Classifications

• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/21—Synthetic spices, flavouring agents or condiments containing amino acids
  • A23L27/215—Synthetic spices, flavouring agents or condiments containing amino acids heated in the presence of reducing sugars, e.g. Maillard's non-enzymatic browning
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
  • A23L27/20—Synthetic spices, flavouring agents or condiments
  • A23L27/26—Meat flavours
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  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
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  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/145—Fungi isolates
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  • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
  • C12P17/02—Oxygen as only ring hetero atoms
  • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/42—Hydroxy-carboxylic acids
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters
  • C12P7/6445—Glycerides
  • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
  • C12P7/6436—Fatty acid esters
  • C12P7/6445—Glycerides
  • C12P7/6481—Phosphoglycerides
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  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
  • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
  • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/30—Organic components
  • C12N2500/36—Lipids
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  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/72—Candida
  • C12R2001/73—Candida lipolytica
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/785—Mucor
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/645—Fungi ; Processes using fungi
  • C12R2001/85—Saccharomyces
  • C12R2001/865—Saccharomyces cerevisiae

Definitions

• the present invention broadly relates to use of microbial biomass (e.g. Mortierella spp. biomass), or extracted lipid therefrom, comprising or in combination with one or more lactones and/or one or more 4-hydroxy fatty acids in a food product, beverage product or feedstuff, to compositions comprising biomass and one or more lactones, and to food products, beverage products or feedstuffs comprising the biomass.
• microbial biomass e.g. Mortierella spp. biomass
• extracted lipid therefrom comprising or in combination with one or more lactones and/or one or more 4-hydroxy fatty acids in a food product, beverage product or feedstuff
• compositions comprising biomass and one or more lactones
• food products, beverage products or feedstuffs comprising the biomass.
• the present invention further relates to said compositions and food products, beverage products or feedstuffs for producing food-like aromas and/or flavours when heated, in particular for undergoing Maillard reactions.
• the present invention further relates to
• the aroma and flavour characteristics of cooked meat are important factors for the eating quality of meat, correlating highly with acceptance and preference by consumers.
• the aroma and flavour characteristics come from a large number of volatile and non-volatile compounds which are produced during heating of the meat such as by cooking or roasting (see, for example, the reviews by Dashdorj et al. (2015) and Mottram (1998)).
• These compounds result from several types of chemical reactions, namely Maillard reactions of amino acids or peptides with reducing sugars, lipid oxidation, the interaction between the Maillard reaction products with the lipid-oxidation products, and degradation of other compounds such as some sulphur-containing compounds during cooking or roasting.
• reaction products are organic and of low molecular weight, including aldehydes, ketones, alcohols, esters, aliphatic hydrocarbons, thiazoles, oxazoles and pyrazines as well as oxygenated heterocyclic compounds such as lactones and alkylfurans. Many of these compounds do not arise during the cooking of meat-substitutes made with plant proteins and fats such as coconut, soy and palm oils, leading to less consumer acceptance of these non-animal products.
• the present invention is predicated on, at least in part, the unexpected determination that certain biomasses or lipid extracts therefrom comprising or combined with a lactone, in particular a y- lactone, can impart a strong and pleasant food-like, and in particular meat -like, aroma and/or flavour to a food.
• a lactone in particular a y- lactone
• This can be achieved using relatively little amounts of biomass or extracted lipid, thus provided an efficient and cost-effective way to enhance the aroma and flavour of food, feedstuff and beverages.
• the inventors have demonstrated that various yeast and fungal isolates, and in particular Mortierella spp., are effective as flavour and aroma enhancers.
• compositions capable of producing a food-like aroma and/or flavour when heated comprising: a) Mortierella spp. biomass or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy fatty acids; c) one or more sugars, sugar alcohols, sugar acids, or sugar derivatives; and d) one or more amino acids or derivatives or salts thereof, or a compound comprising an amino group (e.g. thiamine).
• the one or more lactones comprise one or more y-lactones.
• Suitable y-lactones include, for example, y-hexalactone, y-heptalactone, y-octalactone, y- nonalactone, y-decalactone, y-docecalactone and y-undecalactone.
• the one or more 4-hydroxy fatty acids may be selected from 4 -hydroxyhexanoic acid, 4-hydroxyheptanoic acid, 4-hydroxyoctanoic acid, 4-hydroxynonanoic acid, 4-hydroxydodecanoic acid, 4-hydroxyundecanoic acid and 4-hydroxydecanoic acid.
• the biomass comprises the lactones and/or the one or more 4-hydroxy fatty acids, while in other examples the biomass is combined with the lactones.
• the composition comprises less than 5% by weight protein, other than protein provided by the Mortierella spp. biomass.
• the composition comprises at least about 0.05 mg/mL or mg/g dry Mortierella spp. biomass, based on the volume or weight of the composition excluding the Mortierella spp. biomass. In one example, the composition comprises at least about 1 mg/mL or mg/g dry Mortierella spp. biomass, based on the volume or weight of the composition excluding the Mortierella spp. biomass. In other examples, the composition comprises from about 1 mg/mL or mg/g to about 50 mg/mL or mg/g dry Mortierella spp. biomass or an equivalent amount of wet biomass, based on the volume or weight of the composition excluding the Mortierella spp. biomass. In particular embodiments, the food-like aroma and/or flavour is a meaty aroma and/or flavour.
• the Mortierella spp. is Mortierella alpina, Mortierella elongata or Mortierella isabellina.
• the biomass or extracted lipid comprises phospholipids, optionally wherein those phospholipids comprise one or more esterified co6 fatty acids, e.g. arachidonic acid (ARA), dihomo-gammalinolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-co6 (DPA-co6) or y-linolenic acid (GLA).
• ARA arachidonic acid
• DGLA dihomo-gammalinolenic acid
• EDA eicosadienoic acid
• DTA docosatetraenoic acid
• DPA-co6 docosapentaenoic acid-co6
• GLA y-linolenic acid
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the composition in amounts sufficient to produce a food-like aroma and/or flavour when the composition is heated.
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the composition in amounts sufficient to produce one or more volatile compounds selected from 1,3 -dimethyl benzene; p-xylene; ethylbenzene; 2-Heptanone; 2-pentyl furan; Octanal; 1,2-Octadecanediol; 2,4-diethyl-l-Heptanol; 2-Nonanone; Nonanal; l-Octen-3-ol; 2-Decanone; 2-Octen-l-ol, (E)-; 2,4-dimethyl-Benzaldehyde; 2, 3,4,5- Tetramethylcyclopent-2-en-l-ol, 1-octanol, 2-heptanone, 3-octanone, 2, 3 -octanedione, 1-pentanol, 1- hexanol, 2-eth
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the composition in amounts sufficient to produce one or more volatile compounds selected from 2- heptanone, 3-octanone, 2,3-octanedione, 1 -pentanol, 1 -hexanol, 2 -ethyl- 1 -hexanol, 1 -octanol, trans-2- octen-l-ol and 1 -nonanol when the composition is heated.
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives are present in the composition in an amount of from about 5 mmol to about 100 mmol per kg or per L of composition, based on the volume or weight of the composition excluding the Mortierella spp. biomass or extracted lipid.
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives are present in the composition in an amount of at least about 15 mmol per kg or per L of composition, based on the volume or weight of the composition excluding the Mortierella spp. biomass or extracted lipid.
• the one or more amino acids or derivatives or salts thereof are present in the composition in an amount of from about 5 mmol to about 100 mmol, based on the volume or weight of the composition excluding the Mortierella spp. biomass or lipid. In one example, the one or more amino acids or derivatives or salts thereof are present in the composition in an amount of at least about 15 mmol per kg or per L of composition, based on the volume or weight of the composition excluding the Mortierella spp. biomass or lipid.
• the one or more sugars, sugar alcohols, sugar acids or sugar derivatives comprise glucose and/or ribose.
• the one or more sugars, sugar alcohols, sugar acids or sugar derivatives comprise ribose and glucose.
• the one or more amino acids or derivatives or salts thereof comprise cysteine and/or cystine.
• the one or more amino acids may also, or alternatively, comprise glutamic acid or a salt thereof.
• the composition comprises glutamic acid or a salt thereof and a further amino acid, derivative or salt thereof.
• compositions may also comprise any one or more of, or any combination of, a source of iron, a yeast extract, thiamine, herbs and/or spices and an aqueous component.
• the composition does not comprise a yeast extract.
• the composition comprises: a) Mortierella spp. biomass or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy acids; c) glucose and/or ribose; d) cysteine and/or cystine; e) yeast extract; f) glutamic acid or a salt thereof; g) thiamine; and h) an aqueous component.
• the composition produces a meaty aroma and/or flavour when heated.
• the composition is in the form of a food product, beverage product or feedstuff. Accordingly, the food product, beverage product or feedstuff produces a meaty aroma and/or flavour when heated.
• the composition may be mixed with, or added to, a food product, beverage product or feedstuff, for example wherein the composition is in the form of a powder, particulate or granulated mix.
• the composition may be mixed with, or be added to, the food product, beverage product or feedstuff prior to heating, after heating the composition, and/or after heating the food product, beverage product or feedstuff.
• a meaty aroma and/or flavour may be produced.
• a food product, beverage product or feedstuff comprising a combination of Mortierella spp. biomass or extracted lipid therefrom and one or more lactones and/or one or more 4-hydroxy acids, or a composition or the invention, optionally wherein the food product, beverage product or feedstuff comprises less than 5% dry Mortierella spp. biomass by weight, or an equivalent amount of wet biomass.
• the food product, beverage product or feedstuff has a meaty aroma and/or flavour.
• the food product, beverage product or feedstuff produces a meaty aroma and/or flavour when heated.
• the Mortierella spp. is Mortierella alpina, Mortierella elongata or Mortierella exigua.
• the food product, beverage product or feedstuff is free from any animal or animal-derived ingredients.
• the food product, beverage product or feedstuff comprises an animal or animal -derived ingredient, optionally wherein the animal or animal -derived ingredient is meat.
• the food product, beverage product or feedstuff comprises one or more sugars, sugar alcohols, sugar acids, or sugar derivatives; and one or more amino acids or derivatives or salts thereof, or a compound comprising an amino group (e.g. thiamine).
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the food product, beverage product or feedstuff in amounts sufficient to produce a food-like aroma and/or flavour when the food product, beverage product or feedstuff is heated.
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the food product, beverage product or feedstuff in amounts sufficient to produce one or more volatile compounds selected from 1,3-dimethyl benzene; p-xylene; ethylbenzene; 2-Heptanone; 2-pentyl furan; Octanal; 1,2-Octadecanediol; 2, 4-diethyl-l -Heptanol; 2-Nonanone; Nonanal; l-Octen-3-ol; 2- Decanone; 2-Octen-l-ol, (E)-; 2,4-dimethyl-Benzaldehyde; 2,3,4,5-Tetramethylcyclopent-2-en-l-ol, 1- octanol, 2-heptanone, 3-octanone, 2,3-octanedione, 1 -pentanol
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the food product, beverage product or feedstuff in amounts sufficient to produce one or more volatile compounds selected from 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-l -hexanol, 1- octanol, trans-2-octen-l-ol and 1-nonanol when the food product, beverage product or feedstuff is heated.
• the food product, beverage product or feedstuff comprises an extracted lipid from Mortierella spp. comprising phospholipids.
• the food product, beverage product or feedstuff may comprise about 2.5% or less dry Mortierella spp. biomass by weight, or an equivalent amount of wet biomass.
• a method of producing a food product, beverage product or feedstuff comprising combining a) a combination of Mortierella spp. biomass or extracted lipid therefrom and one or more lactones and/or one or more 4-hydroxy acids, or b) a composition of the present invention; with one or more additional consumable ingredients.
• a method of imparting a food-like aroma and/or flavour to a food product, beverage product or feedstuff comprising contacting the food product, beverage product or feedstuff with a combination of Mortierella spp. biomass or extracted lipid therefrom and one or more lactones and/or one or more 4-hydroxy acids, or a composition of the invention, and heating the food product, beverage product or feedstuff and the combination or composition.
• a method of increasing food-like aromas and/or flavours associated with a food product, beverage product or feedstuff comprising contacting the food product, beverage product or feedstuff with a combination of Mortierella spp. biomass or extracted lipid therefrom and one or more lactones and/or one or more 4-hydroxy acids, or a composition of the invention, and heating the food product, beverage product or feedstuff and the combination or composition.
• the food product, beverage product or feedstuff is a meat or meat-like product.
• the Mortierella spp. biomass is present in the food product, beverage product or feedstuff or is contacted with the food product, beverage product or feedstuff in an amount of less than 5% dry Mortierella spp. biomass by weight, or an equivalent amount of wet biomass.
• the food-like aroma and/or flavour is a meaty aroma and/or flavour.
• the composition, food product, beverage product or feedstuff is heated to at least about 130°C and/or for at least about 1 hour.
• the use is for imparting a food-like (e.g. a meaty or meat-like) aroma and/or flavour to said composition, food product, beverage product or feedstuff.
• the Mortierella spp. is Mortierella alpina, Mortierella elongata or Mortierella exigua.
• the biomass or extracted lipid comprises phospholipids, optionally wherein the phospholipids comprise one or more esterified co6 fatty acids (e.g. arachidonic acid (ARA), dihomo-gammalinolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-co6 (DPA-co6) or y-linolenic acid (GLA)).
• esterified co6 fatty acids e.g. arachidonic acid (ARA), dihomo-gammalinolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-co6 (DPA-co6) or y-linolenic acid (GLA)
• the food product, beverage product or feedstuff is a meat or meat -like product, e.g. a burger, sausage, hot dog, mince or ground meat, steak, streak, strip, fillet, roast, breast, thigh, wing, meatloaf, finger, nugget, cutlet, cube, bacon, soup, gravy, sliced meat, meatballs, fish, fried fish or seafood or imitation thereof.
• the food product, beverage product or feedstuff is free from any animal or animal -derived ingredients.
• the food product, beverage product or feedstuff comprises an animal or animal-derived ingredients, optionally wherein the an animal or animal -derived ingredient is meat.
• the food product, beverage product or feedstuff comprises: one or more sugars, sugar alcohols, sugar acids, or sugar derivatives; and one or more amino acids or derivatives or salts thereof, or a compound comprising an amino group (e.g. thiamine).
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof (or the compound comprising an amino group) are present in the composition, food product, beverage product or feedstuff in amounts sufficient to produce a food-like aroma and/or flavour when the composition, food product, beverage product or feedstuff is heated.
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the food product, beverage product or feedstuff in amounts sufficient to produce one or more volatile compounds selected from 1,3-dimethyl benzene; p-xylene; ethylbenzene; 2-Heptanone; 2-pentyl furan; Octanal; 1,2-Octadecanediol; 2, 4-diethyl-l -Heptanol; 2-Nonanone; Nonanal; l-Octen-3-ol; 2-Decanone; 2- Octen-l-ol, (E)-; 2,4-dimethyl-Benzaldehyde; 2,3,4,5-Tetramethylcyclopent-2-en-l-ol, 1-octanol, 2- heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hex
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and the one or more amino acids or derivatives or salts thereof are present in the composition, food product, beverage product or feedstuff in amounts sufficient to produce one or more volatile compounds selected from 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2- ethyl- 1-hexanol, 1-octanol, trans-2-octen-l-ol and 1-nonanol when the composition, food product, beverage product or feedstuff is heated.
• composition, food product, beverage product or feedstuff that comprise Mortierella spp. biomass further comprises an extracted lipid from Mortierella spp. comprising phospholipids.
• the composition, food product, beverage product or feedstuff comprises about 2.5% or less dry Mortierella spp. biomass by weight, or an equivalent amount of wet biomass.
