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WO2025036979A1 - Production durable de biomasse - Google Patents

Production durable de biomasse Download PDF

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Publication number
WO2025036979A1
WO2025036979A1 PCT/EP2024/073010 EP2024073010W WO2025036979A1 WO 2025036979 A1 WO2025036979 A1 WO 2025036979A1 EP 2024073010 W EP2024073010 W EP 2024073010W WO 2025036979 A1 WO2025036979 A1 WO 2025036979A1
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WIPO (PCT)
Prior art keywords
yeast
jadinii
saccharomycetales
cyberiindnera
wickerhamomyces
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PCT/EP2024/073010
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WO2025036979A8 (fr
Inventor
Mickel Leonardus August JANSEN
Carol Andrea ROA-ENGEL
Evert Tjeerd VAN RIJ
Andre VENTE
Koen Johannes Anthonius VERHAGEN
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DSM IP Assets BV
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DSM IP Assets BV
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Publication of WO2025036979A8 publication Critical patent/WO2025036979A8/fr
Pending legal-status Critical Current
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • C12N1/16—Yeasts; Culture media therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J1/00—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites
    • A23J1/18—Obtaining protein compositions for foodstuffs; Bulk opening of eggs and separation of yolks from whites from yeasts
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/20—Proteins from microorganisms or unicellular algae
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/142—Amino acids; Derivatives thereof
    • A23K20/147—Polymeric derivatives, e.g. peptides or proteins
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K50/00—Feeding-stuffs specially adapted for particular animals
    • A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; 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
    • C12N1/32—Processes using, or culture media containing, lower alkanols, i.e. C1 to C6
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
    • C12P1/02—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by 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
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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
    • C12R2001/72—Candida
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
    • C12Y203/03009—Malate synthase (2.3.3.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/03—Oxo-acid-lyases (4.1.3)
    • C12Y401/03001—Isocitrate lyase (4.1.3.1)

Definitions

  • the present invention relates to a method for cultivating a microorganism capable of utilizing ethanol as feedstock.
  • the invention is directed to a method for the production of biomass, in particular single cell protein, wherein the yeast single cell protein product comprises Saccharomycetales yeast cells, as well as to an animal feed comprising such biomass.
  • SCPs single cell proteins
  • feedstock like waste-based feedstock.
  • farmers and food producers still require alternative solutions for protein production to be able to provide low carbon footprint diets while ensuring an environmental-friendly use of the planet's resources.
  • the present invention relates to a Method for cultivating a microorganism capable of producing at least 40% protein, comprising the steps of (i) supplying microorganism to a reactor, (ii) feeding EtOH as feedstock, (iii) controlling the feed rate, (iv) controlling the temperature, (v) controlling pH and (vi) controlling the growth rate.
  • the feed rate is 0.179 - 0.536 g e thanoi/gbiomass/h, more preferably 0.179 - 0.469 Qethanol/ Q biomass/h .
  • the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°C, most preferably between 30 and 34°C.
  • the pH is kept between 3.5 and 5.5, more preferably between 3.5 and 5.0, most preferably between 3.5 and 4.5.
  • the present invention also relates to a yeast single cell protein product, wherein the yeast single cell protein product comprising >40% protein on dry cell weight, wherein the protein comprises >0.1 %, preferably >0.2% isocitrate lyase and/OR malate synthase.
  • the microorganism is a Saccharomycetales yeast.
  • the Saccharomycetales yeast is a yeast from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces.
  • the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, more preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.
  • the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841 , Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576 or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621 , Cyberlindnera jadinii CBS841 , Saccharomyces cerevisiae GHP1
  • the present invention relates to an animal feed comprising up to 20% (w/w), preferably up to 10% (w/w), yeast single cell protein (SCP) product, wherein the yeast single cell protein product comprises >40% protein on dry cell weight, wherein the protein comprises >0.1 %, preferably >0.2% isocitrate lyase and/OR malate synthase.
  • SCP yeast single cell protein
  • the yeast single cell protein product comprises ethanol fed Saccharomycetales yeast cells.
