WO2025248558A1 - Procédé de production d'un produit de fermentation par levure et composition nutritive - Google Patents

Procédé de production d'un produit de fermentation par levure et composition nutritive

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Publication number
WO2025248558A1
WO2025248558A1 PCT/IN2025/050807 IN2025050807W WO2025248558A1 WO 2025248558 A1 WO2025248558 A1 WO 2025248558A1 IN 2025050807 W IN2025050807 W IN 2025050807W WO 2025248558 A1 WO2025248558 A1 WO 2025248558A1
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Prior art keywords
amount ranging
protein
loog
ranging
nutrient composition
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PCT/IN2025/050807
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English (en)
Inventor
Jeevarathinam SESHAJALAM
Subramani Ramachandrappa
Ramanan Thirumoorthy
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Fermbox Bio Private Ltd
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Fermbox Bio Private Ltd
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Publication of WO2025248558A1 publication Critical patent/WO2025248558A1/fr
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    • 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
    • C12P21/00—Preparation of peptides or proteins
    • 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; 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
    • C12N1/06—Lysis of microorganisms
    • C12N1/063—Lysis of microorganisms of 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; 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
    • 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; 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • 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
    • C12R2001/78—Hansenula
    • 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
    • C12R2001/84—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
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the present disclosure relates to the field of fermentation technology.
  • the present disclosure particularly relates to an efficient process for producing fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules by utilizing nutrient composition extracted from cell biomass.
  • the present disclosure relates to a continuous fermentation process to produce the fermentation product by utilizing nutrient composition extracted from the cell biomass.
  • the present disclosure also relates to nutrient composition extracted from the cell biomass of a fermentation process.
  • Fermentation process plays an important role in production of a wide range of bio-based products, including biofuels, pharmaceuticals, food additives, and biochemicals.
  • the fermentation process involves use of microorganisms, such as bacteria, yeast or fungi to convert organic substrates into desired products through metabolic pathways.
  • microorganisms such as bacteria, yeast or fungi to convert organic substrates into desired products through metabolic pathways.
  • fermentation processes often encounter challenges related to low yield efficiencies and environmental sustainability.
  • the present disclosure aims to provide a fermentation process with two-fold advantage, i.e., i. utilization of nutrients from cell biomass for effective production of fermentation product; and ii. effective management of fermentation effluent.
  • the present disclosure relates to a simple, economical and energy efficient process for production of fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules , wherein the fermentation process employs nutrient composition extracted from the cell biomass.
  • object of the present disclosure is extracting nutrient composition from cell biomass and utilizing the extracted nutrient composition in the fermentation process for efficient production fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules.
  • efficient production fermentation product such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules.
  • the present disclosure relates to a process for producing fermentation product, said process comprising-
  • the present disclosure also relates to a nutrient composition extracted from the cell biomass of fermentation process.
  • the nutrient composition comprises minerals, amino acid, vitamins and/or micronutrients.
  • the nutrient composition aids in improving growth of microorganisms during fermentation, as a result enhances the yield of fermentation product.
  • exemplary or ‘exemplary embodiment’ as used herein refers to ‘serving as an example, instance, or illustration.’ Any embodiment of implementation of the present subject matter described herein as ‘exemplary’ is not necessarily to be construed as preferred or advantageous over other embodiments.
  • the term ‘extracted nutrients’ or ‘nutrient extract’ are used interchangeable and refers to nutrient composition extracted from cell biomass of the fermentation process.
  • cell biomass or ‘microorganism biomass’ can be used interchangeably and refers to biomass obtained after each batch of fermentation process.
  • the cell biomass is employed for extracting nutrient composition for use in subsequent fermentation process for efficient production of fermentation product.
  • the present disclosure relates to simple, economical, energy efficient and improved process for producing fermentation product, such as proteins, enzymes, small organic molecules including but not limited vitamins, antibiotics, APIs and building block molecules.
  • the process of the present disclosure for producing fermentation product is environmentally friendly.
  • the process of the present disclosure provides higher yield of fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules when compared to the fermentation process not employing extracted nutrient composition from cell biomass as described in the present disclosure.
  • the inventors of the present disclosure have particularly identified that adding extracted nutrient composition from cell biomass to the culture medium comprising microorganism and providing optimum condition for growth of the microorganism leads to efficient production of fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules.
  • fermentation product such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules.