• an isolated strain of Mortierella sp. selected from: i) yNI0125 deposited under V21/019953 on 12 October 2021 at the National Measurement Institute Australia; ii) yNI0126 deposited under V21/019951 on 12 October 2021 at the National Measurement Institute Australia; iii) yNI0127 deposited under V21/019952 on 12 October 2021 at the National Measurement Institute Australia; and iv) yNI0132 deposited under V21/019954 on 12 October 2021 at the National Measurement Institute Australia.
• Figure 1 shows polyunsaturated fatty acid biosynthesis pathways.
• Figure 3 shows the profile of volatile compounds released by heating extracted lipids with a mixture of ribose and cysteine as in Example 5, Experiment 3, as measured by gas chromatographymass spectrometry (GC-MS). levels of each of the identified compounds are shown as the area percentage (%) of total identified compounds.
• GC-MS gas chromatographymass spectrometry
• Figure 4 shows the profile of volatile compounds released by Maillard reactions of mixtures comprising 2.5 or 5.0 mg of 18:0/18:1- phosphatidylcholine (PC) or ARA-PC as described in Example 5, Experiment 5, as measured by gas chromatography-mass spectrometry (GC-MS).
• PC phosphatidylcholine
• ARA-PC ARA-PC
• Figure 5 shows the results of a sensory evaluation of meatiness of food samples comprising textured vegetable protein and varying amounts of Mortierella alpina biomass.
• Figure 7 shows the combined meatiness and pleasantness results of a sensory evaluation of food samples comprising textured vegetable protein and varying amounts of Mortierella alpina biomass.
• Figure 8 shows the meatiness results of a sensory evaluation of samples comprising a Maillard reaction matrix at varying concentrations and Mortierella alpina biomass.
• Figure 9 shows the pleasantness results of a sensory evaluation of samples comprising a Maillard reaction matrix at varying concentrations and Mortierella alpina biomass.
• Figure 10 shows the combined meatiness and pleasantness results of a sensory evaluation of samples comprising a Maillard reaction matrix at varying concentrations and Mortierella alpina biomass.
• Figure 11 shows the combined meatiness and pleasantness results of a sensory evaluation of samples comprising a Maillard reaction with Mortierella alpina biomass or Mortierella isabellina biomass.
• Figure 12 shows the results of a sensory evaluation of samples comprising a Maillard reaction with Mortierella alpina biomass and varying amounts of cystine.
• Figure 13 shows the results of a sensory evaluation of food samples comprising a Maillard reaction with Mortierella alpina biomass and varying amounts of cystine.
• Figure 14 shows the results of a sensory evaluation of samples comprising a Maillard reaction with Mortierella alpina biomass and varying amounts of dextrose.
• Figure 16 shows the results of a sensory evaluation of food samples comprising a Maillard reaction with Mortierella alpina biomass and varying combinations of cysteine, cystine, ribose and dextrose.
• a and “an” refer to one or to more than one (i.e. to at least one) of the grammatical object of the article.
• an element means one element or more than one element.
• phospholipid refers to an amphipathic molecule, having a hydrophilic head and a hydrophobic tail, that has a glycerol backbone esterified to a phosphate “head” group and two fatty acids which provide the hydrophobic tail.
• the phosphate group can be modified with simple organic molecules such as choline, ethanolamine or serine. Due to their charged headgroup at neutral pH, phospholipids are polar lipids, having some solubility in solvents such as ethanol in addition to solvents such as chloroform. Phospholipids are a key component of all cell membranes. They can form lipid bilayers because of their amphiphilic characteristic.
• the three acyl groups esterified in a TAG molecule are referred to as being esterified in the sn-1, sn-2 and sn-3 positions, referring to the positions in the glycerol backbone of the TAG molecule.
• the sn-1 and sn-3 positions are chemically identical, but biochemically the acyl groups esterified in the sn-1 and sn-3 positions are distinct in that separate and distinct acyltransferase enzymes catalyse the esterifications.
• a “meat-like flavour and/or aroma”, or a “meat-associated flavour and/or aroma” or a “meaty flavour and/or aroma” refers to flavours and/or aromas that are the same as or are similar to one or more meats, such as beef, steak, chicken, for example roasted chicken or chicken skin, pork, lamb, duck, venison, chicken or other meat soup, meat broth or liver.
• Such aromas are typically detected by human volunteers, for example by a qualified sensory panel.
• Meat -like or meat-associated flavours and/or aromas can also be detected by assessing volatile compounds arising after the cooking of the composition or food.
• Volatile compounds indicative of meat -like or meat-associated aromas and flavours are known in the art and include those exemplified herein, including but not limited to 1,3- dimethyl benzene; p-xylene; ethylbenzene; 2-Heptanone; 2-pentyl furan; Octanal; 1,2 -Octadecanediol; 2, 4-diethyl-l -Heptanol; 2-Nonanone; Nonanal; l-Octen-3-ol; 2-Decanone; 2-Octen-l-ol, (E)-; 2,4- dimethyl-Benzaldehyde; 2,3,4,5-Tetramethylcyclopent-2-en-l-ol, 1 -octanol, 2-heptanone, 3-octanone, 2,3-
• compositions for food and beverage products and feedstuff s
• compositions of the invention may include food products, beverage products or feedstuffs.
• compositions encompasses non-food compositions and compositions that are food products, beverage products or feedstuffs.
• the compositions are concentrated liquid or solid “flavouring compositions”, which can be added to other ingredients to produce a food product, beverage product or feedstuff with a desired flavour.
• the term composition is used interchangeably with food product, beverage product or feedstuff.
• the invention relates to a composition that is capable of producing a food-like aroma and/or flavour when heated, the composition comprising: a) biomass, e.g. Mortierella spp. biomass, or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy fatty acids; c) one or more sugars, sugar alcohols, sugar acids, or sugar derivatives; and d) one or more amino acids or derivatives thereof, or a compound comprising an amino group (e.g. thiamine).
• compositions, food products, beverage products or feedstuffs of the present disclosure are suitable for human or animal consumption, typically at least human consumption.
• the present invention relates to compositions as well as to food products, beverage products or feedstuffs, including food products, beverage products or feedstuffs comprising compositions of the present invention.
• the compositions of the present invention may be incorporated into food products, beverage products or feedstuffs to provide a desired food-like aroma.
• the food products, beverage products or feedstuff are suitable for human or animal consumption, typically at least human consumption.
• a food product, beverage product or feedstuff is a preparation for human or animal consumption which when taken into the body (a) serves to nourish or build up tissues or supply energy; and/or (b) maintains, restores or supports adequate nutritional status or metabolic function.
• a “food product” may be generally considered to include solid, semi-solid, or savoury liquid products
• a “beverage product” may be generally considered to include liquid drinkable products
• feedstuff’ may be considered to generally include animal, such as livestock food. It will be appreciated that there is overlap in the meaning of the terms “food product”, “beverage product” and “feedstock” and the terms may, in some circumstances, be used interchangeably.
• the food or beverage product or feedstuff is a meat or fish substitute product, i.e. a food or beverage product intended to imitate a food or beverage product which typically would contain meat or fish, for example for use in a vegetarian or vegan diet.
• the food or beverage product or feedstuff may be a product which includes meat or fish, and a composition of the present invention may be included to provide additional or alternative flavours or aromas to the product.
• the food or beverage product or feedstuff product may comprise meat obtained from an animal and/or cultivated or cultured meat (i.e. meat that has been produced by cultivating animal cells in vitro).
• the food or beverage product or feedstuff product is a blend of meat (e.g. meat obtained from an animal and/or cultivated or cultured meat) and non-animal protein (e.g. plant or mcrobial protein).
• Suitable food or beverage products or feedstuffs include but are not limited to meat or fish substitutes or meat or fish-based products, soup bases, stew bases, snack foods, bouillon powders, bouillon cubes, flavour packets, seasoning or frozen food products.
• the food or beverage product may be, or may be intended to imitate, for example, burgers, sausages, hot dogs, mince or ground meat, steaks, streaks, strips, fillets, roasts, breasts, thighs, wings, meatloaf, fingers, nuggets, cutlets, cubes, bacon, soup, gravy, sliced meat, meatballs, fish, fried fish or seafood.
• Biomass and/or lipids extracted from the biomass disclosed herein and/or compositions of the present invention may be used to modulate the flavour and/or aroma of a food or beverage product or feedstuff, by enhancing or altering the flavour and/or aroma of the food or beverage product or feedstuff.
• biomass and/or any extracted lipids disclosed herein and/or compositions of the present disclosure may enhance or alter the flavour and/or aroma of a food or beverage product or feedstuff, such as by enhancing meaty, fishy or vegetable flavour and/or aromas or by introducing such flavour and/or aromas to food or beverage products or feedstuffs.
• the biomass and/or extracted lipids disclosed herein, or the compositions, food or beverage products or feedstuffs of the present disclosure are intended to be added as an ingredient to a separate product to enhance or modulate the taste and/or aroma of the separate product to which it is added, for example by enhancing the meatiness or fishiness of the separate product or by altering the aroma or flavour of a product.
• Biomass and/or extracted lipids disclosed herein, or compositions, food or beverage products or feedstuffs of the present disclosure can be used to modulate, by enhancing or altering, the taste and/or aroma profile of, for example, meat replicas, meat substitutes, tofu, instantan, mock duck or a gluten based vegetable product, textured vegetable protein such as textured soy protein, pork, fish, lamb, or poultry products such as chicken or turkey products, and can be applied to the other food product before or during cooking.
• using the biomass and/or extracted lipids disclosed herein, or compositions, food or beverage products or feedstuffs described herein can provide a particular meaty taste and smell, for example, the taste and smell of beef, to a non-meat product or to a poultry product.
• compositions e.g. concentrated flavouring compositions of the present disclosure comprise less than 20% protein derived from a source other than the Mortierella spp. (or other microbial) biomass of the invention, optionally less than 15%, less than 10%, less than 5% or no protein other than protein provided by the Mortierella spp. (or other microorganism) biomass.
• food products, beverage products and feedstuffs of the present disclosure may optionally comprise added protein in an amount of greater than 5%, 10%, 15%, 20%, 25%, 30%, 35% or 40%.
• Suitable sugars, sugar alcohols, sugar acids, or sugar derivatives will be well known to a person skilled in the art.
• the sugars, sugar alcohols, sugar acids, or sugar derivatives are suitable for use in Maillard reactions for food, beverage or feed uses.
• the sugars, sugar alcohols, sugar acids, or sugar derivatives are a component other than the microorganism or a component thereof, and the amino acids or derivatives or salts thereof, even if the biomass or component thereof itself comprises sugars, sugar alcohols, sugar acids, or sugar derivatives.
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives are present in the composition at an amount of per kg of dry compositions or slurries, or per L in the case of liquid compositions, from about 1 mmol to about 1000 mmol, for example from about 5 mmol to about 500 mmol, about 5 mmol to about 300 mmol, about 20 mmol to about 500 mmol, about 20 mmol to about 300 mmol, about 5 mmol to about 200 mmol, about 5 mmol to about 100 mmol, about 5 mmol to about 80 mmol, from about 5 mmol to about 70 mmol, about 10 mmol to about 70 mmol, about 15 mmol to about 70 mmol, or about 30 mmol to about 60 mmol, the amount being measured based on the weight or volume of the composition excluding/before addition of biomass or any extracted lipids.
• amino acids or derivatives or salts thereof used in the present invention are suitable for use in Maillard reactions for food, beverage or feed uses.
• the amino acids or derivatives or salts thereof are a component other than the microorganism (e.g. Mortierella spp. biomass) or a component thereof, and the sugar, sugar alcohol, sugar acid, or sugar derivative, even if the biomass or component thereof itself comprises amino acids or derivatives or salts thereof.
• the one or more amino acids or derivatives or salts thereof contain a free amino group.
• reference to an amino acid or derivative means a free amino acid that is not present in the context of a peptide or protein.
• Suitable amino acids and derivatives thereof include cysteine, cystine, homocysteine, selenocysteine, a cysteine sulfoxide, allicin, selenocysteine, methionine, isoleucine, leucine, lysine, phenylalanine, threonine, tryptophan, 5-hydroxytryptophan, valine, arginine, histidine, alanine, asparagine, aspartate, glutamate or glutamic acid, glutamine, monosodium glutamate, glycine, proline, serine, taurine and tyrosine.
• the amino acid is cysteine and/or cystine.
• compositions comprise cysteine.
• the composition, food product, beverage product or feedstuff comprises glutamic acid or a salt thereof.
• the composition, food product, beverage product or feedstuff comprises glutamic acid or a salt thereof (e.g. monosodium glutamate, or MSG) in addition to the one or more amino acids or derivatives or salts thereof; for example, compositions, food products, beverage products or feedstuffs comprise, according to some embodiments, glutamic acid or a salt thereof and cysteine (or cystine) or a salt thereof.
• the one or more amino acids or derivatives or salt thereof comprises a sulfur-containing amino acid (e.g.
• cysteine methionine, homocysteine, or taurine
• Salts of amino acids which are suitable for human or animal consumption and therefore for incorporation into compositions, food products, beverage products or feedstuffs of the present disclosure will be familiar to and readily selected by a person skilled in the art.
• amino acid “derivative” is intended to encompass amino acids which include a chemical modification, for example by introducing a group in a side chain of an amino acid, such as a nitro group in tyrosine or iodine in a tyrosine, by conversion of a free carboxylic group to an ester group or to an amide group, by converting an amino group to an amide by acylation, by acylating a hydroxy group rendering an ester, by alkylation of a primary amine rendering a secondary amine, or linkage of a hydrophilic moiety to an amino acid side chain.
• Other derivatives may be obtained by oxidation or reduction of the side -chains of the amino acid.
• each of the one or more amino acids or derivatives or salts thereof are present in the composition at an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, from about 1 mmol to about 500 mmol, from about 1 mmol to about 300 mmol, from about 1 mmol to about 200 mmol, from about 2 mmol to about 200 mmol, from about 2 mmol to about 100 mmol, from about 2 mmol to about 200 mmol, from about 5 mmol to about 100 mmol, from about 5 mmol to about 80 mmol, from about 5 mmol to about 70 mmol, from about 10 mmol to about 70 mmol, from about 15 mmol to about 70 mmol, from about 30 mmol to about 60 mmol, from about 1 mM to about 50 mM, or from about 1 30 mM, the amount being calculated based on the weight or volume of the composition excluding/before
• the one or more amino acids or derivatives or salts thereof are present in the composition at an amount of per kg of dry compositions or slurries, or per L in the case of liquid compositions, of at least about 1 mmol, for example at least about 5 mmol, for example at least about 10 mmol, for example at least about 15 mmol, for example at least about 20 mmol, the amount being measured based on the weight or volume of the composition excluding/before addition of biomass and/or any extracted lipids.
• the one or more amino acids comprises cysteine or cystine.
• each of the one or more amino acids or derivatives or salts thereof are present in the food, feedstuff or beverage at a total amount of, per kg of dry composition or slurry, or per L in the case of liquid foods (e.g.
• the one or more amino acids comprises cysteine and/or cystine.
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and one or more amino acids or derivatives or salts thereof or a compound comprising an amino group are present in the compositions of the present disclosure or the food products, beverage products or feedstuffs of the present disclosure in amounts sufficient to product food-like aromas, such as meat-like aromas, when heat is applied to the compositions, food products, beverage products or feedstuffs.
• a compound comprising an amino group e.g. thiamine
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and one or more amino acids or derivatives or salts thereof are present in the compositions of the present disclosure or the food products, beverage products or feedstuffs of the present disclosure in amounts sufficient to produce one or more volatile compounds selected from 1,3-dimethyl benzene; p-xylene; ethylbenzene; 2-Heptanone; 2-pentyl furan; Octanal; 1,2-Octadecanediol; 2,4-diethyl-l -Heptanol; 2- Nonanone; Nonanal; l-Octen-3-ol; 2-Decanone; 2-Octen-l-ol, (E)-; 2,4-dimethyl-Benzaldehyde; 2,3,4,5-Tetramethylcyclopent-2-en-l-ol, 1-octanol, 2-heptanone, 3-octanone, 2,3-octaned
• the one or more sugars, sugar alcohols, sugar acids, or sugar derivatives and one or more amino acids or derivatives or salts thereof are present in the compositions of the present disclosure or the food products, beverage products or feedstuffs of the present disclosure in amounts sufficient to produce one or more volatile compounds selected from 2-heptanone, 3-octanone, 2,3- octanedione, 1 -pentanol, 1 -hexanol, 2 -ethyl- 1 -hexanol, 1 -octanol, trans-2-octen-l-ol and 1 -nonanol when heat is applied to the composition, food product, beverage product or feedstuff.