  • the Saccharomycetales yeast cells are yeast cells from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces, preferably wherein the Saccharomycetales yeast cells are from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, preferably wherein the Saccharomycetales yeast cells are or are derived from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindner
  • the Saccharomycetales yeast cells are capable of producing with ethanol as carbon source 34% (w/w) or more protein per gram dry weight of said Saccharomycetales yeast cells, preferably 41 % (w/w) or more protein per gram dry weight of said Saccharomycetales yeast cells, more preferably 42.5% (w/w) or more protein per gram dry weight of said Saccharomycetales yeast cells.
  • the yeast SCP product comprises all essential amino acids.
  • Saccharomycetales yeast cells are not genetically engineered.
  • the yeast SCP product comprises 34% (w/w) or more protein per gram dry weight of Saccharomycetales yeast cells, preferably 41 % (w/w) or more protein per gram dry weight of Saccharomycetales yeast cells, more preferably 42.5% (w/w) or more protein per gram dry weight of Saccharomycetales yeast cells.
  • the yeast SCP product comprises dried Saccharomycetales yeast cells, preferably wherein said dried Saccharomycetales yeast cells are intact or disrupted or a mixture of intact and disrupted cells.
  • the animal feed further comprises i) further less than 25% (w/w) plant-based protein products, ii) further 5% (w/w) or less fish meal, iii) no fish meal, and/or iv) from 1 to 25% (w/w) oil.
  • the animal feed is a feed for poultry, pigs, horses, camels, cows, sheep or companion animals.
  • the animal feed is a feed for aquatic species, wherein said aquatic species are preferably selected from crustaceans or fish, preferably wherein said crustaceans are shrimps and/or preferably wherein said fish are warm water fish or cold water fish, preferably wherein said warm water fish are selected from catfish, tilapia, seabream, seabass, or carp and/or preferably wherein said cold water fish are selected from cod, salmon or rainbow trout.
  • said aquatic species are preferably selected from crustaceans or fish, preferably wherein said crustaceans are shrimps and/or preferably wherein said fish are warm water fish or cold water fish, preferably wherein said warm water fish are selected from catfish, tilapia, seabream, seabass, or carp and/or preferably wherein said cold water fish are selected from cod, salmon or rainbow trout.
  • the present invention also relates to the use of the animal feed according to the present invention for feeding an animal.
  • the present invention also relates to the use of the animal feed according to the present invention for increasing body weight of an animal.
  • the animal is a poultry, pig, horse, camel, cow, sheep or companion animal or said animal are aquatic species, preferably wherein said aquatic species are selected from crustaceans or fish, preferably wherein said crustaceans are shrimps and/or preferably wherein said fish are warm water fish or cold water fish, preferably wherein said warm water fish are selected from catfish, tilapia, seabream, seabass, or carp and/or preferably wherein said cold water fish are selected from cod, salmon or rainbow trout.
  • said aquatic species are selected from crustaceans or fish, preferably wherein said crustaceans are shrimps and/or preferably wherein said fish are warm water fish or cold water fish, preferably wherein said warm water fish are selected from catfish, tilapia, seabream, seabass, or carp and/or preferably wherein said cold water fish are selected from cod, salmon or rainbow trout.
  • the method of the invention is an aerobic fermentation for the production of biomass.
  • This aerobic fermentation of the method for producing a biomass comprises cultivating a microorganism, a yeast.
  • the microorganism in the aerobic fermentation uses the ethanol, which is fed to the aerobic fermentation, as feedstock for the production of biomass.
  • Saccharomycetales yeast cells when grown with ethanol as carbon source, produce high protein, i.e. 34% (w/w) or more protein per gram dry weight of such Saccharomycetales yeast cells, preferably 41 % (w/w) or more protein per gram dry weight of such Saccharomycetales yeast cells, more preferably 42.5% (w/w) or more protein per gram dry weight of such Saccharomycetales yeast cells (see Figure 1).
  • Saccharomycetales yeast cells are preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces, and more preferably the Saccharomycetales yeast cells are from the genus Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus. Further, surprisingly, it was found that Saccharomycetales yeast cells appear to produce more protein, when grown with ethanol as carbon source than when grown on a sugar substrate (see Figure 2).