  • adding extracted nutrient composition from cell biomass to the culture medium comprising microorganism, during fermentation and providing optimum condition leads to higher yield of the fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs, building block molecules.
  • the fermentation product is selected from a group comprising cellulase enzyme, insulin, insulin analogues, egg proteins and milk proteins.
  • addition of the extracted nutrient composition from the cell biomass to the culture medium comprising microorganism and providing optimum condition for growth of the microorganism increases yield of the fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules by least 22% when compared to the process not including extracted nutrient composition from the cell biomass as described in the present disclosure.
  • the process for producing fermentation product comprises-
  • extraction of nutrient composition comprises-
  • extraction of nutrient composition comprises-
  • microorganism includes but not limited to yeast.
  • the microorganism includes but not limited to Pichia pastoris, Saccharomyces cerevisiae and Hansenulci polymorphci .
  • the lysing the cell biomass is carried out by techniques including but not limited to heat treatment and enzyme treatment.
  • the lysing of the cell biomass including but not limited to Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha is carried out by combination of heat treatment and enzyme treatment.
  • the heat treatment of the cell biomass is carried out at a temperature ranging from about 60 °C to 100 °C, including all the values in the range, for instance, 61 °C, 62 °C, 63 °C, 64 °C and so on and so forth, up until 100 °C.
  • the heat treatment is carried out for a duration ranging from about 30 minutes to 60 minutes, including all the values in the range, for instance, 31 minutes, 32 minutes, 33 minutes, 34 minutes and so on and so forth.
  • the heat treatment of the cell biomass is carried out at a temperature of about 60 °C, about 65 °C, about 70 °C, about 75 °C, about 80 °C, about 85 °C, about 90 °C, about 95 °C or about 100 °C. In an embodiment, the heat treatment of the cell biomass is carried out for a duration of about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes or about 60 minutes.
  • the enzyme treatment of the cell biomass is carried out by employing enzyme including but not limited to cellulase, protease, hemi-cellulase, xylanase, carboxy methyl cellulase, arabinose and amylase.
  • the enzyme treatment of the cell biomass is carried out by enzyme cocktail comprising at least one of enzyme comprising cellulase, protease, hemi-cellulase, xylanase, carboxy methyl cellulase, arabinose and amylase.
  • the enzyme treatment of the cell biomass is carried out by enzyme cocktail comprising cellulase, hemi -cellulase, protease, and amylase.
  • the enzyme treatment of the cell biomass is carried out at a temperature ranging from about 50 °C to 60 °C for a duration ranging from about 60 minutes to 120 minutes.
  • the enzyme treatment of the cell biomass is carried out at a temperature of about 50 °C, about 51 °C, about 52 °C, about 53 °C, about 54 °C, about 55 °C, about 56 °C, about 57 °C, about 58 °C, about 59 °C or about 60 °C.
  • the enzyme treatment of the cell biomass is carried out for a duration ranging from about 60 minutes to 120 minutes, including all the values in the range, for instance, 61 minutes, 62 minutes, 63 minutes, 64 minutes and so on and so forth, up until 120 minutes.
  • the enzyme treatment of the cell biomass is carried out for a duration of about 60 minutes, about 70 minutes, about 80 minutes, about 90 minutes, about 100 minutes, about 110 minutes or about 120 minutes.
  • the enzyme treatment of the cell biomass is carried out at a pH ranging from about 5 to 7, including all the values in the range, for instance, 5.1, 5.2, 5.3, 5.4 and so on and so forth, up until 7. In an embodiment, the enzyme treatment of the cell biomass is carried out at a pH of about 5, about 5.5, about 6, about 6.5 or about 7.
  • the processing/treating of the extracted nutrients is subjecting the extracted nutrients to separation techniques for separating the nutrient composition from undesired components, such as solid mass.
  • lysis of the cell biomass for extraction of the nutrient composition comprises- subjecting the cell biomass to heat treatment at a temperature ranging from about 60 °C to 100 °C, for a duration ranging from about 30 minutes to 60 minutes; and subjecting the heat treated cell biomass to enzyme treatment at a temperature ranging from about 50 °C to 60 °C and at a pH ranging from about 5 to 7, for a duration ranging from about 60 minutes to 120 minutes.
  • the enzyme treated cell biomass is subjected to cooling to a temperature ranging from about 20 °C to 40 °C, including all the values in the range, for instance, 21 °C, 22 °C, 23 °C, 24 °C and so on and so forth, up until 40 °C.
