or parameter affect the position or distribution of the other for example measured component or parameter.

[77] In some embodiments the aqueous medium is inoculated with microorganisms such as one or more microalgae, a yeast, a fungi or bacteria. The term “inoculated” as used herein refers to introducing or adding microorganisms such as microalgae, a yeast, a fungi or bacteria or combinations thereof, into the aqueous medium. In some embodiments the aqueous medium is exposed to a light source. It would be appreciated by those of skill in the art that the presence of a light source or incubation of microorganisms as contemplated herein would affect such as enhance the growth of microorganisms. In some embodiments, the inoculated medium is exposed to a light source to enhance growth and protein synthesis by microorganisms such as microalgae. Different sources and properties of light such as wavelength, polarization and electromagnetic spectrum would be familiar to those of skill in the art and these are encompassed by the present invention.

[78] 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 microalgae, a yeast, a fungi and bacteria.

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

[80] As used herein, the term “fermentation mixture” means a symbiotic microbial culture comprising microalgae, a yeast, a fungi or bacteria species. Preferably, microalgae metabolise a carbon source, oxygen and other chemicals to produce proteins, peptides, single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Preferably, microalgae metabolise a carbon source, oxygen and other chemicals to produce single-cell protein. Preferably, yeast, metabolise a carbon source, oxygen and other chemicals to produce carbon dioxide, proteins, peptides, single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Preferably, yeast, metabolise a carbon source, oxygen and other chemicals to produce single-cell protein. Preferably, fungi, metabolise a carbon source, oxygen and other chemicals to produce proteins, peptides, single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Preferably, fungi metabolise a carbon source, oxygen and other chemicals to produce single-cell protein. Preferably, bacterial metabolise a carbon source, oxygen and other chemicals to produce proteins, peptides, single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Preferably, bacteria metabolise a carbon source, oxygen and other chemicals to produce single-cell protein.

[81] Preferably the microorganism is selected from microalgae, a yeast, a fungi and bacteria. Preferably the microorganism such as the propagated started culture comprises at least one of the strains of microalgae, a yeast, a fungi or bacteria. Preferably the microorganism such as the propagated started culture comprises a symbiotic mixture of at least one microalgae, at least one fungi, at least one yeast and at least one bacteria 11 or a combination thereof. Preferably the microorganism such as the propagated started culture comprises a symbiotic mixture of at least two strains of microalgae, at least two strains of fungi, at least two strains of yeast and at least two strains of bacteria.

[82] 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 dairy industry microbial culture.

[83] In some embodiments, the inoculum is incubated to generate between about 102-5xl06 viable cells / ml or more. According to some embodiments the inoculum can be produced or propagated using microfluidic systems.

[84] In some embodiments, the inoculated microbial mixture is propagated at a first incubation temperature (ITc). In some embodiments the inoculated microbial mixture is propagated at a first incubation temperature (ITc) to generated between about 10’-5x10 cells / ml or more. In some embodiments the inoculated microbial mixture is incubated at a first incubation temperature (ITc) to generate a propagated starter culture between about IO3-5xIO10 viable cells / ml or more.

[85] In some embodiments, the propagated starter culture in incubated at a first incubation temperature (ITc) for at least 8 hrs or more. In some embodiments, the propagated starter culture in incubated at a first incubation temperature (ITc) for at least 12 hrs or more. In some embodiments, the propagated starter culture in incubated at a first incubation temperature (ITc) for at least 16 hrs or more. In some embodiments, the propagated starter culture in incubated at a first incubation temperature (ITc) for at least 24 hrs or more. In some embodiments, the propagated starter culture in incubated at a first incubation temperature (ITc) for at least 48 hrs or more.

[86] In some embodiments, the propagated starter culture comprises at least one microalgae, a yeast, a fungi or bacteria.

[87] In some embodiments, the propagated starter culture comprises a symbiotic mixture of at least one typc of microalgae, a yeast, a fungi or bacteria.

[88] Most microalgae, a yeast, a fungi or bacteria species can be exploited in the context of the present methods. Usually, microalgae, a yeast, a fungi or bacteria which produce increased levels of protein are well suited in the present methods.

[89] Preferably, the microalgae is selected from the group of genera comprising Anabaena, Bortycossus, Chaetoceros, Chlorella, Dunaliella, Haematocissus, Isocgrysis, Nannochloropsis, Nostoc, Nodontella, Oscillatoria, Chamudomonas, Parachlorella, Arthrospira, Porphyridium, Rhodomonas, Phaeodactydum, Scenedesmus and Tetraselmis.

[90] In some embodiments of the present nivention, the fungi is selected from the group of genera comprising Fusarium, Paecilomyces, Rhizobium, Aspergillus, Cladosporidium, Monascus, Penicillium, Trichoderma and Alternaria.

[91] In some embodiments of the present invention, the yeast is selected from the group of genera consisting of 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, Toru lopsis. Torulaspora, Schizosaccharomyces, Yarrowia and Kluyveromyces. In some embodiments of the present invention, the yeast is selected from the group of yeast species comprising 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 andKloeckera apiculate.