  • Saccharomycetales yeast cells especially when grown with ethanol feed stock, appear to be suitable as a sustainable single cell protein product, which is a source for, e.g., protein with the aim of at least partially, preferably fully replacing animal-derived protein sources, such as fish meal in animal feed.
  • the protein content of the biomass can be improved by adjusting the growth rate of the microbial cells at the end of the fermentation process.
  • the fermentation must be halted at the appropriate growth rate.
  • the ethanol concentration inside the fermenter itself is always close to zero ( ⁇ 0.1 g/L), as any ethanol that is fed is virtually immediately consumed by the yeast.
  • the main parameter of the fermentation process, that determines the growth rate is the ethanol feed rate. This feed rate has to be started low, as the biomass concentration at the start of fermentation is low and is subsequently increased exponentially to match the exponential growth rate of the biomass.
  • the ethanol feed rate should be defined by the substrate uptake rate of the organism (the q s ) calculated by the amount of substrate that is consumed (g e thanoi) per amount of biomass in the fermenter (gbiomass) per hour, of 0.179 - 0.536 g e thanoi/gbiomass/h , more preferably 0.179 - 0.469 g e thanoi/gbioma SS /h .
  • the biomass yield decreases with higher temperatures, causing the protein yield (the amount of protein produced per gram of ethanol) to decrease with increasing temperatures (see Figure 3).
  • the temperature is kept between 30 and 40°C, preferably between 30 and 38°C, more preferably between 30 and 36°, most preferably between 30 and 34°C.
  • Protein content does change with the pH level. Protein content is increased at lower pH levels. The biomass yield however drops significantly at pH levels of 3.5 and lower. Preferably, the pH is kept between 3.5 and 5.5, preferably between 3.5 and 5.0, more preferably between 3.5 and 4.5 (see Figure 4).
  • the method for producing biomass may further comprise a step of recovering the biomass from the aerobic fermentation by suitable methods known in the art. Recovering biomass may comprise centrifugation or filtration.
  • the method for producing biomass may further comprise a step of drying the biomass by suitable methods known in the art.
  • Drying biomass may comprise convective/direct drying technologies (like spray drying, fluidized bed) or contact/indirect technologies (like drum drying, vacuum drying, falling film) or supercritical drying (using superheated steam) or natural air/sun drying or even freeze drying.
  • yeast feed with up to 20% (w/w) yeast single cell protein product was well eaten by animals, particularly by aquatic species, such as fish or crustaceans.
  • animal feed with up to 10% (w/w) yeast single cell protein product has a beneficial effect on the increase of the body weight of animals, particularly aquatic species, such as fish or crustaceans.
  • animal feed comprising up to 20% (w/w) or 10 % (w/w) yeast single cell protein product can advantageously fully replace animal-derived protein, such as fish meal.
  • animal refers to any animal except humans.
  • animals are non-ruminants and ruminants. Ruminant animals include, for example, animals such as horses, sheep, goats, cattle, e.g. beef cattle, cows, dairy cows, and young calves, deer, yank, camel, llama and kangaroo.
  • Non-ruminant animals include monogastric animals, including but not limited to companion animals (including, but not limited to cats and dogs), pigs or swine (including, but not limited to, piglets, growing pigs, and sows); poultry such as turkeys, ducks, quail, guinea fowl, geese, pigeons (including squabs) and chicken (including but not limited to broiler chickens (referred to herein as broilers), chicks, layer hens (referred to herein as layers)); horses (including but not limited to hotbloods, coldbloods and warm bloods); crustaceans (including but not limited to shrimps and prawns); and fish including but not limited to warm water fish and cold water fish and thus, fish include but not limited to amberjack, arapaima, barb, bass, bluefish, bocachico, bream, bullhead, cachama, carp, catfish, catla, chanos, char, cichlid
  • the microbial biomass is a yeast and the terms “single cell protein product” and “yeast single cell protein product” are therefore used interchangeably.
  • the protein yield is defined as the amount of protein produced per gram of ethanol.