• the composition comprises comprise glutamic acid or a salt or derivative thereof (e.g. MSG) in addition to the one or more amino acids or derivatives or salts thereof.
• the glutamic acid or salt thereof is present in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, from about 1 mmol to about 200 mmol or from about 2 mmol to about 100 mmol, for example 2 mmol to about 50 mmol, for example from about 2 mmol to about 40 mmol, for example from about 2 mmol to about 40 mmol, for example from about 5 mmol to about 40 mmol, for example from about 5 mmol to about 30 mmol, the amount being calculated based on the volume of the composition excluding/before addition of biomass and/or any extracted lipid.
• the glutamic acid or salt thereof is present in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, at least about 1 mmol, for example at least about 2 mmol, for example at least about 3 mmol, for example at least about 4 mmol, for example at least about 5 mmol, for example at least about 7 mmol, for example at least about 10 mmol, the amount being measured based on the weight or volume of the composition excluding/before addition of biomass and/or any extracted lipids.
• the glutamic acid salt is monosodium glutamate.
• the food or beverage product or feedstuff of the invention comprises glutamic acid or a salt or derivative thereof (e.g. MSG) in addition to one or more other amino acids or derivatives or salts thereof, and the glutamic acid is present in an amount of, per kg of dry composition or slurry, or per L in the case of liquid compositions (e.g.
• beverages from about 0.1 mmol to about 20 mmol, about 0.1 mmol to about 15 mmol, about 0.3 mmol to about 15 mmol, about 0.5 mmol to about 10 mmol, about 0.5 mmol to about 5 mmol, or about 1 mmol to about 5 mmol, the amount being calculated based on the volume of the food, feedstuff or beverage excluding/before addition of microbial biomass and/or lipids.
• the composition comprises glutamic acid or a salt thereof and a further amino acid or salt or derivative thereof selected from cysteine and cystine (or a salt or derivative therof), wherein the glutamic acid or salt thereof is present in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, from about 1 mmol to about 200 mmol or from about 2 mmol to about 100 mmol, for example 2 mmol to about 50 mmol, for example from about 2 mmol to about 40 mmol, for example from about 2 mmol to about 40 mmol, for example from about 5 mmol to about 40 mmol, for example from about 5 mmol to about 30 mmol; and the cysteine or cystine (or a salt or derivative therof) is present in an amount of from about 5 mmol to about 200 mmol or from about 5 mmol to about 100 mmol, for example from about 5 mmol to about 80 m
• the composition comprises glutamic acid or a salt thereof and a further amino acid or salt or derivative thereof selected from cysteine and cystine, wherein the glutamic acid or salt thereof is present in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, at least about 1 mmol, for example at least about 2 mmol, for example at least about 3 mmol, for example at least about 4 mmol, for example at least about 5 mmol, for example at least about 7 mmol, for example at least about 10 mmol, and the cysteine or cystine (or a salt or derivative therof) is present in an amount of at least about 5 mmol, for example at least about 10 mmol, for example at least about 15 mmol, for example at least about 20 mmol, the amount being calculated based on the weight or volume of the composition excluding/before addition of biomass and/or any extracted lipid.
• the food or beverage product or feedstuff comprises glutamic acid or a salt thereof and a further amino acid or salt or derivative thereof selected from cysteine and cystine (or a salt or derivative therof), wherein the glutamic acid or salt thereof is present in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, from about 0.1 mmol to about 20 mmol or from about 0.2 mmol to about 10 mmol, for example 0.2 mmol to about 5 mmol, for example from about 0.2 mmol to about 4 mmol, for example from about 0.5 mmol to about 4 mmol, for example from about 0.5 mmol to about 3 mmol; and the cysteine or cystine (or a salt or derivative therof) is present in an amount of from about 0.5 mmol to about 50 mmol or from about 0.5 mmol to about 20 mmol, for example from about 0.5 mmol to about 10
• the composition comprises glutamic acid or a salt thereof and a further amino acid or salt or derivative thereof selected from cysteine and cystine (or a salt or derivative therof), wherein the glutamic acid or salt thereof is present in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid, at least about 0.1 mmol, for example at least about 0.2 mmol, for example at least about 0.3 mmol, for example at least about 0.4 mmol, for example at least about 0.5 mmol, for example at least about 0.7 mmol, for example at least about 1 mmol, and the cysteine or cystine (or a salt or derivative therof) is present in an amount of at least about 0.5 mmol, for example at least about 1 mmol, for example at least about 1.5 mmol, for example at least about 2 mmol, the amount being calculated based on the weight or volume of the composition excluding/before addition of biomass and/or any extracted lipid.
• Compositions, food products, beverage products or feedstuffs of the present invention may, according to some preferred embodiments, comprise a source of iron.
• Iron may enhance the meaty flavour and/or aromas produced by compositions, food products, beverage products or feedstuffs of the present invention.
• the source of iron is an iron salt, preferably a ferrous salt. Any iron salt suitable for consumption may be used, and such salts will be familiar to a person skilled in the art, for example a chelated form of iron.
• the source of iron is iron (II) fumarate. Iron (II) fumarate is available, for example, as iron tablets from APOHEALTH Pty Ltd (NSW, Australia).
• the source of iron is a component other than the biomass or a component thereof, the amino acid or salt or derivative thereof, and the sugar, sugar alcohol, sugar acid, or sugar derivative, even if the biomass or component thereof itself comprises iron.
• the compositions of the present invention comprise a source of iron in an amount equivalent to, per kg of dry compositions or slurries, or per L in the case of liquid compositions, up to about 100 mg of elemental iron.
• the compositions comprise a source of iron in an amount equivalent to up to about 50 mg, for example from about 20 to about 50 mg, for example from about 30 to about 40 mg, the concentration being calculated based on the volume of the composition excluding/before addition of biomass and/or any extracted lipid.
• compositions, food products, beverage products or feedstuffs of the present disclosure comprise an aqueous component. Presence of some moisture in the compositions facilitates production of food-like flavour and/or aromas upon heating.
• An aqueous component may be water.
• the aqueous component may be, for example, an aqueous buffer such as a phosphate buffer.
• the compositions, food products, beverage products or feedstuffs of the present disclosure comprise an aqueous component aside from any water contained incidentally in other components, such as any moisture present in microorganism biomass.
• Compositions of the present disclosure are, in some preferred embodiments, not dry or substantially dry.
• the composition, food product, beverage product or feedstuff is a dry composition.
• the composition, food product, beverage product or feedstuff is a liquid composition.
• the composition, food product, beverage product or feedstuff is in the form of a powder, solution, suspension, slurry or emulsion.
• the composition, food product, beverage product or feedstuff is provided excluding an aqueous component (i.e. a dry composition), and an aqueous component (such as water) is added to the composition, food product, beverage product or feedstuff prior to or together with heating.
• compositions, food products, beverage products or feedstuffs of the present disclosure may further comprise an aqueous buffer.
• a buffer maintains the pH of the composition, and provides moisture to the composition, food product, beverage product or feedstuff which, as discussed above, facilitates production of food-like flavour and/or aromas upon heating.
• the buffer may be a phosphate buffer.
• the buffer may be a buffer at a pH of from about 5.0 to about 7, for example from about 5 to about 6, for example at about 5.3 or about 6.0.
• the buffer is a phosphate buffer at a pH of about 6.0.
• compositions, food products, beverage products or feedstuffs of the present invention may further comprise one or more additional components.
• additional components may include oils (for example vegetable oils), free fatty acids, alpha-hydroxy acids, dicarboxylic acids, nucleosides, nucleotides, vitamins, peptides, protein hydrolysates, extracts, phospholipids, lecithin, carbohydrates, and organic molecules.
• the compositions of the present invention which may be flavouring compositions (e.g.
• a food-like flavour such as a meat-like flavour
• a food-like flavour such as a meat-like flavour
• the compositions, food products, beverage products or feedstuffs comprise thiamine or derivatives thereof.
• the thiamine can be present as the compound containing an amino group and thus enable the Maillard reaction.
• the compositions, food products, beverage products or feedstuffs of the present invention may comprise the a) the biomass, b) sugars, sugar alcohols, sugar acids, or sugar derivatives and c) thiamine.
• the compositions, food products, beverage products or feedstuffs of the present invention may comprise a) the biomass, b) sugars, sugar alcohols, sugar acids, or sugar derivatives, c) one or more amino acids or derivatives or salts thereof, and d) thiamine.
• Thiamine may therefore enhance the meaty aroma and/or flavour produced by compositions, food products, beverage products or feedstuffs of the present invention.
• thiamine may be present in the compositions, per kg of dry compositions or slurries, or per L in the case of liquid compositions, in an amount of from about 0.1 to about 20 mmol, for example from about 0.1 to about 10 mmol, for example from about 0.5 to about 5 mmol, for example from about 0.5 to about 3 mmol.
• thiamine is present in an amount of at least about 0.1 mmol, for example at least about 0.2 mmol, for example at least about 0.3 mmol, for example at least about 0.4 mmol, for example at least about 0.5 mmol, for example at least about 0.7 mmol, the concentration being calculated based on the weight or volume of the composition excluding/before addition of biomass and/or any extracted lipid.
• thiamine may be present in the food, feedstuffs or beverages, per kg of dry composition or slurry, or per L in the case of liquid compositions (e.g.
• beverages in an amount of from about 0.01 to about 2 mmol, for example about 0.01 to about 1 mol, for example from about 0.05 to about 0.5 mmol, or about 0.1 to about 0.3 mmol, the amount being calculated based on the weight or volume of the food, feedstuff or beverage excluding/before addition of microbial biomass and/or lipids.
• thiamine is present in the food, feedstuff or beverages in an amount of at least about 0.01 mmol, for example at least about 0.02 mmol, for example at least about 0.03 mmol, for example at least about 0.04 mmol, for example at least about 0.05 mmol, for example at least about 0.07 mmol, the concentration being calculated based on the weight or volume of the food, feedstuff or beverage excluding/before addition of biomass and/or any extracted lipid.
• the compositions, food products, beverage products or feedstuffs further comprise a yeast extract.
• a “yeast extract” is generally understood to refer to the water-soluble portion of autolyzed yeast and is available commercially from various suppliers; see, for example Sigma Aldrich, Catalog No. Y1625 Yeast Extract.
• a yeast extract does not contain yeast whole cell biomass. Presence of a yeast extract may enhance meaty aromas and/or flavours produced by the composition, food product, beverage product or feedstuff when heated.
• the yeast extract may be a general unflavoured yeast extract, or may be, for example, a beef flavoured or roast chicken skin flavoured yeast extract.
• the composition, food product, beverage product or feedstuff is suitable for producing food-like aromas and/or flavours which are meat-like aromas and/or flavours
• the composition, food product, beverage product or feedstuff comprises a yeast extract.
• the presence of a yeast extract may enhance meaty aromas and/or flavours produced by compositions, food products, beverage products or feedstuffs of the present disclosure, as observed in the Examples below.
• the yeast extract is present in the composition in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, from about 10 g to about 200 g, for example from about 15 g to about 200g, for example from about 20 g to about 200g, for example from about 30 g to about 200g, for example from about 40 g to about 200g, for example from about 50 g to about 200g, for example from about 50 g to about 180 g, for example from about 60 g to about 180 g, the amount being calculated based on the volume of the composition excluding/before addition of biomass and/or any extracted lipid from microorganisms.
• the yeast extract is present in the composition in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, at least about 5g, for example at least about 7 g, for example at least about 10 g, for example at least about 15 g, for example at least about 20 g, for example at least about 25 g, for example at least about 30 g, for example at least about 40 g, for example at least about 50 g, for example at least about 60 g.
• the yeast extract is present in the composition in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, at least about 30 g.
• the yeast extract is present in the composition in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, at least about 5g, for example at least about 7 g, for example at least about 10 g, for example at least about 15 g, for example at least about 20 g, for example at least about 25 g, for example at least about 30 g, for example at least about 40 g, for example at least about 50 g, for example at least about 60 g.
• the yeast extract is present in the composition in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, at least about 30 g.
• the yeast extract is present in the food, feedstuff or beverage in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid, at least about 0.5g, for example at least about 0.7 g, for example at least about 1 g, for example at least about 1.5 g, for example at least about 2 g, for example at least about 2.5 g, for example at least about 3 g, for example at least about 4 g, for example at least about 5 g, for example at least about 6 g.
• the yeast extract is present in the food, feedstuff or beverage in an amount of, per kg of dry compositions or slurries, or per L in the case of liquid compositions, at least about 3 g.
• the composition, food product, beverage product or feedstuff does not comprise a yeast extract. Since the presence of a yeast extract may enhance meaty aromas and/or flavours produced by the composition, food product, beverage product or feedstuff, a yeast extract maybe omitted when, for example, an alternative food-like flavour and/or aroma is desired, such as a fishy or a vegetable or herby aroma and/or flavour. The absence of a yeast extract may prevent the potential masking of the desired aroma and/or flavour such as a fish-like aroma and/or flavour by meatlike aromas and/or flavours enhanced by the presence of a yeast extract.
• the food-like aroma and/or flavour is a fish-like aroma and/or flavour, a vegetable, and/or a herby aroma and/or flavour
• the composition, food product, beverage product or feedstuff does not comprise a yeast extract.
• the compositions, food products, beverage products or feedstuffs further comprise one or more herbs and/or spices.
• compositions comprising herbs such as for example Fenugreek (Trigonella foenum-graecum), were found in some instances to enhance vegetable, soupy and/or herby flavour and/or aromas produced by the compositions of the present invention.
• a herb and/or spice is understood in the art to refer to a plant part or extract possessing aromatic properties.
• a herb is understood to refer to leafy, green or flowering parts of a plant
• a spice is typically understood to refer to other parts of a plant (usually dried), including seeds, bark, roots and fruit.
• the herb or spice may be in the form of whole plant parts, or chopped, ground or rolled plant parts, or dried, for example as a powder.
• the one or more herbs and/or spices comprise Fenugreek.
• Fenugreek has also been claimed to contain several bioactive components and can bring health benefits to consumers.
• the one or more herbs and/or spices comprise Fenugreek leaf.
• compositions, foods, feedstuffs or beverages comprise: a) Mortierella spp. biomass (or other microbial biomass) or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy fatty acids; c) glucose and/or ribose; d) cysteine and/or cystine and/or methionine and/or thiamine; and e) an aqueous component.
• compositions, foods, feedstuffs or beverages comprise: a) Mortierella spp. biomass (or other microbial biomass) or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy fatty acids; c) glucose and/or ribose; d) cysteine and/or cystine and/or methionine and/or thiamine; e) glutamic acid or a salt thereof; and f) an aqueous component.
• the compositions comprise: a) Mortierella spp. biomass (or other microbial biomass) or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy fatty acids; c) glucose and/or ribose; d) cysteine and/or cystine; e) yeast extract; f) glutamic acid or a salt thereof; g) thiamine; and h) an aqueous component.
• the compositions comprise: a) Mortierella spp. biomass (or other microbial biomass) or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy fatty acids; c) glucose and/or ribose; d) cysteine and/or cysteine; e) a source of iron, for example an iron salt; f) glutamic acid or a salt thereof; g) thiamine; h) an aqueous component, for example as an aqueous buffer, for example a phosphate buffer, for example having a pH of from about 5 to about 6, for example of about 5.3 or about 6.0; and i) optionally a yeast extract.