[92] In some embodiments of the present invention, the bacteria is selected from the group of general group consisting of Lactococcus, Leuconostoc, Allobacullum, Bifidobacterium, Propionobacterium, Ruminococcus, Gluconobacter, Gluconacetobacter, Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Abiotrophia, Aerococcus, Aerosphaera, Agitococcus, Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes, Bavariicoccus, Camobacterium, Camococcus, Catellicoccus, Chungangia, Convivina, Desemzia, Dolosicoccus, Dolosigranulum, Enterococcus, Eremococcus, Facklamia, Floricoccus, Fructobacillus, Globicatella. Granulicatella, Ignavigranum, Isobaculum, Jeotgalibaca, Lacticigenium, Lactovum, Lachancea, Marimlactibacillus, Melissococcus, Metschnikowia, Oenococcus, Okadaella. Pilibacter, Pisciglobus. Sharpea, Komagataeibacter, Brevibacterium, Pediococcus. Nguyenibacter, Sporolactobacillus, Tetragenococcus, Torulaspora, Trichococcus, Thermus, Streptococcus, Staphylococcus, Vagococcus and Weissella.

[93] In some embodiments of the present invention, the symbiotic mixture comprises at least one of the strains of Bortycossus braunii, Pichia pastoris, Aspergillus ochraceus or Lactobacillus delbruckii.

[94] In some embodiments of the present invention, the symbiotic mixture comprises Bortycossus braunii, Pichia pastoris, Aspergillus ochraceus and Lactobacillus delbruckii.

[95] In some embodiments of the present invention, the symbiotic mixture comprises Bortycossus braunii, Pichia pastoris, Saccharomyces cerevisiae and Lactobacillus delbruckii ssp. bulgaricus.

[96] In some embodiments, the propagated starter culture contains between about 103-5xl010 viable cells / ml. cells / ml. cells / ml. cells / ml. cells / ml. In some embodiments, the propagated In some embodiments, the propagated In some embodiments, the propagated In some embodiments, the propagated In some embodiments, the propagated starter culture contains between about starter culture contains between about starter culture contains between about starter culture contains between about starter culture contains between about 103-5xl09 viable 103-5xl08 viable I03-5x 107 viable I03-5x 106 viable IO'-5xlO5 viable cells / ml.

[97] In some embodiments, the propagated starter culture contains about 1010 viable cells / ml. In some embodiments, the propagated starter culture contains about 109 viable cells / ml. In some embodiments, the propagated starter culture contains about 108 viable cells / ml. In some embodiments, the propagated starter culture contains about 107 viable cells / ml. In some embodiments, the propagated starter culture about 10b viable cells / ml. In some embodiments, the propagated starter culture contains about 105 viable cells / ml.

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

[99] 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.

[100] In some embodiments of the present invention, the growth medium described herein is supplemented with a microbial substrate comprising at least one organic compound such as one or more selected from the group of lactose, glucose, sucrose, fructose, galactose, maltose, raffinose or combinations thereof. In some embodiments of the present invention, the at least one organic compound is selected from the group of glucose, sucrose, fructose, galactose, lactose, maltose, raffinose comprises between about 0.1% v / v and about 15% of the growth medium.

[101] In a preferred embodiment, at least one organic compound comprises lactose. In some embodiments, 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 8 % 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.

[102] 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. Incubation, apparatus and systems

[103] In order to tty and address some of tire limitations of the current systems, for example speed of growth of the microorganism and appropriately controlled SPC production, 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, vessel or photoreactor), resulting in increased advection and chaotic mixing of microorganism, e.g. microalgae, a yeast, a fungi or bacteria, comprising aqueous growth medium and microbial substrate.

[104] 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.

[105] For liquid microbial cultures, such as microalgae, a yeast, a fungi or bacteria, as contemplated herein, rapid and healthy growth may depend on factors or parameters including but not limited to: (1) aeration, so that the symbiotic microbes in the mixture have access to for example where aerobic, fresh oxygen for growth; (2) substrate and nutrient a\ ailability. where samples are thoroughly mixed to provide nutrients homogenously throughout the culture to facilitate optimal SPC production; (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; (4) optimal interaction of symbiotic microbes in the mixture with metabolites to achieve optimal functional protein production; (5) minimisation or complete elimination of the production of by-product such as acids; (6) Temperature and / or duration of incubation of the symbiotic microbes; and (7) optimal level of production of parameters such as physico-chemical or chemical parameters selected from and not limited to pH, lactose, sucrose, esters, polyphenols, minerals, lactic acid, citric acid, ascorbic acid, acetic acid, glucuronic acid and amino acids.

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

[107] 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.

[108] The devices or techniques include and are not limited to visual imaging, infrared (IR) / thermal imaging, laser techniques, oxygen levels, acid level, microbial substrate, lactose levels and other physicochemical and / or chemical parameters which can deploy devices or techniques such as HPLC, MALDITOF (MS), Energy Dispersive X-ray Spectroscopy, EDX analysis and SEM (Scanning Electron Microscopy) studies using FESEM (Field Emission Scanning Electron Microscope).