  • a common manner, as stated in US3151038, of determining protein content is to analyze for total nitrogen by the Kjeldahl procedure and then multiply by 6.25, the standard factor according to accepted practice. Hawk, Philip B., Oser, Bernard L., and Summerson, William H, 1947, Practical Physiological Chemistry, 12th edition, The Blakiston Company, Philadelphia and Toronto, state as follows on pages 213 and 214: The usual factor employed for the calculation of protein from the nitrogen content is 6.25 and is based on the assumption that proteins contain on the average 16 percent of nitrogen.
  • the protein content is defined as the amount of protein in the biomass based on dry matter.
  • any animal referred to herein is preferably not a wildlife animal.
  • said animal is preferably a farming animal and/or a livestock animal.
  • the animal is preferably an animal of an aquaculture.
  • aquaculture relates to aquafarming and thus, the farming of aquatic species such as fish or crustaceans in a variety of environments, including but not limited to tanks, lakes, ponds, or any other natural or man-made aquatic reservoirs that may be suitable for breeding, hatchery, rearing and harvesting of the aquatic species.
  • animal feed refers to any compound, preparation, or mixture suitable for, or intended for intake by an animal (e.g., a fish).
  • Animal feed for a monogastric animal typically comprises concentrates as well as vitamins, minerals, enzymes, direct fed microbial, amino acids and/or other feed ingredients (such as in a premix)
  • animal feed for ruminants generally comprises forage (including roughage and silage) and may further comprise concentrates as well as vitamins, minerals, enzymes direct fed microbial, amino acid and/or other feed ingredients (such as in a premix).
  • An animal feed additive e.g., fish feed additive
  • a formulated enzyme product which may further comprise e.g. vitamins, minerals, enzymes, amino acids, preservatives and/or antibiotics; i.e. a premix.
  • the animal feed additive/premix is typically mixed in a feed mill with concentrates and/or forage such as vegetable protein, legumes or other plant material. Further, the animal feed is typically fed as a pelleted feed to mono-gastric animals.
  • single cell protein refers to a protein obtained by and/or derived from a (unicellular) microorganism.
  • an SCP may refer to a protein purified and/or isolated from a microorganism’s cell culture for example.
  • SCPs may refer to the dead dried cells of microorganisms.
  • an “single cell protein product” or “SCP product” may or may not comprise one or more selected from the group of intact (unicellular) microorganism cells, disrupted (unicellular) microorganism cells, isolated proteins obtained from one or more (unicellular) microorganism(s), isolated proteins derived from one or more (unicellular) microorganism(s), purified proteins obtained from one or more (unicellular) microorganism(s), and purified proteins derived from one or more (unicellular) microorganism(s).
  • an (unicellular) microorganism may relate to a bacterium, a fungus like yeast and/or an algae
  • said (unicellular) microorganism is yeast according to the present invention.
  • SCP products from yeast offer the advantage of providing comparatively high protein contents, while at the same time said products can be produced on industrial scale at comparatively low cost, independent from seasonal effects and with comparatively low harvesting efforts. Thus, yeast SCP products are highly advantageous.
  • yeast refers to a eukaryotic, unicellular microorganism classified as a member of the fungus kingdom that mostly reproduce asexually by mitosis. Further herein, said term preferably relates to yeast cells, which can be grown under artificial and/or lab conditions, e.g. as in vitro culture conditions, and in particular under standard laboratory conditions. Said term preferably also embraces yeast cells of a single type that have been grown in the laboratory for several generations and thus, said term preferably embraces also potential mutants of a yeast cell and/or strain.
  • yeast is preferably Saccharomycetales yeast.
  • a “yeast cell” is a cell of a yeast, preferably a cell of a yeast as described herein.
  • Saccharomycetales refers to the order Saccharomycetales within the phylum Ascomycota. Members of Saccharomycetales are also known and sometimes referred to as budding yeasts.
  • the term “”w/w” is intended to be understood as "weight by weight” and thus refers to the proportion of a particular substance within a mixture, as measured by weight or mass.
  • SCP product producer may vary in their ability to use and/or utilize ethanol as carbon source for SCP production.
  • the yeast SCP product preferably comprises Saccharomycetales yeast cells, wherein said Saccharomycetales yeast cells are Saccharomycetales yeast cells from one or more Saccharomycetales yeast genera, species and/or strains that are capable of using ethanol as carbon source.