• the compositions comprise: a) Mortierella spp. biomass (or other microbial biomass) or extracted lipid therefrom; b) one or more lactones and/or one or more 4-hydroxy fatty acids; c) ribose; d) cysteine; e) a source of iron, for example an iron salt; f) glutamic acid or a salt thereof; g) thiamine; h) an aqueous component, for example as an aqueous buffer for example a phosphate buffer, for example having a pH of from about 5 to about 6, for example of about 5.3 or about 6.0; and i) optionally a yeast extract.
• the composition comprises (aside from the biomass and/or any extracted lipid) the components set out in “matrix A” or “matrix B” in Table 1 below, or “matrix C” in Table 2 below (as prepared from the stock ingredients set out below), or the components of Matrix A, B or CB in equivalent concentrations if otherwise prepared.
• matrix A or “matrix B” in Table 1 below
• matrix C in Table 2 below
• the present disclosure further relates to a method of producing a food product, beverage product or feedstuff comprising combining biomass and/or any extracted lipid therefrom disclosed herein, optionally with a lactone and/or a 4-hydroxy fatty acid, with one or more additional consumable ingredients; or a composition of the present disclosure with one or more additional consumable ingredients.
• Suitable additional ingredients which may be included in such food products, beverage products or feedstuffs are discussed below.
• the biomass and any optional extracted lipids comprising phospholipids disclosed herein or composition can be combined with the other consumable ingredient by mixing, applying it to the surface of the other ingredient, or by soaking/marinating the other ingredient.
• the food, feedstuff or beverage product is prepared by (a) heating biomass and/or any extracted lipid therefrom with a lactone and/or 4-hydroxy fatty acid, or a composition of the invention and (b) mixing the products from (a) with other food, feedstuff or beverage consumable ingredients, or by (a) mixing biomass and/or extracted lipid therefrom with a lactone and/or 4-hydroxy fatty acid; or a composition of the present disclosure with other food, feedstuff or beverage consumable ingredients and (b) heating the mixture resulting from (a).
• the food product, beverage product or feedstuff may either be in a solid or liquid form, and may be intended to be kept frozen, refrigerated or at room temperature prior to cooking.
• the food product, beverage product, feedstuff or composition is provided as a dry product excluding an aqueous component, and an aqueous component (such as water) is added to the food product, beverage product or feedstuff or composition prior to, during or subsequent to heating, especially prior to heating.
• the composition may be in a solid or liquid form, to be admixed with, or added to a food or beverage product or feedstuff pror to heating, or after heating one or both of the composition and the food or beverage product or feedstuff.
• the compositon may be in solid or liquid form, and may represent, for a example, a concentrated mix, to be mixed with or added to a food or beverage product or feedstuff.
• the mix may be, for example, in the form of a powder, particulate or granulated mix.
• the food or beverage product or feedstuff may include edible macronutrients, protein, carbohydrate, vitamins, and/or minerals in amounts desired for a particular use.
• the amounts of these ingredients will vary depending on whether the composition is intended for use with normal individuals or for use with individuals having specialized needs, such as individuals suffering from metabolic disorders and the like.
• the food or beverage product or feedstuff of the present invention contains no components derived from an animal.
• at least some of the ingredients are plant material or material derived from a plant.
• Such embodiments are advantageously suitable for a vegan or vegetarian diet.
• the food or beverage product or feedstuff can be soy-free, wheat-free, yeast-free, MSG-free, and/or free of protein hydrolysis products.
• the food or beverage product or feedstuff preferably has a food-like taste or aroma, such as a meaty or fishy aroma, as imparted by the biomass and any extracted lipids comprising phospholipids disclosed herein or composition of the present disclosure.
• suitable additional ingredients with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates and proteins.
• suitable additional ingredients with nutritional value include, but are not limited to, macronutrients such as edible fats, carbohydrates and proteins.
• suitable edible fats other than phospholipids contained in compositions of the present disclosure include, but are not limited to, palm oil, canola oil, corn oil, sunflower oil, safflower oil, coconut oil, borage oil, fungal oil, black current oil, soy oil, blends thereof and mono- and diglycerides.
• examples of carbohydrates include (but are not limited to): glucose, edible lactose, and hydrolyzed starch.
• proteins include (but are not limited to) soy proteins, mycoproteins (e.g Rhiza my coproteins), instantan, pea protein, potato protein, electrodialysed whey, electrodialysed skim milk, milk whey, or the hydrolysates of these proteins.
• the protein is a textured or structured protein product, which comprises protein fiber networks and/or aligned protein fibers that produce meat -like textures.
• vitamins and minerals may be added to the food or beverage product or feedstuff of the present invention: calcium, phosphorus, potassium, sodium, chloride, magnesium, manganese, iron, copper, zinc, selenium, iodine, and Vitamins A, E, D, C, and the B complex. Other such vitamins and minerals may also be added.
• Food and beverage products and feedstuffs described herein also can include a natural coloring agent such as turmeric or beet juice, or an artificial coloring agent such as azo dyes, triphenylmethanes, xanthenes, quinines, indigoids, titanium dioxide, red #3, red #40, blue #1, or yellow #5.
• a natural coloring agent such as turmeric or beet juice
• an artificial coloring agent such as azo dyes, triphenylmethanes, xanthenes, quinines, indigoids, titanium dioxide, red #3, red #40, blue #1, or yellow #5.
• Food and beverage products and feedstuffs described herein also can include meat shelf-life extenders such as carbon monoxide, nitrites, sodium metabisulfite, Bombal, vitamin E, rosemary extract, green tea extract, catechins and other anti-oxidants.
• meat shelf-life extenders such as carbon monoxide, nitrites, sodium metabisulfite, Bombal, vitamin E, rosemary extract, green tea extract, catechins and other anti-oxidants.
• the components utilized in the food or beverage product or feedstuff of the present invention can be of semi-purified or purified origin.
• semi-purified or purified is meant a material which has been prepared by purification of a natural material or by de novo synthesis.
• compositions, food products, beverage products and feedstuffs described herein can be assessed for flavour and aroma using human panelists. It will be appreciated that assessment of aromas by panellists will involve a certain degree of subjectivity, and that precise descriptions of aromas and whether they are appealing/unappealing may differ somewhat between panellists. Nonetheless, trends and the general nature of aromas can be effectively assessed by panellists.
• the evaluations can involve eyeing, feeling, chewing, smelling and tasting of the product to judge product appearance, color, integrity, texture, flavour, and mouth feel, etc., preferably at least smelling the composition, food or beverage product or feedstuff to assess aroma. Panelists can be served samples under red or under white light. A scale can be used to rate the overall acceptability or quality of the food or specific quality attributes such meatiness, texture, and flavour.
• the compositions, food products, beverage products and feedstuffs can also be presented to animals such as pet animals to assess their attractiveness to those animals.
• a food product, beverage product or feedstuff or composition described herein can be compared to another product (e.g., meat or meat substitute) based upon olfactometer readings.
• the olfactometer can be used to assess odor concentration and odor thresholds, odor suprathresholds with comparison to a reference gas, hedonic scale scores to determine the degree of appreciation, or relative intensity of odors.
• volatile chemicals identified using GCMS can be evaluated. For example, a human can rate the experience of smelling the chemical responsible for a certain peak. This information could be used to further refine the profile of flavour and aroma compounds produced by the food products, beverage products, feedstuffs or compositions of the present invention.
• the present invention further relates to methods of producing a composition, food product, beverage product or feedstuff, by combining a biomass with any one or more of the ingredients described above, optionally in the amounts as described above.
• compositions, food products, beverage products or feedstuffs of the present disclosure produce a food-like flavour and/or aroma, preferably a meat-like flavour and/or aroma, when heated.
• Heating refers to increasing the temperature of the composition, food products, beverage products or feedstuffs, for example to above room temperature, to any temperature and for any amount of time sufficient to produce food-like flavour and/or aromas.
• the temperature is raised high enough and long enough for Maillard reactions to occur between amino groups and sugars in the composition, with additional reactions occurring with lipids, preferably phospholipids, in the composition, food products, beverage products or feedstuffs to produce the food-like flavour and/or aromas.
• a suitable temperature and period of time may be readily carried out by the skilled person.
• “heated” or “heating” or similar is to be understood as meaning heating under conditions sufficient for producing a food-like aroma, unless otherwise specified.
• the heat may be applied to the composition of the invention prior to it being contacted with the food product, beverage product or feedstuff or after the application to the food product, beverage product or feedstuff or both.
• Such heating of the composition, or the food product, beverage product or feedstuff may take place for example in an oven, frypan, wok or similar, or in a barbeque.
• compositions or food products, beverage products or feedstuffs Whilst the precise temperature to which a composition, food product, beverage product or feedstuff should be heated to produce a food-like flavour and/or aroma, preferably a meat -like flavour and/or aroma, may vary depending on, for example, the precise composition and the time for which the composition is heated and the amount of composition being heated, in some embodiments, the compositions or food products, beverage products or feedstuffs produce a food-like flavour and/or aroma when heated to a temperature of at least about 100°C, for example at least about 110°C, for example at least about 120°C or at least about 130°C, or at least about 140°C. In particular embodiments, the compositions or food products, beverage products or feedstuffs produce a food-like flavour and/or aroma when heated to about 140°C.
• compositions and food products, beverage products or feedstuffs of the present disclosure may produce a food-like flavour and/or aroma, preferably a meat-like flavour and/or aroma when heated for varying amounts of time, depending on, for example, the temperature to which the compositions or food products, beverage products or feedstuffs are heated, the precise nature of the composition, food product, beverage product or feedstuff and the amount of composition, food product, beverage product or feedstuff being heated. Nonetheless, in some embodiments the compositions, food products, beverage products or feedstuffs may produce a food-like flavour and/or aroma when heated for at least 5 or at least 10 minutes, for example at least 15 minutes.
• the compositions, food products, beverage products or feedstuffs may produce a food-like flavour and/or aroma when heated for at least about 30 minutes, for example at least about 45 minutes. In some embodiments, the compositions, food products, beverage products or feedstuffs may produce a food like flavour and/or aroma when heated for at least about 1 hour, for example about 1 hour. Preferably, the heat is applied for a length of time whereby a burnt flavour and/or aroma is not produced, as is understood by a person of skill in the art.
• the compositions, food products, beverage products or feedstuffs of the present invention may produce a food-like flavour and/or aroma, preferably a meat-like flavour and/or aroma, when heated for at least 5 or at least 10 minutes at a temperature of at least about 100°C.
• the compositions, food products, beverage products or feedstuffs of the present invention may produce a food-like flavour and/or aroma when heated for at least 30 minutes at a temperature of at least about 100°C.
• the compositions, food products, beverage products or feedstuffs of the present invention may produce a food-like flavour and/or aroma when heated for at least 30 minutes at a temperature of at least about 120°C.
• the food-like flavours and/or aromas produced by compositions, food products, beverage products or feedstuffs of the present disclosure may, according to preferred embodiments, include a meat-like flavour and/or aroma.
• the food-like flavour and/or aroma may be an aroma of cooked meat or meat-based foods.
• the food-like flavour and/or aroma may be of beef, steak, chicken, for example roasted chicken or chicken skin, pork, lamb, duck, venison, chicken or other meat soup, meat broth, liver, or generally “meaty”.
• the meat -like flavour or arma is a chicken (e.g. roast chicken or pan-fried chicken), beef (e.g. roast or pan-fried beef), or pork (e.g.
• roast or pan-fried pork flavour or aroma.
• aromas are typically detected by human volunteers, for example by a qualified sensory panel.
• a composition, food product, beverage product or feedstuff is said to produce a food-like or meat-like flavour and/or aroma when at least one third, for example at least one half, of the number of volunteers on a tasting/smelling panel detect a food-like or meat -like flavour and/or aroma in a double-blind test of the composition, food product, beverage products or feedstuff.
• the food-like flavours and/or aromas produced by compositions, food products, beverage products or feedstuffs of the present disclosure may, according to some embodiments, include a fishlike flavour and/or aroma, for example a cooked fish flavour and/or aroma, for example a fried fish flavour and/or aroma.
• the food-like flavours and/or aromas produced by compositions, food products, beverage products or feedstuffs of the present disclosure may include a vegetable and/or herbal flavour and/or aroma, for example a cooked vegetable and/or herby flavour and/or aroma, for example a soup, mushroom, onion, vegetable, herbal or roasted vegetable flavour and/or aroma.
• the composition, food product, beverage product or feedstuff includes ribose and the food-like flavour and/or aroma includes a meaty, for example cooked meat -like flavour and/or aroma, and/or a fishy, for example a cooked or fried fish-like flavour and/or aroma.
• volatile compounds indicative of meat-like or meat-associated aromas and flavours include, for example volatile compounds such as 1,3-dimethyl benzene; p-xylene; ethylbenzene; 2-Heptanone; 2-pentyl furan; Octanal; 1,2-Octadecanediol; 2,4-diethyl-l -Heptanol; 2- Nonanone; Nonanal; l-Octen-3-ol; 2-Decanone; 2-Octen-l-ol, (E)-; 2,4-dimethyl-Benzaldehyde; 2,3,4,5-Tetramethylcyclopent-2-en-l-ol, 1-octanol, 2-heptanone, 3-octanone, 2,3-octanedione, 1- pentanol, 1-hexanol, 2-ethyl-l -hexanol, trans-2-octen-l-
• volatile compounds indicative of meat-like or meat-associated aromas and flavours include 2-heptanone, 3-octanone, 2,3-octanedione, 1-pentanol, 1-hexanol, 2-ethyl-l -hexanol, 1-octanol, trans-2-octen-l-ol and 1-nonanol are produced.
• volatile compounds indicative of meat-like or meat-associated aromas and flavours include 1-pentanal, 3-octanone, 2- octen-l-ol, 1-nonanol and 1-octanol, and optionally 1, 3 -bis( 1,1 -dimethylethyl) -benzene are produced.
• the composition, food product, beverage product or feedstuff includes glutamic acid, for example glutamic acid in addition to a further amino acid or salt or derivative thereof such as cysteine, and the food-like flavour and/or aroma includes a meaty, for example cooked meat-like, and/or a fishy, for example a cooked or fried fish-like flavour and/or aroma.
• the composition, food product, beverage product or feedstuff includes a yeast extract and the food-like flavour and/or aroma includes a meaty, for example cooked meat-like flavour and/or aroma.
• the composition, food product, beverage product or feedstuff does not include a yeast extract and the food -like flavour and/or aroma includes a fish-like, for example cooked fish or fried fish-like, vegetable and/or herby aroma and/or flavour.
• the microorganism is Mortierella spp., for example Mortierella alpina, Mortierella elongata or Mortierella exigua and the food-like flavour and/or aroma includes a meat-like flavour and/or aroma, such as a beef-like flavour and/or aroma.
• the composition, food product, beverage product or feedstuff includes one or more herbs and/or spices, for example fenugreek, for example fenugreek leaf, and the food-like flavour and/or aroma includes a vegetable, soupy and/or herby flavour and/or aroma.
• herbs and/or spices for example fenugreek, for example fenugreek leaf
• the food-like flavour and/or aroma includes a vegetable, soupy and/or herby flavour and/or aroma.
• compositions, food products, beverage products or feedstuffs of the present disclosure may produce food-like flavours as well as food-like aromas.
• Such food-like flavours may be flavours corresponding to the food-like aromas disclosed herein.
• reference to aromas herein may be understood, according to certain aspects, to also refer to aromas and/or flavours where appropriate.
• the combination of biomass or extracted lipid therefrom and one or more lactones and/or one or more 4-hydroxy fatty acids, or composition of the present invention is incorporated into the food or beverage product or feedstuff prior to or during heating, such that when the food or beverage product is heated (for example during cooking), the biomass and lactone combination and any optional extracted lipids comprising phospholipids disclosed herein or composition produces the associated food-like aromas (by way of Maillard and associated reactions).