[109] 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 form example during the process of microbial fermentation.

[110] It is anticipated that micro-oxygenation can improve the overall health of microorganisms, such as microalgae, a yeast, a fungi or bacteria during fermentation. It is further suggested that micro-oxygenation can lead to improved protein production such as SCP and stability while also helping in the development of more complex characteristics, a reduction in off-odours such as sulphur. Moreover, it is anticipated that micro-oxygenation can enhance the stability of a complex SCP composition. [Ill] In some embodiments of the present invention, the method further comprises the step of microoxygenation of the fermenting mixture. In some embodiments of the present invention, micro-oxygenation is carried out at about 0.3 to 5 m3 L / min for between 1 to 10 mins, preferably at about 0.5 to 10 m3 L / min for between 1.5 to 10 mins, at about 0.75 to 3 m3 L / min for between 2 to 10 min, at about 1 to 3 m3 L / min for between 3 to 10 mins. As used herein, the term “micro-oxygenation” means introducing an amount of oxygen such as a measured amount of oxygen into the fermented medium. 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 medium. It would be appreciated that temperature, total and free SO2, taste and malolactic fermentation must be controlled.

[112] 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 such as for example a photobioreactor as show in Figure 1, large-scale cooling and large-scale purification systems.

[113] In some embodiments of the present invention, the content of the growth medium comprises at least 30% by weight of lactose, preferably at least 40% by weight of lactose, preferably at least 50% by weight of lactose or more. In some embodiments of the present invention, the solids content of the growth medium comprises at least 30% by weight of lactose, preferably at least 40% by weight of lactose, preferably at least 50% by weight of lactose or more.

[114] In some embodiments of the present invention, the content of the whey waste comprises at least 30% by weight of lactose, preferably at least 40% by weight of lactose, preferably at least 50% by weight of lactose or more. In some embodiments of the present invention, the solids content of the whey waste comprises at least 30% by weight of lactose, preferably at least 40% by weight of lactose, preferably at least 50% by weight of lactose or more.

[115] 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, esters and acids such as acetic acid. Therefore, ongoing fermentation must be stopped after, such as, soon or immediately after, the preferred characteristics and SCP levels in the fermentation mixture have been reached. In some embodiments, treating the fermenting mixture leads to stabilisation of the fermented microbial growth medium such as whey. In some embodiment, treating the fermenting mixture substantially eliminates or completely prevents the production of spoiling compounds and molecules such as alcohol, carbon dioxide, esters and acids such as acetic acid, enzyme activity-, after completion of the fermentation and the preferred characteristics and SCP levels in the fermentation mixture have been reached.

[116] 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 SCP. It is anticipated that treating the fennenting 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 functional protein.

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

[118] 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.

[119] In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation when predetermined criteria are reached. In some embodiments of the present invention, the predetermined criteria comprise a level of biomass yield. In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation when the predetermined criteria reach a level of biomass yield greater than 20% w / w, preferably greater than 30% w / w, preferably greater than 40% w / w, preferably greater than 50% w / w, preferably greater than 60% w / w, preferably greater than 70% w / w, preferably greater than 80% w / w or more.

[120] In some embodiments of the present invention, the fermenting mixture is treated to stop fermentation when the level of lactose is between about 3% and 1%.

[121] In some embodiments of the present invention, the fermentation mixture is lyophilised.

[122] In some embodiments of the present invention, the fermentation mixture is subjected to further treatment. In some embodiments of the present invention, treating the fermented mixture comprises 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. In some embodiments of the present invention, treating the fermented mixture comprises rapidly cooling the fermented mixture to stop fermentation.

[123] In some embodiments the fermentation mixture comprises the microbiota, microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals.

[124] 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. Preferably, treatment of the fermented mixture does not adversely affect or modify the physico-chemical properties or test parameters of the composition. Preferably, treatment of the fermented mixture does not adversely affect or modify the organoleptic properties of the composition. Preferably, treatment of the fermented mixture does not adversely affect or modify the microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals such as calcium of the composition.

[125] Preferably, treatment of the fermented mixture enhances the organoleptic properties of the composition. Preferably, treatment of the fermented mixture enhances the microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals such as calcium of the composition.

[126] In one aspect of the present invention, there is provided a propagated starter culture. In some embodiments of the present invention, the propagated starter culture is lyophilised. In some embodiments of the present invention, the propagated starter culture is used in fermentation. In some embodiments of the present invention, the propagated starter culture is used in producing a food product or SCP.

[127] In one aspect of the present invention, there is provided a use of dairy-derived whey waste as a food source for a microorganism which microorganism is a source of single cell protein.

[128] In one aspect the present invention, there is provided a composition comprising single cell protein (SCP) obtained from microorganisms that have been cultured in a medium comprising dairy-derived whey waste. In some embodiments, there is provided a product or supplement comprising the composition of the present invention.