  • the Saccharomycetales yeast cells may be Saccharomycetales yeast cells from one or more genera selected from the group consisting of Cyberiindnera, Kluyveromyces, Wickerhamomyces, Yarrowia, Pichia and Saccharomyces.
  • the yeast SCP product comprises Saccharomycetales yeast cells, wherein said Saccharomycetales yeast cells are preferably Saccharomycetales yeast cells selected from the group consisting of Pichia anomala, Yarrowia lipolytica, Wickerhamomyces anomalus, Cyberiindnera jadinii, Saccharomyces cerevisiae and/or Kluyveromyces lactis.
  • the animal feed according to the present invention comprises up to 20% (w/w) or up to 10% (w/w) yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, and wherein said Saccharomycetales yeast cells are yeast cells from the genus Wickerhamomyces, Cyberiindnera, Saccharomyces, Kluyveromyces, Yarrowia and/or Pichia, preferably from Cyberiindnera, Saccharomyces, Kluyveromyces and/or Wickerhamomyces.
  • yeast cells from said genera are capable of growing on a culture medium comprising ethanol as carbon source as also shown herein in the Examples (see, e.g., Figure 1).
  • the animal feed according to the present invention comprises up to 20% (w/w) or up to 10% (w/w) yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, and wherein said Saccharomycetales yeast cells are preferably from Cyberiindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala and/or Yarrowia lipolytica, more preferably from Wickerhamomyces anomalus, Cyberiindnera jadinii, Saccharomyces cerevisiae and/or Kluyveromyces lactis.
  • SCP products from said species can have, e.g., 34% (w/w) or more, preferably 41% (w/w) or more, more preferably 42.5% (w/w) or more, protein per dry matter (see Figure 1) and moreover, can fully replace animal-derived protein, such as fish meal, in an animal feed according to the present invention.
  • the animal feed according to the present invention comprises up to 20% (w/w) or up to 10% (w/w) yeast SCP product, wherein the yeast SCP product comprises Saccharomycetales yeast cells, and wherein said Saccharomycetales yeast cells are derived and/or are from Cyberiindnera jadinii ATCC 26387, Cyberiindnera jadinii FERM-BP1656, Cyberiindnera jadinii CBS621 , Cyberiindnera jadinii CBS841, Saccharomyces cerevisiae GHP1 , Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberiindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576 and/or Yarrowia lipolytica CBS 7504, preferably from Cyberi
  • the term “derived from” preferably refers to yeast cells, which were originally obtained from a given yeast strain and thus originate from said given yeast strain. Such derived cells may differ from said given yeast strain due to naturally occurring and/or artificially introduced alterations like genetic mutations, but preferably have similar characteristics as cells from the yeast strain they originated from. Such similar characteristics are preferably the capability to produce with ethanol as carbon source 34% (w/w) or more protein per gram dry weight of yeast cells, preferably 41% (w/w) or more protein per gram dry weight of yeast cells, more preferably 42.5% (w/w) or more protein per gram dry weight of yeast cells.
  • cells that are derived from a given strain may have, preferably on genome level, a sequence identity of 80% or more, preferably of 85% or more, more preferably of 90% or more, even more preferably of 95% or more to the respective strain that can be seen as reference.
  • a derived cell may have a sequence identity of at least, e.g., 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to the respective reference, preferably on genome level.
  • sequence identity denotes a property of sequences that measures their similarity or relationship.
  • sequence identity or “identity” as used in the present disclosure means the percentage of pair-wise identical residues - following (homologous) alignment of a sequence of nucleotide and/or amino acids with a respective sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical nucleotides and amino acid residues, respectively, by the total number thereof and multiplying the product by 100.
  • BLAST Altschul et al., 1997)
  • BLAST2 Altschul et al., 1990
  • FASTA Pearson and Lipman, 1988
  • GAP Needleman and Wunsch, 1970
  • Smith-Waterman Smith and Waterman, 1981
  • Wisconsin GOG Package for determining sequence identity using standard parameters.