• heating causes cyclisation of the 4- hydroxy fatty acids to form lactones for the production of said food-like aromas.
• the biomass or extracted lipid therefrom and lactone and/or 4-hydroxy fatty acid combination, or composition of the present invention is heated prior to incorporation in or addition to a food or beverage product or feedstuff.
• the biomass or extracted lipid therefrom and lactone and/or 4- hydroxy fatty acids combination have been heated prior to incorporation into the food, such as in the presence of a sugar and an amino acid or derivative, under conditions suitable to produce one or more (e.g.
• volatile compounds indicative of meat-like or meat-associated aromas and flavours for example volatile compounds such as 1,3-dimethyl benzene; p-xylene; ethylbenzene; 2- Heptanone; 2-pentyl furan; Octanal; 1,2-Octadecanediol; 2,4-diethyl-l -Heptanol; 2-Nonanone; Nonanal; l-Octen-3-ol; 2-Decanone; 2-Octen-l-ol, (E)-; 2,4-dimethyl-Benzaldehyde; 2, 3,4,5- Tetramethylcyclopent-2-en-l-ol, 1-octanol, 2-heptanone, 3-octanone, 2, 3 -octanedione, 1-pentanol, 1- hexan
• free C6 fatty acid upon entering the fungal cell, can be activated with coenzyme A by the acyl-CoA synthases Fatlp and Faalp-Faa4p.
• the C6-C0A can serve as a substrate for glycerolipid pathway acyl transferase enzymes like glycero-3-phosphate acyltransferase, 1- acylglycerol-3- phosphate acyltransferase, diacylglycerol acyltransferase, phospholipid diacylglycerol acyltransferase and be incorporated into triglycerides or phospholipids.
• the microorganism used in accordance with the present invention may be a Mortierella spp.
• the microorganism may be Mortierella elongata, Mortierella alpina, Mortierella exigua or Mortierella isabellina.
• Other Mortierella spp. include M. humilis, M. camargensi, M. lignicola, M. zonata, M. sepedonioides, M. stylospora, M. polycephala, M. alliacea, M. claussenii, M. globalpina, M. globulifera, M. pusilia, M. strangulata, M. rostafinskii, M. bainieri, M.
• the microorganism is a fungus other than a Mortierella spp., and in particular a fungus having arachidonic acid present in an amount of at least or about 10%, 20%, 30%, 40% or 50% of the total fatty acid content of the polar lipid of the yeast.
• fungi include Pithium spp., such as Pithium ultimum, Pithium debaryanum, and Pithium insidiosum.
• the microorganism is Yarrowia lipolytica strain W29 or genetically- modified derivatives thereof. As demonstrated by the Examples below, such microorganisms are particularly effective in producing food-like, in particular meaty, aromas.
• the microorganism is an alga, such as a microalga, or Bacillariophyceae. More particularly, the microorganism is an algae with arachidonic acid esterified in polar lipids, preferably esterified in phospholipids, e.g. where arachidonic acid is present in an amount of at least or about 10%, 20%, 30%, 40% or 50% of the total fatty acid content of the polar lipid.
• the microrganisms utilised in the present invention comprise arachidonic acid.
• the arachidonic acid is esterified in polar lipids, preferably esterified in phospholipids.
• the microorganism, such as the Mortierella spp comprises arachidonic acid esterified in polar lipids, preferably esterified in phospholipids, where arachidonic acid is present in an amount of at least or about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% of the total fatty acid content of the polar lipid.
• arachidonic acid is present in an amount of about 10% to about 60% (e.g. 20% to 50%), or is present as at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% or at least about 55%) of the total fatty acid content of the polar lipid.
• the microorganism biomass or microorganism from which the extracted lipid is extracted may be Yarrowia lipolytica, for example strain W29, and may be prepared by a culturing process, in particular a fermentation process comprising feeding with arachidonic acid.
• the present invention involves the use of microorganism biomass, such that compositions, food products, beverage products and feedstuffs of the present invention comprise microorganism biomass.
• the microorganisms typically Mortierella spp.
• dry biomass may be approximately 25% of the mass of “wet” biomass).
• biomass encompasses matter containing at least some whole cells of the microorganisms, rather than only components which have been separated therefrom, but may contain both whole cells and cell components.
• biomass also encompasses "low- TAG biomass", as described below.
• Microorganisms/biomass such as obtained by a fermentation process, may have been processed by, for example, washing, drying, heat inactivation, freezing and/or freeze drying, but typically will still contain at least some, preferably most, of the whole cell material of the microorganism.
• biomass may be referred to as “whole cell biomass”, but it will be appreciated that the microorganism cells contained in compositions of the present invention may be present in a disrupted form, for example having undergone physical or chemical lysis; the biomass/microorganism will typically still contain substantially all of the cell material.
• biomass and “microorganism” do not refer to, for example, oils or proteins extracted or isolated from microorganisms and separated from the other components of the cells.
• compositions comprising microorganisms comprising phospholipids i.e. microorganism biomass
• have been found to be particularly effective in producing an enhanced foodlike aroma such as meaty or fishy aromas when heated.
• the biomass used herein is low-TAG biomass.
• the term “low-TAG biomass” refers to biomass from a microorganism (e.g. Mortierella spp.), wherein the biomass has been processed to remove some, most, substantially all, or all of the triacylglycerol (TAG) (e.g. at least about 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 95% of the TAG has been removed), while retaining most, substantially all, or all of the polar lipids (including phospholipids) and other cellular material (e.g. proteins and carbohydrates).
• TAG triacylglycerol
• low-TAG biomass can be produced by delipidating whole cell biomass, and then adding most, substantially all, or all of the polar lipids (including phospholipids) back to the delipidated biomass (as demonstrated herein).
• “low-TAG biomass” is typically equivalent to whole cell biomass but without all, substantially all or most of the TAG that is present in the whole cell biomass.
• the low-TAG biomass comprises less than about 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.005% (w/w), triacylglycerol TAG.
• “low-TAG biomass’” encompasses an absence of all or substantially all of the TAG present in whole cell biomass (i.e. “TAG-free biomass”).
• TAG-free biomass refers to biomass from a microorganism, wherein the biomass does not include TAG or includes less than about 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.005% (w/w) TAG.
• the microorganism included in compositions of the present disclosure may be in suspension, frozen, dried or any other suitable form.
• the microorganism cells may be alive or dead, or a mix of living and dead cells, for example at least 99% of the cells being dead.
• the cells may have been heat- treated in order to render them incapable of replicating.
• the extracted lipid can be a relatively crude composition obtained by, for example, lysing the microbial cells (e.g. Mortierella sp.) and separating the lipid, or a more purified composition where most, if not all, of one or more or each of the water, nucleic acids, proteins and carbohydrates derived from the cells have been removed.
• microbial cells e.g. Mortierella sp.
• a more purified composition where most, if not all, of one or more or each of the water, nucleic acids, proteins and carbohydrates derived from the cells have been removed.
• Lipid extraction and purification methods are well known in the art.
• An extracted lipid may comprise, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95% (w/w) lipid by weight of the composition.
• an extracted lipid comprises between about 10% and 95% lipid by weight, for example between about 10% and about 50%, or between about 50% and 95%, lipid by weight.
• the lipid may be solid or liquid at room temperature (25 °C), or a mixture of the two; when liquid it is considered to be an oil, when solid it is considered to be a fat.
• the extracted lipid may comprise only the lipid extracted from the microorganism (e.g. Mortierella sp.), or may be blended with another lipid. In an embodiment, extracted lipid has not been blended with another lipid produced from another source, for example, animal lipid.
• the microorganism e.g. Mortierella sp.
• extracted lipid has not been blended with another lipid produced from another source, for example, animal lipid.
• the extracted lipid may contain all lipids initially present in the microbial cell, or may contain only a fraction of lipids initially present in the microbial cell.
• an extracted lipid may have been processed to remove some or all of a particular type of lipid, for example to remove some or all neutral lipid (such as triacylglycerols (triglycerides, ‘TAG’) and to retain polar lipids (such as phospholipids, and/or other polar lipids such as cephalins, sphingolipids (sphingomyelins and glycosphingolipids).
• the extracted lipid may have been processed to remove some or all of polar lipids (such as phospholipids) and retain only or mostly neutral lipid (such as triacylglycerols (triglycerides, ‘TAG’).
• the extracted lipid comprises one or more sterols such as, for example from yeast cells, ergosterol and/or ergosterol esters.
• the extracted lipid comprising polar lipids and non-polar lipids.
• the non-polar lipids are present in the extracted lipid in a lower amount than the polar lipids.
• the extracted lipid comprises polar lipids, and in particular, phospholipids.
• Phospholipids are amphipathic molecules, having a hydrophilic head and a hydrophobic tail, comprising a glycerol backbone esterified to a phosphate “head” group and two fatty acids which provide the hydrophobic tail.
• the phospholipids of the present invention comprise one or more esterified co6 fatty acids. Biosynthesis of co6 fatty acids in organisms such as microalgae, mosses and fungi usually occurs as a series of oxygen -dependent desaturation and elongation reactions ( Figure 1).
• the polar lipid comprises a total fatty acid (TFA) content which comprises co6 fatty acids, wherein at least some of the co6 fatty acids are esterified in the form of phospholipids in the polar lipid, the co6 fatty acids comprising arachidonic acid (ARA), dihomo-gammalinolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-co6 (DPA-co6) or y- linolenic acid (GLA), or any combination thereof,
• the phospholipids in the polar lipid comprise phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS), each comprising one or more of ARA, DGLA, EDA, DTA, DPA-co6 and GLA, and optionally one or more
• the polar lipid comprises a total fatty acid (TFA) content which comprises 06 fatty acids, wherein at least some of the 06 fatty acids are esterified in the form of phospholipids in the polar lipid, and wherein the 06 fatty acids comprise two, three, four or more fatty acids selected from the group consisting of arachidonic acid (ARA), dihomo-gammalinolenic acid (DGLA), eicosadienoic acid (EDA), docosatetraenoic acid (DTA), docosapentaenoic acid-06 (DPA-06) and y-linolenic acid (GLA),
• ARA arachidonic acid
• DGLA dihomo-gammalinolenic acid
• EDA eicosadienoic acid
• DTA docosatetraenoic acid
• DPA-06 docosapentaenoic acid-06
• GLA y-linolenic acid
• the polar lipid comprises a total monounsaturated fatty acid content comprising oleic acid and palmitoleic acid (Cl 6: 1 A9cis).
• the polar lipid comprises a total saturated fatty acid content comprising palmitic acid and stearic acid
• the phospholipids form part of a polar lipid (whether contained within microorganism biomass or extracted from a microorganism as an extracted polar lipid or broader lipid), which may comprise, consist essentially of or consist of phospholipids, wherein:
• the polar lipid comprises a total monounsaturated fatty acid content comprising oleic acid and palmitoleic acid (Cl 6: 1 A9cis).
• the polar lipid comprises DPA-06
• one or more or all of GLA, DGLA, EDA, ARA and DTA are also present.
• the polar lipid comprises EDA and one, two or all three of arachidonic acid (ARA), dihomo-gammalinolenic acid (DGLA) and y-linolenic acid (GLA) esterified in the polar lipid, and wherein the level of EDA in the polar lipid is at least about 1 % of the total fatty acid content of the polar lipid.
• ARA arachidonic acid
• DGLA dihomo-gammalinolenic acid
• GLA y-linolenic acid
• the polar lipid lacks one, two, three or all four of C16:2, C16:3co3, EPA and DHA. In a preferred embodiment, the polar lipid lacks C16:3co3, EPA and DHA. In a further embodiment, the polar lipid also lacks ALA or has less than 1 % ALA.
• the extracted lipid comprises three, four or more fatty acids selected from the group consisting of ARA, DGLA, EDA, DTA, DPA-06 and GLA, such as a combination of ARA, DGLA and GLA, or a combination of fatty acids other than ARA, DGLA and GLA, preferably a combination of ARA, DGLA, GLA and at least one of EDA, DTA and DPA-06.
• the phospholipids comprise at least two, at least three or all four of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and phosphatidylserine (PS), each comprising one, two, three or more than three of ARA, DGLA, EDA, DTA, DPA-06 and GLA, and optionally one or more or all of phosphatidic acid (PA), phosphatidylglycerol (PG) and cardiolipin (Car), each comprising one, two, three or more than three of ARA, DGLA, EDA, DTA, DPA-06 and GLA.
• PC phosphatidylcholine
• PE phosphatidylethanolamine
• PI phosphatidylinositol
• PS phosphatidylserine
• PA phosphatidic acid
• PG phosphatidylglycerol
• Car cardiolipin
• the polar lipid comprises myristic acid (C14:0) in an amount of less than about 2% by weight of the total fatty acid content of the polar lipid. In a preferred embodiment, the polar lipid comprises myristic acid (C14:0) in an amount of less than about 1% by weight of the total fatty acid content of the polar lipid.
• stearic acid is present at a level of less than about 14% or less than about 12% or less than about 10% of the total fatty acid content of the polar lipid. In preferred embodiments, stearic acid is present at a level of less than about 7% or less than about 6% or less than about 5%, preferably less than 4% or less than 3%, of the total fatty acid content of the polar lipid.
• ARA is present in an amount of about 10% to about 60%, about 10% to about 30%, about 10% to about 25%, about 15% to about 60%, about 20% to about 60%, or about 30% to about 60%, by weight of the TFA content of the polar lipid. In preferred embodiments, ARA is present in an amount of about 20% to about 60%, or about 30% to about 60%, or about 40% to about 60%, or about 50% to about 60%, by weight of the TFA content of the polar lipid. In some embodiments, ARA is present in an amount of at least or about 10%, 15%, 20%, 25% or 30% by weight of the TFA content of the polar lipid.
• the polar lipid comprises one or more or all of EDA, DTA and DPA- 06.
• the saturated fatty acid content of the polar lipid comprises one or more or all of lauric acid (C12:0), myristic acid (C14:0), a C15:0 fatty acid, C20:0, C22:0 and C24:0, preferably comprising C14:0 and C24:0 or C14:0, C15:0 and C24:0, more preferably comprising C14:0, C15:0 and C24:0 but not C20:0 and C22:0.
• Cl 5:0 is absent from the polar lipid, or C15:0 is present in the polar lipid in an amount of less than about 3%, preferably less than about 2% or less than about 1%, of the total fatty acid content of the polar lipid.
• palmitic acid is present in the polar lipid in an amount of about 3% to about 45%, or about 10% to about 40%, or about 20% to about 45%, of the total fatty acid content of the polar lipid.
• Phospholipids may be separated from a broader lipid fraction extracted from microorganisms by any suitable method, for example by use of solvent extraction as described in Example 2 below.
• lipids may be extracted from a lipid source by dissolving in ethanol or another alcohol such as isopropanol, evaporating the ethanol or other alcohol, and phospholipids then further separated from neutral lipids by precipitation of phospholipids from cold acetone.
• Lipid extracted from the microbial cells may be subjected to normal oil processing procedures.
• the term "purified" when used in connection with lipids disclosed herein means that that the extracted lipid has been subjected to one or more processing steps of increase the purity of the lipid component.
• a purification step may comprise one or more or all of the group consisting of: degumming, deodorising, decolourising, drying and/or fractionating the extracted oil, as described below.
• the term “purified” does not include a transesterification process or other process which alters the fatty acid composition of the lipid or oil of the invention so as to change the fatty acid composition of the total fatty acid content.
• the fatty acid composition of the purified lipid is essentially the same as that of the unpurified lipid.
• the oil is typically transferred to a glass container and flushed with argon before being stored under refrigeration. This treatment improves the colour of the oil and removes a majority of the volatile substances or odorous compounds including any remaining free fatty acids, monoacylglycerols and oxidation products.
• yeast extract is understood in the art to generally refer to the water-soluble portion of autolyzed yeast and typically does not contain phospholipid fractions (see, for example, Sigma Aldrich, Catalog No. Y1625 Yeast Extract).