[129] In one aspect the present invention, there is provided a method of identifying a strain of microorganism, or mixture of strains of microorganisms, capable of efficiently using dairy-derived whey waste as a food source to produce biomass as a source of single cell protein, which method comprises cultivating one or more microorganisms in a cell culture medium comprising dairy-derived whey waste and selecting a microorganism, or mixture thereof, based on one or more predetermined criteria that indicate efficient use of the whey waste to produce said biomass, such as growth rate, biomass yield, overall nutritional content, protein content and / or resistance to contaminants. In some embodiments, the predetermined criteria include biomass yield. In some embodiments, the predetermined criteria include biomass yield greater than 20% w / w, preferably greater than 30% w / w, preferably greater than 40% w / w, preferably greater than 50% w / w, preferably greater than 60% w / w, preferably greater than 70% w / w, preferably greater than 80% w / w or more. In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation when the predetermined criteria reach a level of biomass yield greater than 50% w / w.

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

[131] In some embodiments, the treated fermentation mixture such as SCP is subjected to filtration such as large-scale filtration. Methods of incubating a Microorganism culture

[132] 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. In some embodiments, incubating the microorganism culture is conducted at a first incubation temperature (ITc) and / or second incubation temperature (2Tc) in the range of 30°C to 38°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of between about 35-39°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of preferably between about 35.5-38.5°C. In some embodiments, incubating the microorganism culture is conducted at a temperature in the range of preferably between about 36-38°C.

[133] In some embodiments, the first incubation temperature (ITc) is the same as the second incubation temperature (2Tc).

[134] In some embodiments, the microorganism mixture is incubated at the first incubation temperature (ITc) for at least about 30 mins, preferably at least about 45 mins, preferably at least about 1 hour, preferably at least about 1.5 hours, preferably at least about 2 hours, preferably at least about 2.5 hours, preferably at least about 3 hours, preferably at least bout 4 hours, preferably at least about 5 hours, preferably at least about 6 hours, preferably at least about 6.5 hours, preferably at least about 7 hours, preferably at least about 7.5 hours, preferably at least about 8 hours, preferably at least about 8.5 hours, preferably at least about 9 hours, preferably at least about 9.5 hours, preferably at least about 10 hours, preferably at least about 10.5 hours, preferably at least about 11 hours, preferably at least about 11.5 hours, preferably at least about 12 hours, preferably at least about 12.5 hours, preferably at least about 13 hours, preferably at least about 13.5 hours, preferably at least about 14 hours or more.

[135] In one embodiment the mixture is incubated at the first incubation temperature (ITc) for 12 hours.

[136] 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).

[137] 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 microalgae, a yeast, a fungi or bacteria. In some embodiments, the propagated starter culture comprises a symbiotic mixture of at least two strains of microalgae, at least two strains of fungi, at least two strains of yeast and at least two strains of bacteria.

[138] In some embodiments, the microorganism mixture is incubated at second incubation temperature (2Tc) for at least about 24 hrs, at least 36 hrs, at least 48 hrs, at least 72 hrs or more.

[139] 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).

[140] 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.

[141] It is contemplated that cessation of growth of the microorganism culture leads to cessation of fermentation and concomitant preservation of the desired SCP, pH, lactose, sucrose, esters, polyphenols, minerals, minerals, lactic acid, citric acid, ascorbic acid, acetic acid, glucuronic acid and amino acids levels in the fermented mixture. Quorum sensing

[142] 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.

[143] In general and without wishing 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 microalgae, a yeast, a fungi or bacteria.

[144] 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 and single cell protein.

[145] By exposing the microalgae, a yeast, a fungi or bacteria microorganism cultures such as the symbiotic mixture of at least one type of microalgae, at least one fungi, at least one yeast and at least one bacteria or a combination thereof, 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 functional protein attributes or factors of the fermented SCP. Propagated starter culture and uses thereof

[146] 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.

[147] In one aspect of the present invention, there is provided a propagated starter culture. In some embodiments of the present invention, the propagated starter culture is lyophilised. In some embodiments of the present invention, the propagated starter culture is used in fermentation. In some embodiments of the present invention, the propagated starter culture is used in producing a food product or SCP. In some embodiments of the present invention, the propagated starter culture is used in producing a function food.

[148] In one aspect of the present invention, there is provided a use of dairy-derived whey waste as a food source for a microorganism which microorganism is a source of single cell protein.

[149] In one aspect the present invention, there is provided a composition comprising single cell protein (SCP) obtained from microorganisms that have been cultured in a medium comprising dairy-derived whey waste. In some embodiments, there is provided a product or supplement comprising the composition of the present invention.

[150] In one aspect the present invention, there is provided a method of identifying a strain of microorganism, or mixture of strains of microorganisms, capable of efficiently using dairy-derived whey waste as a food source to produce biomass as a source of single cell protein, which method comprises cultivating one or more microorganisms in a cell culture medium comprising dairy-derived whey waste and selecting a microorganism, or mixture thereof, based on one or more predetermined criteria that indicate efficient use of the whey waste to produce said biomass, such as growth rate, biomass yield, overall nutritional content, protein content and / or resistance to contaminants. In some embodiments, the predetermined criteria include biomass yield. In some embodiments, the predetermined criteria include biomass yield greater than 20% w / w, preferably greater than 30% w / w, preferably greater than 40% w / w, preferably greater than 50% w / w, preferably greater than 60% w / w, preferably greater than 70% w / w, preferably greater than 80% w / w or more. In some embodiments of the present invention, the fermenting mixture is treated in order to stop fermentation when the predetermined criteria reach a level of biomass yield greater than 50% w / w.