  • the percentage of sequence identity can, for example, be determined herein using the program BLASTP, version 2.2.5, November 16, 2002 (Altschul et al., 1997), calculating the percentage of numbers of “positives” (homologous amino acids) from the total number of amino acids selected for the alignment.
  • percent (%) sequence identity with respect to cells and/or strains described herein is preferably defined on nucleic acid level and thus, as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent nucleotides sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publically available computer software such as BLAST, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared. The same is applicable to amino acid sequences, mutatis mutandis.
  • nucleic acid sequences are provided by the local homology algorithm of Smith and Waterman, (1981), Advances in Applied Mathematics 2: 482-489. This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl. 3:353-358, National Biomedical Research Foundation, Washington, D.C., USA, and normalized by Gribskov (1986), Nucl. Acids Res. 14(6): 6745-6763. An exemplary implementation of this algorithm to determine percent identity of a sequence is provided by the Genetics Computer Group (Madison, Wis.) in the "BestFit" utility application.
  • a preferred method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, Calif). From this suite of packages the Smith-Waterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • the following illustrative example may be considered.
  • the resulting yeast cells may be genetically identically to the deposited genetic material of the initial yeast strain.
  • the cells “are” the cells from the deposited strain and have 100% sequence identity with the deposited material on genome level. However, a portion of the cells or even all cells may show some degree of genetic and/or epigenetic variation, for example due to one or more mutations.
  • the cells under study “are derived” from the initial strain the genetic material and/or cells of which is deposited.
  • Such mutations may be naturally occurring during cultivation and propagation of cells. Alternatively or additionally, though preferred, such mutations may be artificially introduced, e.g., by genetic engineering. Consequently, derived cells may potentially exhibit, e.g., a variation in the protein per dry matter (%) (w/w), the amount of essential amino acids and/or composition of essential amino acids, compared to the initial yeast strain the cells are derived from.
  • the obtained yeast cells may also be referred to as mutants compared to the cells and/or strain they are derived from.
  • genetic engineering is used in its broadest sense for methods known to the person skilled in the art to modify desired nucleic acids in vitro and in vivo, e.g. by targeted mutagenesis and/or recombinant DNA technology. Accordingly, said methods may comprise cloning, sequencing and transformation of recombinant nucleic acids, and appropriate vectors, primers, enzymes, host cells and the like are known by the skilled artisan.
  • genetically engineered cells are genetically engineered in view of high protein per dry matter (%), suitable essential amino acid composition, efficient ethanol usage as carbon source and the like in the context of the present invention.
  • the Saccharomycetales yeast cells are not genetically engineered. This is advantageous to ensure that the SCP product comprised in the animal feed comprises only well- defined and/or well characterized yeast cells but no mutated and thus, potentially undefined and/or uncharacterized yeast cells. This is also advantageous for a constant SCP product quality.
  • the yeast SCP product comprised in the animal feed according to the present invention comprises all essential amino acids. This is advantageous, as an animal feed comprising a yeast SCP product comprising all essential amino acids is capable of fully replacing currently used animal- and/or plant-derived protein sources in animal feed. Thus, said SCP product can represent an alternative protein source that does not require any supplementation in view of essential amino acids.
  • essential amino acid preferably refers to amino acids that cannot be synthesized by an animal from metabolic intermediates. Thus, such amino acids have to be supplied from an exogenous diet as they are required, e.g., for growth.
  • nine amino acids are considered essential: phenylalanine, valine, tryptophan, threonine, isoleucine, methionine, histidine, leucine, and lysine.
  • said nine essential amino acids are obtainable by a single complete protein containing all the essential amino acids.
  • Such complete proteins can be derived from animal-based sources of nutrition, whereas plant-based foods represent commonly a source for essential amino acids in the form of incomplete proteins.
  • the animal feed according to the present invention is preferably a feed for poultry, horses, camels, pigs, cows, such as beef cattle or dairy cows, sheep or companion animals, such as cats or dogs.
  • said animal feed is preferably a feed for aquatic species.
  • said species are preferably selected from crustaceans or fish.
  • the animal feed may be a feed for crustaceans and/or fish.
  • the animal feed is a feed for crustaceans, said crustaceans are preferably shrimps.
  • said fish may preferably be warm water fish or cold water fish.