• yeast extract includes a composition that is sold commercially and labelled as a yeast extract. These are water-soluble fractions of yeast cells comprising amino acids, carbohydrates, vitamins and minerals and are typically sold in a dry powdered form.
• microorganisms may be genetically modified by suitable methods, to contain a desired amount or profile of phospholipids, for example increased amounts of phospholipids/polar lipids and/or increased amounts of co-6 fatty acid esterified in phospholipids.
• an “elongase” refers to the polypeptide that catalyses the condensing step in the presence of the other members of the elongation complex, under suitable physiological conditions. It has been shown that heterologous or homologous expression in a cell of only the condensing component ("elongase") of the elongation protein complex is required for the elongation of the respective acyl chain.
• YPD medium is a rich medium which contains 10 g/L yeast extract (Sigma Aldrich, Catalog No. Y1625), 20 g/L peptone (Sigma Aldrich, Catalog No. P0556) and 20 g/L glucose (Sigma Aldrich, Catalog No. G7021).
• YPD plates contain, in addition, 20 g/L agar.
• SD-Ura medium contained Yeast Synthetic Drop-out Medium (Sigma Catalog No. Y1501) at the recommended amount per litre. This medium was supplemented with uracil as required.
• SD agar plates contained 6.7 g/L yeast nitrogen base, 20 g/L glucose and 20 g/L agar.
• the medium used for preparing seed cultures for larger scale yeast cultures was a defined medium (DM-Gluc), having a base medium (BM) containing per litre 10.64 g potassium di-hydrogen orthophosphate (KH2PO4), 4.0 g di-ammonium hydrogen orthophosphate ((NFLOzHPO ⁇ and 1.7 g citric acid (monohydrate). These ingredients were dissolved in about 70% of the required volume of water that had been purified by reverse osmosis, adjusted to pH 6.0 with 2 M NaOH, and made up to the required volume using purified water.
• the BM was sterilised at 121°C for 20 min and cooled to room temperature.
• the fermentation medium (FM) for yeast cultures of 2 L or more in volume also used the BM as base medium unless otherwise stated.
• the required volume was added to the bioreactor and sterilised at 121 °C for a 60 min fluid cycle for an autoclavable bioreactor or 30 min for a steam-in-place bioreactor, and cooled to 30°C.
• the following ingredients were added, per litre of base medium: 121 ml of 660 g/L glucose (autoclaved), giving a final concentration of 80 g/L, 5 ml of IM magnesium sulphate heptahydrate (autoclaved), 5 ml of Trace metal solution (see below, filter sterilised), 5 ml 15 g/L thiamine hydrochloride (filter sterilised) and 50 ml of 200 g/L ammonium chloride (filter sterilised).
• the glucose, magnesium, trace metal solution and thiamine solution were mixed and added to the bioreactor together.
• the pH was checked, normally slightly less than 6.0.
• a pH controller was used to add ammonia solution to the medium and bring the pH to 6.0.
• Small scale (50 ml) and larger scale yeast cultures of 2 L or more for inducing more TAG synthesis were also grown in a defined medium containing glycerol at 8% (w/v) and having a lower nitrogen content (DM-Glyc-LowN).
• This medium was the same as DM-Gluc except that the glucose was replaced with 80 g/L glycerol (final concentration) as carbon source and the (NH4)2HPO4 content was reduced to 2.0 g/L or even 0.5 g/L, as stated.
• starter cultures were grown in either YPD medium or SD-Ura medium, with addition of uracil and any amino acids if required, for 24-48 h. A sample of the starter culture was centrifuged and the cells used to inoculate the larger culture. These cultures were incubated for 48-96 h and the pH maintained at 6.0 unless otherwise stated.
• the Trace metal stock solution (TMS) used in the media described above contained, per litre: 2.0 g CuSO 4 .5H 2 O, 0.08 g Nal, 3.0 g MnSO 4 .H 2 O, 0.2 g NaMoO 4 .2H 2 O, 0.02 g H3BO3, 0.5 g COC1 2 .6H 2 O, 7.0 g ZnCl 2 , 22.0 g FeSO 4 .7H 2 O, 0.50 g CaSO 4 .2H 2 O, and 1 ml of sulphuric acid.
• the reagents were added in the listed order. Addition of the sulphuric acid resulted in dissolution of the calcium sulphate.
• the trace metal solution was filtered sterilised through a 0.2 pm filter and stored at 2-8 °C in a bottle wrapped in aluminium foil.
• yNI0121 The fungal strain described herein as yNI0121 (Mucor hiemalis) has been deposited with National Measurement Institute, Port Melbourne, VIC 3207, Australia on 4 February 2021 under the Budapest Treaty and has been designated the following Deposit Number: yNI0121 Deposit Accession number V22/001757.
• lipid substrates such as ARA, DGLA, y-linolenic acid (GLA) or other fatty acids
• cell pellets were washed successively with 1 ml of 1% tergitol (v/v), 1 ml of 0.5 % tergitol and a final wash with 1 ml water to remove any remaining substrate from the exterior of the cells and freeze-dried as described above.
• total cellular lipid was extracted from yeast cells such as S. cerevisiae or Y. lipolytica by using a method modified from Bligh and Dyer (1959). Approximately 50 mg freeze- dried cells were homogenized with 0.6 ml of a mixture of chloroform/methanol (2/1, v/v) with 0.5 g zirconium oxide beads (Catalog No. ZROB05, Next Advance, Inc., USA) in a 2 ml Eppendorf tube using a Bullet Blender Blue (Next Advance, Inc. USA) at speed 6 for 5 min. The mixture was then sonicated in an ultrasonication water bath for 5 min and 0.3 ml 0.1 M KC1 was added.
• PL and TAG were fractionated from about 100 mg of total lipid by loading the lipid on 18 cm lines on each of eight TLC plates (Silica gel 60; Catalog No. 1.05626.0001, Merck, Darmstadt, Germany) and chromatographed with a solvent mixture consisting of hexane/diethylether/acetic acid (70:30:1, v:v:v). An aliquot of a lipid standard containing TAG, DAG, FFA and MAG (18-6A; NuChek Inc, USA) was run in parallel to assist with identifying the lipid bands.
• fatty acid methyl esters were prepared from total extracted lipid or the purified TAG or polar lipid fractions, including PL samples, by treatment with 0.7 ml 1 N methanolic -HC1 (Sigma Aldrich, Catalog No. 90964) in a 2 ml glass vial having a PTTE-lined screw cap at 80°C for 2 h.
• a known amount of heptadecanoin (Nu-Chek Prep, Inc., Catalog No. N-7-A, Waterville, MN, USA) dissolved in toluene was added to each sample before the treatment as an internal standard for quantification.
• the individual FAMEs were identified and quantified by GC using an Agilent 7890A GC (Palo Alto, California, USA) with a 30 m SGE-BPX70 column (70% cyanopropyl polysilphenylene- siloxane, 0.25 mm inner diameter, 0.25 pm film thickness), a split/splitless injector and an Agilent Technologies 7693 Series auto sampler and injector, and a flame ionisation detector (FID). Samples were injected in split mode (50:1 ratio) at an oven temperature of 150°C.
• the ethanol from the combined supernatants was evaporated using a SR- 100 rotary evaporator (Buchi, Switzerland) operating at 400 rpm with a vacuum of 15 mbar, with the chiller set at -16°C and the waterbath at 37°C.
• the lipid recovered from the krill oil probably still contained a small amount of solvent. Nevertheless, the recovery of essentially 100% indicated that the krill oil from the capsules was highly enriched for PL to begin with.
• Example 4 Feeding omega-6 fatty acids to yeasts and incorporation into polar lipids
• ARA was added aseptically by overpressure to the medium in the form of 12.5 g ARA (NuChek) as free fatty acid in 300 ml of 17% Triton-X-100 to give a final concentration in the fermenter of 0.5 g/L ARA and 0.2% Triton-X-100, with further addition of 100 ml of unhydrolyzed ARA oil to provide a concentration of 0.4% (v/v) unhydrolyzed ARA oil in addition to the FFA.
• a seed culture was prepared in 400 ml YM medium at 29°C with shaking at 180 rpm overnight, providing an inoculum having an GD600 of 4.23. When the medium temperature was 29°C, 400 mL of the seed culture was transferred to the fermenter by overpressure, providing an initial cell density (OD600) of 0.07 by calculation.
• the culture was terminated at late logarithmic growth phase to maximise polar lipid content and ARA incorporation and was not heat treated at the end of the fermentation.
• the cells were budding as observed by light microscopy and there were very few that stained with Methylene Blue, so the oil content and therefore the TAG content was low as intended.
• a final yield of 294 g of wet paste was obtained from the 21 L of culture, with approximately 72% water content i.e. approximately 28% w/w solids.
• the cell paste was frozen and then freeze dried in 3 batches to yield 73 g of dry yeast cake.
• the dry yeast cake was milled to a fine powder and dispensed as 3 portions - a 3 g portion for lipid analysis, a 35 g portion for food application trials and a 35 g portion for further processing to yield a crude lipid fraction.
• the cell density (GD600) of the inoculum was 9.19, so 200 mL was added to the 25 L medium in the fermenter to achieve a starting GD600 calculated at 0.08.
• the ARA oil and FFA were added as before.
• the pH dropped from an initial 7.08 at 0 h to 4.63 at 15.68 h but then started to increase in the last 30 min of the culturing.
• the culture might have reached stationary phase and glucose was depleted.
• the cells After the exhaustion of glucose, the cells might have started breaking down phospholipids for maintenance. It was therefore considered important to harvest the culture before it reached stationary phase.
• the culture was run without pH control for 16 hours, the pH naturally falling from pH 6.96 to 5.07 due to acid production from cellular metabolism.
• the cell density (dry weight) was 5.27 g/L at 16 h, while the OD600 increased from 0.07 to 12.1 at 16 h.
• the culture assimilated 4.5 g/L of glucose, which was 51% of the 8.9 g/L glucose supplied in the start medium.
• the harvested cells were heat inactivated at a temperature of 95°C for 3 min as before, yielding 584 g wet weight of biomass corresponding to 114 g dry weight.
• Lipid was extracted from freeze-dried samples and analysed as before. The total lipid content of the 16 h freeze-dried cells was 3.4%.
• the TLC analysis showed that more polar lipid was present than in experiment B012.
• the ARA level in the polar lipid and TAG fractions were 10.2% and 13.3%, respectively.
• the data for the fatty acid compositions are provided in Table 9.
• Table 7 Comparison of Y. lipolytica W29 cultures under different fermentation conditions Table 8. Fatty acid composition of polar lipids and TAG in Y. lipolytica after culturing with ARA, for experiments B005, B009, B012 and B013.
• Polar lipid samples were prepared by extraction from yeast cells supplemented with GLA or ARA and fractionation as described in Example 4. Samples of 8.0 mg of polar lipid from the ARA- fed cells, 7.6 mg from the GLA-fed cells, 9.0 mg from the control cells and 16.0 mg of polar lipid extracted from pork meat, each dissolved in chloroform, were transferred to 20 ml glass vials. The solvent was evaporated under nitrogen flow at room temperature. 2 ml of 0.1 M potassium phosphate buffer, pH 7.2, containing 4.5 mg/ml ribose (Catalog No. R9629, Sigma-Aldrich) and 5.0 mg/ml cysteine (Catalog No.
• Octanal was produced from the samples containing YL ARA, YL and soy lecithin, i.e. all three polar lipid samples, but not produced from the ARA-Oil and no lipid samples.
• the unsaturated alcohol l-octen-3-ol also produced in all the oil-containing samples tested (YL ARA, YL, soy lecithin and ARA Oil) may contribute to an herbaceous aroma resulted from thermal decomposition of methyl linoleate hydroperoxide.
• the vials were subjected to ultrasonication for 1 h at 40°C in a water bath to emulsify the mixtures and then incubated at 140°C for 1 h by placing the vials on aluminium foil inside an oven. After the vials were cooled, the volatile compounds in the headspace of each vial were analysed by solid-phase microextraction coupled with gas chromatography-mass spectrometry (HS-SPME-GCMS) as before. The reaction mixtures including 0.5 mg lipid showed peaks for the volatile compounds but at lower intensities than desired with some compounds being undetected.
• HS-SPME-GCMS gas chromatography-mass spectrometry
• HS-SPME-GCMS analysis of the volatiles produced after the heating step showed the presence of numerous compounds which were either increased or decreased in the mixtures having ARA-PC relative to the mixtures having 18:0/18: 1-PC or were present in one mixture and absent or not detected in the other mixture.
• the compound 2-pentyl thiophene has a characteristic aroma which has been described as chicken, roasted hazelnut or meaty.
• the compound 2-pentyl furan has an aroma described as a fruity, earthy or having a vegetable aroma.
• the inventors nevertheless tested this polar lipid in Maillard reactions, with the conditions as in Experiment 5 except using 15, 30 or 60 mg polar lipid per reaction in 2 ml volumes to increase the amount of ARA -polar lipid.
• the control polar lipid extract had been prepared from Y. lipolytica cells which had not been fed co6 fatty acids in the medium. Control reactions were also set up having aliquots of the polar lipid extracts but lacking the ribose and cysteine.
• the aromas from the reactions were smelled by three volunteers.
• the mixtures having the ARA-PL provided mild aromas that were described as “pork like, pork crackling, meaty, fatty” or “broiled chicken, milder aroma” or “like broiled fish” whereas the control mixtures having the polar lipid from Y. lipolytica not fed the ARA was described as being sulphurous or “burnt” in their aroma.
• the mixtures lacking ribose and cysteine were described as “burnt vegetable”.
• Y. lipolytica strain W29 cells producing polar lipids including PL were grown in 25 L cultures, either in the presence of ARA (Yl-ARA) in the growth medium or in the absence of ARA (Yl).
• the fatty acid composition of the polar lipid in the Y. lipolytica cells is shown in Table 10 for Experiments 2 and 3.
• ARA was present at 16.4% of the total fatty acid content of the polar lipid, with GLA at 1.4% and DGLA at 1.9%.
• the harvested cells were then freeze dried and the dried material milled to a powder. The cells were not heat treated or otherwise treated to kill or inactivate the cells.
• the inventors wished to test the dried yeast cells for the capacity to provide aroma compounds after the cells were heated in the presence of a sugar, for example D-xylose, and an amino acid, for example L-cysteine.
• a series of reactions were prepared to test the effect of different amounts of the sugar, the amino acid and varying amounts of freeze-dried cells (Table 13). Briefly, L-cysteine powder (Catalog No. 30089, Sigma -Aldrich), D-xylose powder (Catalog No. X1500, Sigma- Aldrich), sodium citrate dihydrate (Catalog No. W302600, Sigma- Aldrich), and wheat flour were weighed into 10 ml GC headspace analysis vials (Catalog No.
• vial 13 The loosened cap on vial 13 was presumed to have allowed escape of some of the volatile compounds during heating.
• Duplicate samples for vials 18-20 were prepared without the water, kept at ambient temperature for 5 or 7 days before the addition of water and then heated to 120°C for 60 min. These vials provided the same aroma results as vials 18-20 that had been prepared and heated immediately, then frozen for the week, showing that the mixtures can be stored stably for at least one week at room temperature.
• the heating time was also a factor to consider. Vials 14-16 and 17-19 were designed to compare this variable with 45 or 60 min heating. The shorter heating time resulted in a noticeably lighter coloured mixture while the longer cooking time produced considerably browner colour. This darkening effect also appeared to be correlated with cysteine levels with more cysteine generally resulting in a darker reaction as long as adequate sugar was present.
• Mortierella alpina is a filamentous and saprophytic fungus of the family Zygomycete which is commonly found to inhabit soils from temperate grasslands. Some strains of this species are used commercially to produce oils containing polyunsaturated fatty acids (PUFA), specifically the co6 fatty acids arachidonic acid (C20:4; ARA), linoleic acid (C18:2; LA) and y-linolenic acid (C18:3; GLA).