[151] Although the inventors do not wish to be bound by theon . it is believed that the present invention may benefit from tire quorum sensing capabilities of the microorganisms such as a symbiotic mixture of at least one type of yeast and at least one type of microalgae, a yeast, a fungi or bacteria.

[152] 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, as a result of the controlled conditions and substrate, can produce enhanced quantities of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. In particular, the present inventors surprisingly and unexpectedly observed that when the microbial culture is propagated according to tire method of the present invention, the propagated microorganisms, as a result of the controlled conditions, substrate and growth medium, can produce an increased qualify of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals with particular physico-chemical parameters and / or characteristics. The present inventors also surprisingly and unexpectedly observed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, as a result of the controlled conditions, substrate and growth medium, can produce microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, 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, as a result of the controlled conditions, substrate and growth medium, can provide a long term supply of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, 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, as a result of the controlled conditions, substrate and growth medium, can provide a suitable functional food supplement of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals.

[153] In some embodiments, the propagated starter culture is lyophilised. In some embodiments, the fermentation mixture is lyophilised. As used herein the term “lyophilised” means preserving for example propagated antimicrobial culture or fermentation mixture 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 or fermentation mixture is preserved long-term. In some embodiments, the lyophilised propagated starter culture or fermentation mixture comprises viable microbial cells. In some embodiment the lyophilised propagated starter culture or fermentation mixture comprises viable microalgae, a yeast, a fungi or bacteria. In some embodiment the lyophilised propagated starter culture or fermentation mixture comprises a symbiotic mixture of at least one microalgae, at least one fungi, at least one yeast and at least one bacteria or a combination thereof. 1154] 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 microbial biomass such as SCP. In some embodiment, the reconstituted dosage form can be used for direct addition into an ongoing fermentation process in the preparation of fermentation mixture.

[155] In some embodiments, lyophilisation can be used to prepare a dosage form of a fermentation mixture comprising greater than 20% w / w, preferably greater than 30% w / w, preferably greater than 40% w / w, preferably greater than 50% w / w, preferably greater than 60% w / w, preferably greater than 70% w / w, preferably greater than 80% w / w or more for direct addition into feedstuff.

[156] Statistical Analysis

[157] 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-nomial variables. Data can be presented as mean standard deviation (mSD). Reports and Data Transmission

[158] In some embodiments, the methods and systems disclosed herein further comprise generating one or more reports such as measurement reports of different protein concentration or biomass parameters, characteristics 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.

[159] In some embodiments, the report includes information on the capability of a microorganism to ferment suitable substrate such as whey, for the generation of desirable SCP level, pH, lactose, sucrose, esters, polyphenols, minerals, minerals, lactic acid, citric acid, ascorbic acid, acetic acid, glucuronic acid and amino acids concentrations.

[160] In some embodiments, the report provides recommendations on the selection of suitable symbiotic mixture of at least one type of microalgae, at least one fungi, at least one yeast and at least one bacteria or a combination thereof, growth medium such as whey and in tire production of a particular single cell protein (SCP) or biomass.

[161] In some embodiments, the report provides recommendations on the timing of treatment of the fermentation mixture.

[162] In some embodiments, the report provides direction or instructions for use of dairy-derived whey w aste as a food source for a microorganism which microorganism is a source of single cell protein.

[163] In some embodiments, the report provides recommendations of constituents of composition comprising single cell protein (SCP) obtained from microorganisms that have been cultured in a medium comprising dairy-derived whey waste.

[164] In further embodiments, test parameters of the present disclosure such as those that are characterised with or associated with certain such as desirable for example SCP properties, SCP level, pH, lactose, sucrose, esters, polyphenols, minerals, minerals, lactic acid, citric acid, ascorbic acid, acetic acid, glucuronic acid and amino acids concentrations, 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.

[165] 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. MATERIALS &METHODS and EXPERIMENTAL EXAMPLES

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

[167] 1. Oxygen (O2), Carbon dioxide (CO2) and / or light

[168] 1.1. Oxygen (O2)

[169] 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;

[170] Filtration - filtration increases to 200ppb-300ppb.

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

[172] 1.2. Carbon dioxide (CO2)

[173] 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.

[174] 1.3 Light

[175] It is appreciated that all the optical processes in for example microalgae are dependent on the wavelength of light such as incident light. Visible light covers the wavelengths between approximately 390 nm (violet) to 780 nm (red) of the electromagnetic spectrum, coinciding almost completely with the photosynthetically active radiation (PAR) range (400-700 nm). Different light sources, such as the sun (in case of for example glass reactor), LED lamp or a fluorescent bulb, all have a unique wavelength-dependent intensity distribution (emission spectrum). Incident light menas the available amount of light for photosynthesis. In our experiments, we use LED lamp or a fluorescent bulb as a light source.