  • said fish are warm water fish
  • said fish are preferably selected from the group consisting of catfish, tilapia, seabream, seabass, and carp.
  • said fish are cold water fish with said fish being selected from cod, salmon, or rainbow trout.
  • the animal feed according to the present invention is a feed for shrimps, salmon and/or rainbow trout.
  • Figure 1 shows yeast single cell protein product from different yeast strains when grown with ethanol as carbon source. Shown is the protein on dry matter (%) (w/w) of obtained SCP product (y-axis) per yeast strain (x-axis) of in total 8 genera.
  • Figure 2 left shows the biomass yield (g x g s ' 1 ) for Cyberlindnera jadinii CBS621 and Saccharomyces cerevisiae CEN.PK113-7D with ethanol as feedstock compared to glucose.
  • Figure 3 shows the protein content in the biomass (%DM) for Cyberlindnera jadinii CBS621 and Saccharomyces cerevisiae CEN.PK113-7D with ethanol as feedstock compared to glucose.
  • Figure 3 shows the protein yield for fermentations with Cyberlindnera jadinii FERM- BP1656 at different temperatures.
  • Figure 4 shows the protein content in the biomass for fermentations with Cyberlindnera jadinii FERM-BP1656 at different pH levels.
  • Figure 5 shows the content of malate synthase in the protein for Cyberlindnera jadinii FERM-BP1656 and Saccharomyces cerevisiae GHP1 with ethanol as feedstock compared to glucose.
  • Figure 6 shows the content of isocitrate lyase in the protein for Cyberlindnera jadinii FERM-BP1656 and Saccharomyces cerevisiae GHP1 with ethanol as feedstock compared to glucose.
  • Yeast strain Cyberlindnera jadinii FERM-BP1656 was cultivated in a shake flask (100 ml) for24h at 32 °C and 280 rpm.
  • the shake flask medium was based on Verduyn (Verduyn et al.. 1992), an overview of which is shown in Table 1.
  • a 250 ml seed fermenter was inoculated with 10 ml material, resulting in a starting weight of 100 g.
  • the medium composition of this fermenter is described in Table 1.
  • pH was controlled at 5.0 by addition of 10% (w/w) ammonia.
  • Temperature was controlled at 30 °C.
  • Airflow was controlled at 0.25 nL/min.
  • Dissolved oxygen concentration was controlled at 20% using the agitation rate. After all glucose in the seed fermenter was consumed, the ethanol feed rate was started at 0.615 g/h. This feed rate was subsequently exponentially increased with an exponent of 0. 1 h ⁇ 1 . After a feed rate of 3.0 g/h was reached, the feed rate was kept constant at this value. The feed consisted of 10 % (w/w) ethanol. This cultivation was run for 72h, after which biomass was harvested for inoculation of the main fermenter. At the end of this fermentation, a biomass concentration of 32.35 g dry weight/kg was obtained.
  • the 250 ml main fermenter was inoculated with 0.6 g dry matter or approximately 19 ml culture, resulting in a starting weight of 150 g.
  • the medium composition of this fermenter is described in Table 1.
  • pH was controlled at 5.0 by addition of 10% (w/w) ammonia.
  • Temperature was controlled at 30 °C.
  • Airflow was controlled at 0.25 nL/min.
  • Dissolved oxygen concentration was controlled at 20% using the agitation rate.
  • the ethanol feed rate was started upon inoculation at 2.1 19 g/h. This feed rate was subsequently exponentially increased with an exponent of 0.2 IT 1 . After a feed rate of 7.86 g/h was reached, the feed rate was kept constant at this value.
  • the feed consisted of 10 % (w/w) ethanol.
  • the ethanol feed rate range can also be expressed as a q s range of 0.035 gs/gx/h to 0.450 g e thanoi/gbiomass/h.
  • the oxygen uptake rate (OUR) reached at this feed rate was 190 mmol/kg/h.
  • This cultivation was run for 48h, at which point a biomass concentration of 38.46 g dry weight/kg was obtained, with a sum of hydrolysed amino acids (excluding cysteine and tryptophan) of g/gbiomass and a Kjeldahl protein content (N*6.25) of 57.4 %.