• PUFA polyunsaturated fatty acids
• ARA co6 fatty acids
• linoleic acid C18:2; LA
• y-linolenic acid C18:3; GLA
• Mucor hiemalis is a zygosporic fungus of the Order Mucorales that is ubiquitous in nature and can be found, for example, in unspoiled foods.
• the Biomes of Australian Soil Environments (BASE) project database is a database that contains integrated information about microbial diversity and function for microbial isolates from more than 1 ,400 soil samples taken from 902 locations across Australia. It includes associated metadata for all of the soil samples across extensive environmental gradients, including information from phylogenetic marker sequencing of bacterial 16S rRNA, archaeal 16S rRNA and eukaryotic 18S rRNA genes to characterise the diversity of microorganisms in community assemblages. Fungal diversity was informed by the 18S rRNA gene amplicon sequences.
• ITS internal transcribed spacer
• mycelia from the edge of each colony were transferred through agar slices to fresh MEA plates and incubated at ambient temperatures for 4 days. Colonies that appeared pure through visual inspection were inoculated into 5 ml of malt extract broth and grown at ambient temperature in a static culture for 5 days. A total of 67 fungal strains were thereby isolated from the three soil samples.
• Genomic DNA was isolated from each hyphal biomass using the YeaStar Genomic DNA kit (Zymo research, Catalog No. D2002).
• An internal transcribed spacer (ITS) was amplified through PCR as described by Ho and Chen, (2008) using oligonucleotide primers xMaFl GGAAGTAAAAGTCGTAACAAGG (SEQ ID NO: 2) and xMaF2 TCCCCGCTTATTGATATGC (SEQ ID NO: 3).
• the nucleotide sequence of the ITS from the amplicons from each isolate were determined by Sanger sequencing. The obtained sequences were compared to sequences within the NCBI repository using BLAST. The closest hits, with at least 95% nucleotide sequence identity for each isolate and often at 98% or 99% identity, were used to identify the species for each fungal isolate.
• the ITS regions amplified with primers xMaFl and xMaF2 produced amplicons having a length of between 639 and 647 basepairs for the Mucor hiemalis strains, between 668 and 672 basepairs for the Mortierella alpina strains, between 628 and 652 basepairs for the Mortierella sp. isolates, and between 640 and 659 for the two Mortierella elongata strains. The length of this ITS amplicon was therefore useful in helping to distinguish between the four species. Table 13. Species identities of isolated soil fungi.
• the Mortierella alpina strains yNI0133 to yNI0135 produced abundant ARA as well as GLA and DGLA.
• the ARA level in both the TAG and polar lipids was about 30% by weight of the total fatty acid content in those fractions.
• These M. alpina strains therefore did not exhibit any preference for accumulating the co6 PUFA in polar lipid relative to TAG.
• the GLA and DGLA levels were about 2% and about 6%, respectively, in TAG, and about 4-7% and about 2-4%, respectively, in the polar lipid. Compared to the ARA levels, this indicated that the M.
• alpina strains have efficient A6 elongase and A5 desaturase enzymes. Genes encoding such enzymes have been isolated from other strains of M. alpina (Huang et al., 1999; Knutzon et al., 1998). The Mortierella alpina strains also produced about 4-5% of C24:0 in the TAG fractions
• the presumed Mortierella sp. strains yNI0126 to yNI0130 produced ARA and DGLA in addition to GLA and accumulated these co6 PUFA in both TAG and polar lipids.
• the Mortierella sp. strains accumulated 2- to 4-fold more ARA in their polar lipid than in their TAG. It was concluded that these Mortierella sp. strains, like the Mucor strains, preferentially accumulated their co6 PUFA in the polar lipid relative to the TAG.
• the other SFA present in all strains were myristic acid (C14:0), pentadecanoic acid (C15:0), arachidic acid (C20:0), behenic acid (C22:0) and lignoceric acid (C24:0).
• the monounsaturated fatty acids Cl 6: 1 A7, C17: 1, C18: 1A11 (vaccenic acid) and C22: 1 were present at low but detectable levels in all of the strains.
• strains yNI0121 (Mucor hiemalis), yNI0125 (Mortierella elongata), yNI0127 (Mortierella sp.) and yNI0132 (Mortierella alpina) in order generate larger quantities of fungal biomass to evaluate mycelium disruption methods and to produce sufficient amounts of extracted lipid for food incorporation experimentation.
• yNI0132 had also been isolated from the 102.100.100/14183 soil sample, identified as M. alpina on the basis of ITS homology and exhibited similar fatty acid profile in the polar lipid and TAG fractions as yNI0133, yNI0134 and yNI0135.
• the fungal strains yNI0121, yNI0125, yNI0127 and yNI0132 were freshly propagated by agar slice growth, taking 0.5 x 0.5 cm agar pieces with fungal mycelium from the edge of colonies and placed them in the centre of a fresh MEA plate. The plates were kept at ambient temperature for 3 to 5 days until the new colonies were at least 3 cm in diameter. For each strain, intermediate cultures were then prepared by inoculating six 0.5 x 0.5 cm agar pieces containing mycelium into 10 ml malt extract medium and incubating these with shaking for 3 days at 26°C and then kept stationary for 2 days.
• the complete cultures were then used to inoculate 50 ml of malt extract medium in 250 ml baffled flasks and incubated with shaking at 26°C for 3 days. These cultures were then used to inoculate 600 ml of medium containing (per litre) 60 g glucose, 10 g yeast extract, 5 g malt extract, 4 g KH2PO4, 3 g (NH4)2HPO4 and 0.6 g MgSO4 with the pH adjusted to 6.0 with 2 M NaOH. These larger cultures were incubated with shaking at 26°C, the cultures sampled after 2 days and the biomass harvested by centrifugation after 3 days, freeze dried and then frozen.
• the seed cultures were used to inoculate 600 ml cultures in Medium 1 (per litre): 20 g glucose, 5 g yeast extract, 10 g peptone, incubated at 26°C with shaking at 200 rpm for aeration.
• Parallel cultures of 800 ml were also grown at the same time in a second medium, Medium 2, containing 30 g glycerol, 0.85 g yeast extract, 8.7 g KH2PO4, 1.9 g (NH 4 ) 2 HPO 4 , pH 6.2, cultured at 26°C with shaking at 200 rpm for aeration. Growth was significantly faster in Medium 1, reaching about 14 g/1 dry weight at 70 h.
• Total lipid was extracted from harvested wet fungal biomass (Table 15, Experiment 2) using hexane as solvent, as follows. Most of the water was removed by washing the cell biomass with ethanol, using 2 ml of ethanol per gram of cell biomass (wet weight) followed by centrifugation each time to recover the cell biomass. The pelleted cells were resuspended in hexane, using 5 ml hexane per gram of cell biomass. The suspensions were homogenised and the cells disrupted with the UltraTurrax (IKA, Malaysia) for 3 min followed by sonication for 5 min, which pair of treatments was repeated twice for a total of three times.
• UltraTurrax IKA, Malaysia
• the precipitate was washed 5 times with 30 ml portions of cold acetone (-20°C).
• the residual solvent in the extracted and purified phospholipid preparation was removed in a rotary evaporator at room temperature for 10 h.
• the polar lipid yield was measured gravimetrically and a small aliquot used for FAME analysis. Another aliquot was chromatographed on TLC to check for purity. From an initial input of 45.63 g of M. alpina dry biomass, 1.1 g of relatively pure phospholipid was recovered.
• Mortierella alpina strain yNI0132 was grown in a Braun fermenter in a rich medium containing glucose as the main carbon source, seeking to produce more cell biomass and a suitable polar lipid:TAG ratio having co6 faty acid incorporated into polar lipids.
• the growth medium was based on a rich yeast extract-malt extract medium which favoured biomass production rather than TAG production, even though M. alpina is an oleaginous species that naturally is capable of producing abundant TAG.
• the medium used for the seed culture for inoculation and for the first phase of culture contained (per litre) 60 g glucose, 10 g yeast extract, 5 g malt extract, 3 g (NH ⁇ hSCU, 1 g KH2PO4, 0.6 g MgSCU-VILO, 0.06 g CaCL and 0.001 g of ZnSCU, pH 6.2.
• the second stage of culturing used a feed solution of 5 L containing (per litre) 5 g malt extract, 7.5 g (NH ⁇ hSCU, 1 g KH2PO4, 6.0 g MgSC>4-7H2O, 0.3 g CaCL and 0.005 g of ZnSCL but no yeast extract.
• the first phase culture medium was prepared and sterilised in the fermenter by autoclaving in situ at 121°C for 15 min, then cooled by direct cooling to the fermenter jacket.
• the glucose stock solution (438 g glucose monohydrate plus 563 ml water) was autoclaved separately as a 40% solution and, while still warm at 45°C, was added to the fermenter.
• An inoculum culture was prepared in 4 x 200 ml YM broth in 500 ml flasks using starter cultures from agar plates. The inoculum culture was incubated for 71.5 h at 30°C with shaking at 180 rpm, at which time the inoculum cultures showed luxuriant growth. The inoculum culture was introduced into the fermenter without homogenisation of the culture. In the first phase of culturing with the aim of maximising biomass production, a high aeration rate was maintained at about 0.6 to 1.0 vvm (18-30 1/min) and mixing was low at 50-150 rpm to maintain dissolved oxygen at greater than 1 ppm without excessive shear forces being applied to the culture.
• the nutrient feed solution was added to the fermenter.
• the pH was controlled at 6.0 throughout by addition of NaOH and the temperature was maintained at 30°C.
• the culture was sampled (50 ml) every 24 h post inoculation.
• the parameters that were measured daily were cell density (dry cell weight), glucose level by HPLC, total nitrogen level by the Kjeldahl method, phosphate and sulphate levels by colorimetric strips, and the appearance of the fungus by light microscopy.
• Dry weight dry cell weight was measured by weighing the material collected on a glass microfibre obtained by filtering 20 ml of culture using a Buchner funnel and a vacuum pump before being dried in an oven and then weighed.
• the culture was harvested at 94 h when the cell density had reached 19.5 g/1 (wet weight/w).
• the biomass was harvested by filtration through a nylon gauze (200 micron).
• the biomass was resuspended and washed twice, each time with two volumes of cold water relative to the volume of biomass.
• the mycelial biomass was greywhite in colour.
• Excess water was removed by squeezing the wet mycelial cake through the filter cloth by hand. This yielded 2.27 kg of washed biomass having a dry weight of approximately 590 g.
• the biomass cake was spread to a 1 -2 cm layer in ziploc bags and frozen. Table 16.
• Mortierella alpina strain yNI0132 was grown in a Braun fermenter in a rich medium containing glucose as the main carbon source, and harvested at 65 hrs.
• the medium used for the seed culture for inoculation and for the first phase of culture contained (per litre) 65.6 g glucose, 10 g yeast extract, 5 g malt extract, 3 g (NH zSCL, 1 g KH2PO4, 0.6 g MgSCU- TILO. 0.06 g CaCL and 0.001 g of ZnSCU, pH 6.2, as well as Polyglycol P2000 at 1.0% as an antifoam.
• the second stage of culturing used a feed solution of (per litre) 0.833 g malt extract, 1.25 g (NELOzSCL, 0.167 g KH2PO4, 1 g MgSO4-7H2O, 0.05 g CaCL and 0.0008 g of ZnSCU but no yeast extract.
• the first phase culture medium was prepared and sterilised in the fermenter by autoclaving in situ at 122°C for 30 min, then cooled by direct cooling to the fermenter jacket.
• the glucose stock solution was sterilised separately at 121 °C for 15 min and transferred to the fermenter after the broth and glucose had cooled to 45°C.
• a IL inoculum culture was prepared in YM broth in 5L ml flasks using colonies from agar plates. The inoculum culture was incubated for 24 h at 28°C with shaking at 150 rpm. The fermenter was sterilised with batch medium (no glucose) and antifoam (at 1.0 % in the fermenter). Glucose was separately sterilised and added to the sterile batch medium by peristaltic pump, and the pH was adjusted to pH 6.0 and temperature to 30°C. The fermenter was inoculated with 1000 mL starter culture by peristaltic pump when the bottle showed luxurious growth of pumpable colonies (16-24 hours).
• the fermenter was sampled at TO (after adding inoculum) and everyday (50 mL) and measured for cell density (oven dry weight (DW)), glucose by HPLC and phosphate and sulphate (strips).
• DW was measured by weighing the dry pellet on glass microfibre obtained by filtering approximately 15 g of sample using a Buchner funnel and a vacuum pump before being dried in an oven and then weighed. Nutrient feed without glucose was transferred as bolus at 43.1 hours post inoculation.
• the fermenter was harvested at 65.2 hours post inoculation, and the harvested culture was processed using a wine press (100 kPA, 10 minutes).
• the biomass was resuspended in sterile water and reprocessed using the wine press at lOOkPA for 10 minutes.
• the biomass cake was wrapped in aluminium foil in thin layers, and the paste wet weight in each wrap was recorded and the biomass yield per litre of culture calculated.
• the wrapped biomass samples were placed in ziplock bags and frozen.
• the frozen biomass was rehydrated in sterile water (1:4) and suspended using the Silversson high shear mixer.
• the paste was then homogenised using APV homogeniser at 10 000 psi, 10 minutes until a free-flowing liquid was produced.
• the homogenised sample was pasteurised at >76°C, with a pump rate of 20-30 rpm.
• a portion of the pasteurised sample was packed in sterile containers and frozen, another of the sample was freeze dried.
• the biomass pastes pre -homogenised, homogenised and pasteurised were sub sampled for microtests, with 100 pL of each sample/ treatment plated on YM plates and incubated at 25°C for 96 hours.
• Example 8 Maillard reactions using fungal biomass and extracted lipid
• the experiment also tested a combination of cells and the extracted lipid, all produced as described in Example 7. These reactions had L-cysteine, D-ribose, thiamine hydrochloride, iron fumarate and glutamic acid present in a phosphate buffer at pH 6.0, and either had added yeast extract or lacked the yeast extract.
• the presence or absence of yeast extract was intended to test whether it would either mask, or enhance, the aroma produced by the M. alpina cells or extracted lipid having PL, or have little effect.
• the base medium used for the Maillard reactions designated “Matrix A” lacking yeast extract and “Matrix B” including yeast extract, had the following composition in aqueous buffer at final concentrations: 10 mM L-cysteine, 10 mM D-(-)-ribose, 2 mM thiamine hydrochloride, 35 pg/ml of iron fumarate (Apohealth, NSW, Australia) and 2 mM L-glutamic acid monosodium salt hydrate.
• Reactions #4 to #7 containing M. alpina biomass and/or extracted lipid were described by all five volunteers as having a meaty aroma, but with different aroma notes recorded by the volunteers, whilst the descriptions of the aromas from reactions having the S. cerevisiae biomass were more variable between the volunteers.
• the control reaction mixtures lacking the lipid extract, and the mixtures having the lipid extract without any cell biomass, were generally perceived to have a lower intensity of aromas compared to the corresponding samples that contained biomass or a combination of biomass and extracted lipid from M. alpina.
• Reaction mixtures containing biomass spiked with the extracted lipid from M. alpina were described as having similar or enhanced aromas compared to reactions containing only M. alpina biomass.
• lipolytica cells incorporating ARA in its polar lipid were applied as wet cells at 200 mg per 2 ml reaction in 20 ml glass vials, tightly sealed. Control reactions had the same base media compositions but lacked the Y. lipolytica or M. alpina cells.
• the reaction mixes were sonicated as a batch by placing all vials in a floating foam and placed in a sonicator (Soniclean, Thermoline) set up at a medium power for 30 min and then heat treated in an oven at 140°C for 60 min. The vials containing the reaction mixtures were cooled slowly over about 15 min until warm to the touch. The contents were sniffed in random order by nine volunteers who did not know the composition of each mixture. The reactions had been coded with random 3 -digit numbers to avoid bias, and the volunteers sniffed coffee beans between samples to reset the olefactory senses.