[176] 2, Non-exhaustive list of desirable organic acids

[177] lactic acid, L-lacric acid, Acetic acid, gluconic acid, glucuronic acid, citric acid, malic acid, tartaric acid, malonic acid, oxalic acid, succinic acid, pyruvic acid, usinic acid.

[178] 3.1. Non-exhaustive list of undesirable organic acid intermediate metabolites

[179] Capric acid, Caproic acid, Heptanoic acid, Isovaleric, Lauric, Nanoic acid, succinic acid, undecanoic acid.

[180] 4. Impact of temperature

[181] 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 1.

[182] Table 1. Microbial solution such as whey Parameters being monitored T "C Fermentation higher speed of fermentation, microbial development, light source 20-38 °C Post fermentation treatment low crash temp treatment precipitate protein, microbial flocculation, haze formation and stabilisation of product. Packaging Maintaining low temperature shelf life, stability, evolution of SCP content &CO2 production, development of off flavours and aroma

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

[184] 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 fermentation mixture such as SCP is obtained.

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

[186] Alcohol - needed to start fermentation.

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

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

[189] pH - in brewing - high brewing pH will extract tannins; while in fermentation - low pH will stall fermentation and affect microbial health and protein sy nthesis; low pH affect the protein transport chain in microbial cells as one of the main functions of microbial 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.

[190] sugar concentration - enhances microbial growth and protein synthesis during microbial incubation.

[191] 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, microbial health and bacterial conversion of for example ethanol to acids. Depending on the shape of the fermentation vessels, critical difficulties are encountered when volumes reach about 10,000-12,000 hL. Larger fermentation vessels lead to poor microbial growth, poor diacetyl reduction and poor ester production. The impact of fennentation 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.

[192] 13. Chemical Composition

[193] One of the parameters which is monitored and closely monitored during tire methods of the present invent is the chemical composition. In Table 2 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.

[194] Table 2. TYPE Compound / Parameter / Molecule Average Composition Initial Amino Acids Lactic acid 15 g / 1 70 g / 1 Sugar Glucose 8.36 g / 1 100 g / 1 Fructose H g / 1 100 g / 1 Vitamins Vitamin Bl 0.74 mg / ml 70 g / 1 Vitamin B2 8 mg / ml 70 g / 1 Vitamin B6 0.52 mg / ml 70 g / 1 Vitamin B12 0.84 mg / ml 70 g / 1 Vitamin C 25 mg / 1 70 g / 1 General Composites Proteins 3 mg / ml 100 g / 1 Tea Polyphenols 7.8 Mm GAE 100 g / 1 Minerals Ca, Fe, Mn, Ni, Zn 0.1 - 0.4 ug / ml 70 g / 1 Anions F-, C1-, Br-, NO3- 0.04 - 3.20 mg / g 100 g / 1

[195] 14. Sugar, type of sugar, source of sugar such as plant source used as a microbial substrate and flavours.

[196] Lactose - dairy products for fermented product such as fermented mixture or SCP produced by the method of the present invention; Sucrose - sweetness of fermented product such as fermented mixture or SCP produced by the method of the present invention; Honey - clean, high TA development, mixed saccharides and others.

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

[198] The aqueous solution relied upon in the present invention is whey as a by- or co-product material of the dairy industry. Daily- derived whey is the growth medium. Dairy alternative material can also be used as growth medium as well as plant-based material. The growth medium covers whey from milk and / or milk alternatives such as synthetic milk or plant-based milk.

[199] Alkalinity - affects the amount of acid required to bring down pH;

[200] Calcium - higher i.e. hard water helps in microbial flocculation (settling); Chlorides - higher increases malty; Permanent Hardness = chlorides and sulphates; Iron - higher i.e. hard water helps in microbial flocculation which also helps ester formation; Sulphates - higher produces dryer outcomes and can enhance bitterness; TDS - helps micro metabolism and growth; and Zinc - helps micro metabolism and growth.

[201] 16. Metabolites

[202] 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-O-glucuronide; (R)-2,3-Dihydroxy-3-methylpentanoic acid; 1-O-Vanilloyl-beta-D-glucose; 4-Hydroxy-5-(3',4'-dihyroxyphenyl)-valeric acid; Neuraminic acid; Pilosin; 4-O-beta-D-Glucosyl-Esculetin; 2-Dehydropantolactone; (R)-l,2-dimethyl-5,6-dihydroxy-tetrahydroisequinoline; l-(beta-D-Ribofuranosyl)-1,4-dihydomicotinamide; 7,8-Dihydroxanthopterin; 4-Chioro-L-lysine; 2-Dechloroethylifosfamide; 4-Hydroxycyclohcxylcarboxylic acid; and N-(Carbethoxyacetyl)-4-chloro-L-tryptophan.

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

[204] 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 amino acids and peptides 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).