  • Table 1 Medium composition of the preculture shake flask, seed fermenter and main fermenter.
  • Raw files were analyzed with Spectronaut (Biognosys), version 17, against the proteins of a C.jadinii or S.cerevisiae database. Label-free quantification was performed using the top three peptides measured for each protein. Retention time realignment was done based on non-linear regression and normalization was set to total peptide amount.
  • Single cell protein products were generated using Saccharomycetales yeast cells from different yeast genera using ethanol as carbon source. More specifically, strains of the genera Cyberlindnera, Kluyveromyces, Wickerhamomyces, Yarrowia, Saccharomyces, Pichia, Ogateae and Blastobotrys were investigated.
  • Protein (g) per dry matter (%) (w/w) of SCP product obtained in the Example is given in Figure 1 for SCP products obtained using Saccharomycetales yeast cells from Yarrowia lipolytica, Kluyveromyces marxianus, Blastobotrys adeninivorans, Ogateae polymorpha, Pichia anomala, Pichia pastoris, Saccharomyces cerevisiae, Wickerhamomyces anomalus, Cyberlindnera jadinii and Kluyveromyces lactis.
  • yeast cells Saccharomycetales yeast cells from Wickerhamomyces anomalus, Kluyveromyces lactis, and Cyberlindnera jadinii in view of protein (g) per dry matter (g) given as protein on dry matter (%) with (up to) over 41 % (w/w) observed.
  • the present invention may also be summarized in the following items:
  • Method for cultivating a microorganism capable of producing at least 40% protein comprising the steps of:
  • Saccharomycetales yeast is a yeast from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces.
  • Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus. 10.
  • Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841 , Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus IFO 569, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576 or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841 , Wickerhamomyces anomalus IFO 569, Saccharo
  • yeast single cell protein product wherein the yeast single cell protein product comprising >40% protein on dry cell weight, wherein the protein comprises >0.1 %, preferably >0.2% isocitrate lyase and/OR malate synthase.
  • yeast single cell protein product of claim 11 wherein the yeast is a Saccharomycetales yeast.
  • Saccharomycetales yeast is a yeast from the genus Cyberlindnera, Saccharomyces, Kluyveromyces, Wickerhamomyces, Pichia or Yarrowia, preferably from the genus Cyberlindnera or Saccharomyces or Kluyveromyces or Wickerhamomyces.
  • yeast single cell protein product of any of claim 12 or 13, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii, Saccharomyces cerevisiae, Kluyveromyces lactis, Wickerhamomyces anomalus, Pichia anomala or Yarrowia lipolytica, preferably from Cyberlindnera jadinii or Saccharomyces cerevisiae or Kluyveromyces lactis or Wickerhamomyces anomalus.
  • yeast single cell protein product of any of claims 12 to 14, wherein the Saccharomycetales yeast is a yeast from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841 , Wickerhamomyces anomalus IFO 569, Saccharomyces cerevisiae GHP1, Saccharomyces cerevisiae CEN.PK113-7D, Wickerhamomyces anomalus CBS 1980, Cyberlindnera jadinii ATCC 9950, Kluyveromyces lactis CBS 2896, Wickerhamomyces anomalus CBS 2576 or Yarrowia lipolytica CBS 7504, preferably from Cyberlindnera jadinii ATCC 26387, Cyberlindnera jadinii FERM-BP1656, Cyberlindnera jadinii CBS621, Cyberlindnera jadinii CBS841 , Saccharomyces cerevisiae G
  • An animal feed comprising up to 20% (w/w) of yeast single cell protein product as claimed in any of the preceding claims.

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Abstract

La présente invention concerne un procédé de culture d'un micro-organisme, de préférence une levure Saccharomycetales, capable d'utiliser de l'éthanol comme charge d'alimentation. L'invention concerne un procédé de production de biomasse, en particulier d'une protéine monocellulaire, le produit protéique unicellulaire de levure comprenant des cellules de levure Saccharomycetales, ainsi que des aliments pour animaux comprenant une telle biomasse.
PCT/EP2024/073010 2023-08-16 2024-08-15 Production durable de biomasse Pending WO2025036979A1 (fr)

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