• the aromas from mixtures having glutamic acid were generally described as more associated with meaty aromas compared to the reactions lacking glutamic acid.
• a reaction mixture having glutamic acid was described as providing meaty aroma by 5 of the 9 participants whereas the corresponding sample lacking glutamic acid was described as having a meaty aroma by only 2 participants.
• Addition of fenugreek leaf powder in the reactions was generally described as generating a pleasant, sweet herb or vegetable aroma, but addition of the herb powder also moderated the meaty aroma in the presence of the Y. lipolytica or M. alpina cells.
• the M. alpina biomass as a dried powder was compared to several commercial plant -based and meat flavouring products on the market in Australia, including Deliciou plant-based beef, Deliciou plant-based chicken, Deliciou plant -based pork, Massel plant -based stock cube - beef, Massel plant-based stock cube - chicken, Oxo stock cube-beef, Oxo stock cube-chicken and Bonox beef stock.
• Reaction mixtures were prepared in 2 ml volumes using 150 mg of dry product or 200 mg of product as a wet paste and heated at 140°C for about 60 min.
• the samples containing the M. alpina cell biomass were described as comparable or superior in their meaty aroma to the commercially-available flavouring products.
• reaction mixes were prepared and then dried down by placing the vials in an oven at 115°C for 2 h followed by 82°C for a further 2 h.
• corresponding samples were dried overnight at 70°C.
• all of the samples were reconstituted in 2 ml of water, mixing them well to dissolve the dried powder, and subjected to sniffing by volunteers.
• the samples treated at the higher temperature generally provided a burnt smell, whereas the samples subjected to the lower temperature drying still provided some meaty aromas. This indicated that lower temperature drying was better than the higher temperature for retaining the meaty aroma. Further investigation is carried out to optimise the drying conditions.
• compositions can be used with the yeast or fungal biomass containing co6 fatty acids to enhance meaty aromas when heated, including in the presence of other flavouring components as commonly used in food preparations.
• the inventors further tested the M. alpina cells and the extracted lipid obtained from the cells in further Maillard reactions under modified conditions. From the previous experiments, the samples containing M. alpina biomass were considered to have the strongest meat -like aroma, often described as having a roast meat/BBQ meat aroma. Several volunteers in the aroma tests, however, described that to them the aroma was like an overcooked or even burnt meat with a charred note. A “fatty aroma” was also noted by some. In another experiment, when the mixtures were tasted after heating, some volunteers described a sourness or bitterness in the samples including the matrix bases A and B, in particular bitterness for samples containing M. alpina.
• an alternative base medium was used to compare it to the Matrix B base.
• This alternative medium contained a mixture of amino acids, including cystine (33%), glutamine, alanine, leucine, glutamic acid, lysine, valine, proline and methionine as well as 2.7% dextrose by weight. This mixture was added at 7.5% (w/v) to the aqueous medium, as was an additional 0.5% (w/v) cystine and 0.5% (w/v) dextrose.
• the samples for the Maillard reactions used either 150 mg of dry M. alpina biomass or 300 mg of wet slurry of S. cerevisiae cells. Control samples had only the amino acids and sugars and no cells added.
• the alternate base medium was used at two concentrations: 7.5% (w/v) or 0.75% (w/v).
• Another sample had an additional 100 mg dextrose added per 2 ml mixture.
• Some samples contained 200 mg of extracted polar lipid, mostly PL, from M. alpina.
• a shortened heat treatment of 45 min at 140°C was applied for samples containing M. alpina while the standard heat treatment of 75 min at 140°C was used for other samples.
• the volunteers described that the mixtures having the higher concentration of base medium had a more distinguished meat-like and pleasant aroma compared to the samples prepared at the low concentration. Further, the higher concentration samples had a browny/golden brown colour after the heat treatment, whereas the lower concentration samples did not have that colour.
• Example 10 Food products using fungal biomass and extracted lipid
• the oils and plant-based fats used were canola oil, “Heart Smart” safflower cooking oil and copha vegetable shortening from a supermarket and a plant -based ghee (Emkai Lite Interesterified vegetable fat, Sai food products, Tamil, India).
• the food items tested by applying the taste mixtures were a macro firm tofu obtained from a local supermarket, dried bean curd (tofu skin, Shenzhen Ming Lee Food Manufacturing Co. Ltd., Guandongzhou, China), a plant-based mince (V2 Foods, Australia) and textured vegetable protein high fibre slices (TVP, Lamyong, NSW, Australia).
• the fungal biomasses used were a wet slurry of .S', cerevisiae having about 10% ARA (B013, see Example 4), or M. alpina biomass in either a wet or dry form (Example 7).
• This experiment used the B013 yeast biomass, containing ARA in both the polar lipid and TAG (Example 4).
• a mixture (mixture A) was prepared containing 2 ml of a Matrix B2 base medium.
• Matrix B2 contained one tenth the concentration of thiamine hydrochloride compared to Matrix B but otherwise had an identical composition.
• Mixtures were prepared having 0.5 ml of B013 cell slurry and 0.5 ml of a chicken flavoured yeast extract (2.5 g/3 ml water, Flavex). Control mixtures lacked either the B013 cell slurry or the Matrix B2 base medium.
• Tofu pieces were marinated in the mixtures for 45 min and cooked on a baking tray in an oven set at 180°C for about 6 min.
• all of the tofu pieces had a salty/sweet/umami taste but only the test pieces treated with mixture A exhibited a light roast chicken aroma and taste. It was considered that the umami taste was most likely brought by the flavoured yeast extract whereas the B013 yeast biomass contributed to the chicken aroma.
• the yeast biomass was substituted with 200 mg of M. alpina wet biomass, having about 30% ARA in its lipid.
• the composition of the mixtures and baking conditions were otherwise the same as in Experiment 2 except that the mixtures were heated for 45 min rather than 75 min prior to application to the tofu pieces. After heating them in the oven, the tofu pieces were sniffed and tasted.
• the volunteers described that the control tofu marinated in Matrix B2 without the M. alpina biomass had a pleasant, light meaty aroma, whereas the tofu treated with the mixture having the M. alpina biomass had a strong meaty aroma and taste.
• vials were prepared comprising M. alpina wet biomass (in noncontrol samples, concentrations varying from 0.10% - 10% biomass/TVP w/w%), matrix C (as described above in Table 20) and, in one sample, water instead of matrix C.
• the vials were vortexed at 20000 rpm for 2 minutes before being subjected to a heat treatment at 140°C for 45 minutes.
• the composition of the vials is shown below in Table 23. Table 23. Composition of samples
• 450 g of rehydrated TVP was prepared by adding 320 g of water to 130 g of TVP and leaving to rehydrate for 30 minutes. The rehydrated TVP was then divided into 9 portions, and each portion added to one of the vials of Table 21 and mixed and marinated thoroughly for approximately 5 minutes. Each portion of the marinated TVP was then cooked on a frying pan at a medium heat setting (1000 W) with 3 mF of canola oil for 2 minutes.
• each marinated TVP sample was cooked on an oiled frying pan (3 ml of safflower oil) at 1000 W for 2 minutes, stirring occasionally. Between each sample, the frying pan was cleaned and dried. All cooked samples were stored in individual closed containers and kept at 60 °C for no longer than 1 hour until tasting.
• Cysteine, Ribose Cysteine, Dextrose
• Cystine, Ribose Cystine, Dextrose
• Results are shown in Figure 16. While all combination of amino acid and sugar produced meaty aromas and flavours when combined with the M. alpina biomass, the combination of cysteine and dextrose was the most preferred combination and had the highest meatiness score. This sample was noted with dark chicken meat such as chicken thigh, strong umami notes and slight aftertaste without any strong bitter tastes.
• Yeast Extract when absent: The sample contained a light chicken aroma with more intense vegetable aroma. It was also noted to have similar aroma notes to the positive control (OM) but without the depth/intensity.
• Cysteine when absent: When compared to the positive control (OM), this sample had a sweet aroma but lack of meaty notes. The dextrose in the sample would likely be the main contributor to the sweet aroma and the caramelisation during heating. The sample also had a bitter aftertaste with key taste notes such as mushroom and metallic.
• Dextrose when absent: Bland in aroma without the umami and meaty note, and a stronger salty aroma note. Compared to the OM sample, this sample had a weaker umami taste but still had a sweet aftertaste. The sweet aftertaste was different from the thiamine -removed sample which contained 200mM of dextrose.
• the extraction was repeated a further 3 times (total 4 washes) by adding a further 500 mL ethanol to the filtered biomass and repeating the steps.
• the washed and filtered biomass was transferred to a wash glass and the ethanol dried off.
• the resulting biomass was the ‘defatted biomass’ fraction.
• polar lipid (PL) fraction To produce the polar lipid (PL) fraction, total lipid fraction was weighed and resuspended in 30 volumes acetone (w/v) before being vortexed for 30 sec) and sonicated for 30 secs repeatedly until visibly resuspended. The sample was then precipitated at 4°C for 30 min and then centrifuged at 3900g for 10 min at 4 °C. The supernatant was removed and the precipitate washed twice more with another 30 x volumes ice cold acetone. The precipitate was dried under nitrogen gas until it reached a constant weight, thereby producing the ‘PL’ fraction.
• Benzeneacetaldehyde and Nonanol were not present in SI, S4 or S8 (‘Matrix OM + TAG’) but were present in all other samples.
• Benzeneacetaldehyde has been identified in beef (Specht et al. J Ag Food Chem 1994;42(10):2246-53) and as a desirable aroma for meatiness (Zhang et al. Food Sci Tech. 2017;82:184-91).
• Nonanol is one of the major volatiles in lamb (Luo et al. Food Sci Nutrition. 2019 (7):2796-805) and it has been suggested that is produced via lipid (oleic acid) oxidation.
• TAG fractions are contributing to off-notes there are volatiles that are detected in greater abundance in S6 (‘Matrix OM + biomass minus PL’) and S8 when compared to the whole biomass or sample not containing TAG, such as Tetradecane (waxy), Naphthalene (Mothball) and 2,2,4-trimethyl-l,3-pentanediol diisobutyrate (plasticiser).
• S6 ‘Matrix OM + biomass minus PL’
• S8 when compared to the whole biomass or sample not containing TAG, such as Tetradecane (waxy), Naphthalene (Mothball) and 2,2,4-trimethyl-l,3-pentanediol diisobutyrate (plasticiser).
• the non-lipid components of the biomass also appear to play an important role in the aroma profile, likely interacting with the lipids to modulate their effect on the aroma. This is seen in the PCA plot where S7 and S8 are positioned away from the biomass containing samples S2-6 which are clustered closer together. Additionally, there are volatiles that are detected in S8 or S7 in much higher abundance than in other samples. Examples of these for S7 are Dodecanal; 2-Undecanone; 2-Methyl- 1 -undecanol; 1 -Hexadecanol 2,4-dimethyl-benzaldehyde; and 2 -Ethyl- 1 -hexanol.
• S8 examples of these for S8 are l-(lH-pyrrol-2-yl)-ethanone; 2 -butyl- 1 -octanol; Hexadecane; l-Octen-3-ol; 1-Pentanol and 1 -Heptanol, many of which have been identified as mushroom and/or off -notes (2, 6, 7, 9) which aligns with the sensory data.
• Table 43 List of aroma volatile compounds identified and their aroma description.
• Matrix OM (5 mL) was mixed with either the Mortierella biomass (BM) or fraction thereof according to Table 44 in 20 ml GC headspace vials. The samples were vigorously mixed (2000 rpm) for 2 minutes at room temp (22-24 °C) and subjected to heating at 140 °C for 45 min. Table 44
• the sample with the most pleasant and meaty aroma was the wet M. alpina biomass, with the PL containing samples also demonstrating some meatiness: the ‘biomass minus TAG’ sample showed a meaty note that was not as complex and had less intensity of the wet biomass; and the ‘PL’ sample was also identified as having some meatiness, but at approximately with even less intensity than the wet biomass.
• the TAG-containing fraction sample were not identified as meaty: the ‘biomass minus PL’ was described as floral and pleasant, while the ‘TAG’ alone sample was unpleasant with descriptors of “rancid” and “chemical”. This suggested that the TAG fraction was contributing off- notes that could be mitigated by other fractions in the biomass (as in the biomass or biomass minus PL samples).
• the ‘defatted biomass’ sample was pleasant but lacked the meaty notes of the wet biomass.
• the control Maillard matrix (OM) had the lower acceptance score, lower overall meatiness, roastiness and animalic (farmlike) note but a higher sulfury notes compared to other samples containing the biomass, re-confirming the importance of biomass in the meaty aroma formation. In general, the differences in aroma characteristics of tested biomasses were minimal.
• Sample ATCC32223 had the lowest acceptance score, with the lowest scores in meatiness, sulfury and animalic notes when compared to other biomasses.
• Biomass from SS3 had the highest overall meatiness and roastiness scores, the lowest rancid (off-animal fat) notes compared to ATCC32223 and SI 1-2. SI 1-2 had the lowest roastiness notes and the highest animalic, rancid and sulfury notes.
• a total of six participants (both male and female, aging from 25-65) analysed the TVP food samples by sniffing and tasting. The participants were requested to evaluate both aroma and taste for the meatiness and pleasantness based on a five -point hedonic scale, with the higher score indicating increased meatiness and pleasantness.
• the low-ARA strain M. isabellina
• M. alpina NI0132 The low-ARA strain, M. isabellina, has been shown to produce y-hexalactone following feeding with hexanoic acid (EP 1038971).
• a study was performed to induce production of y-hexalactone in M. alpina NI0132.
• the strain was inoculated on YPD plates (Sigma: Yeast extract lOg/L, peptone 20g/L, dextrose 20g/L, agar 15g/L) and incubated at 28°C for 3-5 days.
• M. alpina was prepared in 250 mL flasks with 50 mL liquid fermentation medium (10 g/L yeast extract, 60 g/L glucose, 5 g/L malt extract, 3 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 0.6 g/L MgSO 4 , 0.06 g/L CaCl 2 , 0.001 g/L ZnSO 4 , pH 6.2) and incubated for 2 days at 30°C, 180 rpm.
• liquid fermentation medium 10 g/L yeast extract, 60 g/L glucose, 5 g/L malt extract, 3 g/L (NH 4 ) 2 SO 4 , 1 g/L KH 2 PO 4 , 0.6 g/L MgSO 4 , 0.06 g/L CaCl 2 , 0.001 g/L ZnSO 4 , pH 6.2
• the inoculum was homogenised (20,000 rpm) and added at a concentration of 10% to 100 mL fermentation medium in 500 mL flasks, in duplicates) before being incubated at 30°C, 180 rpm.
• castor oil (1 g/L) or methyl ricinoleate (1 g/L dissolved in Tween 80, 0.3 g/L) were added to the flasks. These chemicals were filter sterilised before addition to the flasks.
• the harvesting of biomass was conducted at various time intervals (3h, 6h, 8h, and 24h) after the addition of castor oil or methyl ricinoleate by centrifugation (3500 rpm, 10 min).
• the biomass pellet was snap frozen in liquid nitrogen and stored at -80°C freezer.
• Biomass is thawed and extracted using Chloroform:Methanol:Water (1:3:1 or 8:4:3 ratio). Extracts are then cleaned using aminopropyl SPE cartridges to remove TAG and PL. The fatty acid fraction is collected for analysis. The dried fatty acid fraction is derivatised with BSTFA at 45 °C for 30 min and analysed on GCMS on full scan mode. Lactone intermediates (e.g. 4-hydroxy fatty acids) are identified by comparing the mass spectra to NISTMS database. Peak areas of intermediates are normalised to internal standard and quantitated against a calibration curve. Sensory analysis of biomass containing 4-hydroxylated fatty acids is then performed as essentially described above.

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