[205] Table 3. 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:

[206] Table 3. Example methodologies, techniques and assays. Separation technique Type of detector used Description of technique 1. Gas Chromatography (GC) Hydrogen flame ion detector Sensitivity is high Mass Spectrometry Detectors High performance. 2. Liquid chromatography (LC) Conductivity detector Very sensitive and selective, and can be performed quickly and easily in situ. Chemiluminescenc e The wavelength is used to identify substances with high selectivity and detection limits Volt-ampere detector Reproducibility with the simple and rapid procedure Fourier infrared detector The coupling of LC and IR systems can be performed offline or in-line. The on-line analysis Mass Spectrometry More sensitive, selective, and specific, and can determine trace amounts of substances. Evaporative light scattering detector Cheaper than MS methods and also compatible with a wide range of solvents and gradient elusions. Refractive index detector Simplicity of operation, cost, energy consumption, and availability in most QC labs are better than mass spectrometry detectors Diode array detector This technique advantageously allows an easy quantification 3. Thin-layer chromatography (TLC) UV detector Fast, sensitive, highly selective, simple, easy colour development, easy to use 4. Ion exchange chromatography (IEC) Conductivity detector High precision and accuracy with good reproducibility 5. Capillary' zone electrophoresis method UV detector High resolution, high automation, simple operation, fast speed, low chemical consumption 6. UV spectrophotomet er Photomultiplier tube Reaction of organic acids with other substances and measurement of complexes at specific wavelength

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

[208] Example 1

[209] Symbiotic Microbial Mixture 1 - comprising Bortycossus braunii, Pichia pastoris, Aspergillus ochraceus or Lactobacillus delbruckii. This mixture 1 was shown to be able to produce greater than 60% SCP when microbial mixture is incubated in growth medium comprising dairy-derived whey and lactose at 18% at 2Tc of 37°C for up to 48 hrs (data not shown).

[210] Example 2

[211] Symbiotic Microbial Mixture 2 - comprising Bortycossus braunii, Pichia pastoris, Aspergillus ochraceus and Lactobacillus delbruckii ssp. bulgaricus. This mixture 2 was shown to be able to produce greater than 80% SCP when microbial mixture is incubated in growth medium comprising dairy-derived whey and lactose at 25% at 2Tc of 37°C for up to 48 hrs (data not shown).

[212] CONCLUSION

[213] 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, as a result of the controlled conditions and substrate, can produce enhanced quantities of microbial biomass, SPC, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. 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, as a result of the controlled conditions, substrate and growth medium, can produce an increased quality of microbial biomass, SCP, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals with particular physico-chemical parameters and / or characteristics. The present inventors also surprisingly and unexpectedly observed that when the microbial culture is propagated according to the method of the present invention, the propagated microorganisms, as a result of the controlled conditions, substrate and growth medium, can produce microbial biomass, SCP, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, 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, as a result of the controlled conditions, substrate and growth medium, can provide a long term supply of microbial biomass, SCP, functional protein, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, 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, as a result of the controlled conditions, substrate and growth medium, can provide a suitable functional food supplement of microbial biomass, SCP, free single amino acids, short oligopeptides, carbohydrates, lipids, vitamins, and minerals. Furthermore, the present inventors surprisingly and unexpectedly observed that the produced by the methods of the present invention, SCP has further beneficial properties in that it offers functional protein properties characterised with greater gain in muscle mass in the subjects following consumption. * * *

[214] 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.

[215] REFERENCES Patrick F. Fox, Timothy P. Guinee , Timothy M. Cogan , Paul L. H. McSweeney, 2017. Fundamentals of Cheese Science. New York: Springer. Zandona E, Blazic M, Rezek Jambrak A. Whey Utilization: Sustainable Uses and Environmental Approach. Food Tcchnol Bioteclmol. 2021 Jun;59(2): 147-161. doi: 10.17113 / ftb.59.02.21.6968. PMID: 34316276; PMCID: PMC8284110.

Claims

1. A method of producing single cell protein (SCP) or biomass, the method comprising culturing microbial inoculum in a growth medium comprising dairy-derived whey.

2. A method according to claim 1, the method further comprising:v) inoculating growth medium comprising dairy-derived whey with a microbial inoculum to form an inoculated microbial mixture;vi) incubating the inoculated microbial mixture at a first incubation temperature (ITc) to generate a propagated starter culture containing between about 103 - 5xlO10 cells / ml;vii) adding the propagated starter culture to a large scale growth medium comprising daily-derived whey to form a fermentation mixture and incubating the fermentation mixture; and optionallyviii) treating the fermentation mixture to produce a SCP.

3. A method of producing single cell protein according to claim 2, wherein the fermentation mixture is incubated at a second incubation temperature (2Tc) for at least 24 hrs, at least 36 hrs, at least 48 hrs, at least 72 hrs or more.

4. A method of producing single cell protein according to anyone of claims 1 to 3, wherein the microbial inoculum or the propagated starter culture is obtained from a microbiota.

5. A method of producing single cell protein according to any one of the preceding claims whereinat least one of the strains in the microbial inoculum is microalgae, a yeast, a fungi or bacteria.

6. A method of producing single cell protein according to anyone of claims 1 to 5, wherein the propagated starter culture comprises a symbiotic mixture of at least one microalgae, at least one fungi, at least one yeast and at least one bacteria or a combination thereof.

7. A method of producing single cell protein according to anyone of claims 1 to 6, wherein thepropagated starter culture comprises a symbiotic mixture of at least two strains of microalgae, at least two strains of fungi, at least two strains of yeast and at least two strains of bacteria.

8. A method of producing single cell protein according to anyone of claims 5 or 7, wherein the microalgae is selected from the group of genera comprising Anabaena, Bortycossus, Chaetoceros, Chlorella, Dunaliella, Haematocissus, Isocgrysis, Nannochloropsis, Nostoc, Nodontella, Oscillatoria, Chamudomonas, Parachlorella, Arthrospira, Porphyridium, Rhodomonas, Phaeodactylum, Scenedesmus and Tetraselmis', the fungi is selected from the group of genera comprising Fusarium, Paecilomyces, Rhizobium, Aspergillus, Cladosporidium, Monascus, Penicillium, Trichoderma and Alternaria\ 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, Yarrowia and Kluyveromyces: and thebacteria is selected from the genera consisting of Leuconostoc, Lactococcus, Acetobacter, Allobacullum, Bifidobacterium, Leuconostoc, Propionob acterium, Ruminococcus, Gluconobacter, Gluconacetobacter, Lactobacillus, Pediococcus, Lactococcus, Streptococcus, Abiotrophia, Aerococcus, Aerosphaera, Agitococcus, Alkalibacterium, Allofustis, Alloiococcus, Atopobacter, Atopococcus, Atopostipes. Bavariicoccus, Carnobacterium, Camococcus, 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.

9. A method of producing single cell protein according to claim 8, wherein the at least one of thestrains comprises Bortycossus braunii, Pichiapastoris, Aspergillus ochraceus or Lactobacillus delbruckii.

10. A method of producing single cell protein according to claim 8, wherein the symbiotic mixture comprises Bortycossus braunii, Pichia pastoris, Aspergillus ochraceus and Lactobacillus delbruckii.

11. A method of producing single cell protein according to anyone of the preceding claims, wherein the first incubation temperature (ITc) is in the range of between about 20°C to 38°C, preferably between about 22°C to 37°C, preferably between about 24°C to 36°C, preferably between about 26°C to 35°C.

12. A method of producing single cell protein according to anyone of claims, wherein the second incubation temperature (2Tc) is less than 38°C, preferably less than 37°C, preferably less than 36, preferably less than 35°C.

13. A method according to any one of the preceding claims, wherein the solids content of the whey waste comprises at least 30% by weight of lactose, preferably at least 40% by weight of lactose, preferably at least 50% by weight of lactose or more.

14. A method according to any one of the preceding claims, wherein treating the fermented mixturecomprises 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.

15. A method according to any one of the preceding claims, wherein the growth medium is supplemented with microbial substrate.

16. A method according to claim 15, wherein the microbial substrate comprises at least one organic compound such as at least one organic compound selected from the group of glucose, sucrose, fructose, galactose, lactose, maltose, raffinose or combinations thereof17. A method according to any one of the claims 15 or 16, wherein the at least one organic compound selected from the group of glucose, sucrose, fructose, galactose, lactose, maltose, raffinose, comprises between about 1% v / v and about 15% of the growth medium.

18. A propagated starter culture obtained according to the method of any one of claims 1 to 17.

19. A propagated starter culture according to claim 18 wherein the propagated starter culture islyophilised.

20. A method of producing a food stuff which comprises isolating SCP from microorganisms cultured in a medium comprising dairy-derived whey waste and combining the SCP with one or more food ingredients.

21. Use of dairy-derived whey waste as a food source for a microorganism which microorganism is a source of single cell protein.

22. A composition comprising single cell protein (SCP) obtained from microorganisms that have been cultured in a medium comprising dairy-derived whey waste.

23. A product or supplement comprising the composition of claim 22.

24. A method of identifying a strain of microorganism, or mixture of strains of microorganisms, capable of efficiently using dairy-derived whey waste as a food source to produce biomass as a source of single cell protein, which method comprises cultivating one or more microorganisms in a cell culture medium comprising dairy-derived whey waste and selecting a microorganism, or mixture thereof, based on one or more predetermined criteria that indicate efficient use of the whey waste to produce said biomass, such as growth rate, biomass yield, overall nutritional content, protein content and / or resistance to contaminants.

25. A method according to claim 24, where the predetermined criteria include biomass yield greater than 20% w / w, preferably greater than 30% w / w, preferably greater than 40% w / w, preferably greater than 50% w / w, preferably greater than 60% w / w, preferably greater than 70% w / w, preferably greater than 80% w / w or more.

26. A method according to anyone of claims 24 or 25, where the predetermined criteria include biomass yield greater than 50% w / w.33

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