  • the enzyme treatment cell biomass is subjected to cooling to a temperature of about 20 °C, about 25 °C, about 30 °C, about 35 °C or about 40 °C.
  • the cooled enzyme treated cell biomass is subjected to separation technique including but not limited to centrifugation and filtration for extraction of nutrient composition.
  • Liquid extract obtained from the separation techniques comprises combination of nutrients (nutrient composition) including components selected from a group comprising minerals, amino acids, vitamins, micronutrients and combinations thereof.
  • the minerals present in the nutrient composition includes but not limited to potassium, calcium, magnesium, copper, sodium, manganese, boron, cobalt, zinc and iron.
  • the potassium is in an amount ranging from about 100,000 to 300,000 ppb;
  • the calcium is in an amount ranging from about 300 ppb to 900pb;
  • the magnesium is in an amount ranging from about 5000 to 10,000 ppb,
  • the copper is in an amount ranging from about 50 to 200 ppb;
  • the sodium is in an amount ranging from about 2000 to 4000 ppb;
  • the manganese is in an amount ranging from about 50 to 200 ppb,
  • the boron is in an amount ranging from about 20 to 100 ppb;
  • the cobalt is in an amount ranging from about 20 to 100;
  • the zinc is in an amount ranging from about 500 to 2000 ppb;
  • the iron is in an amount ranging from about 1000 to 2500 ppb.
  • the potassium is an amount of about 145127 ppb
  • the calcium is in an amount of about 670.8 ppb
  • the magnesium is in an amount of about 6719.9 ppb
  • the copper is in an amount of about 127 ppb
  • the sodium is in an amount of about 2869.1 ppb
  • the manganese is in an amount of about 89.3
  • boron is in an amount of about 41.4 ppb
  • cobalt is in an amount of about 51.6 ppb
  • the zinc is in an amount of about 1152.4
  • iron is in an amount of about 1547 ppb.
  • the nutrient composition comprises total amino acid content ranging from about 40g/100 g to 75 g/100 g of proteins, including all the values in the range, for instance 41 g/100 g, 42 g/100 g, 43 g/100 g, 44 g/100 g and so forth, up until 75 g/lOOg.
  • the nutrient composition comprises total amino acid content of about 57. 17 g/100 g of proteins.
  • the amino acid in the nutrient composition includes but not limited to aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucin, tyrosine, phenylalanine, histidine, lysine, arginine, proline and tryptophan.
  • the aspartic acid is in an amount ranging from about 2 to 10 g/lOOg of protein
  • threonine is in an amount ranging from about 1 to 5 g/lOOg of protein
  • the serine is in an amount ranging from about 1 to 5 g/lOOg of protein
  • the glutamic acid is in an amount ranging from about 5 to 15 g/lOOg of protein
  • the glycine is in an amount ranging from about 1 to 5
  • the alanine is in an amount ranging from about 2 to 10 g/lOOg of protein
  • cysteine is in an amount ranging from about 0.5 to 5 g/lOOg of protein
  • the valine is in an amount ranging from about 2 to 10 g/lOOg of protein
  • the methionine is in an amount ranging from about 0.5 to 5 g/lOOg of protein
  • the isoleucine is in an amount ranging from about 1 to 5 g/lOOg of protein
  • the leucin is
  • the nutrient composition comprises aspartic acid in an amount of about 5.79 g/lOOg of protein, threonine in an amount of about 2.59 g/lOOg of protein, serine in an amount of about 2.84 g/lOOg of protein, glutamic acid in an amount of about 9.05 g/lOOg of protein, glycine in an amount of about 2.94 g/lOOg of protein, alanine in an amount of about 4.18 g/lOOg of protein, cysteine in an amount of about 0.74g/100g of protein, valine in an amount of about 3.44 g/lOOg of protein, methionine in an amount of about 0.9g/100g of protein, isoleucine in an amount of about 2.9 g/lOOg of protein, leucin in an amount of about 4.09 g/lOOg of protein, tyrosine in an amount of about 2.17 g/100 of protein, phenylalanine in an amount of about 2.
  • the vitamin in the nutrient composition includes but not limited to riboflavin, calciferol, niacin and thiamine.
  • the micronutrient in the nutrient composition includes but not limited to iodine and molybdenum.
  • residual cell biomass solids obtained after the separation technique is subjected to effluent treatment.
  • the residual cell biomass solids would be about 15% of the original cell mass.
  • the culture medium employed for cultivation of microorganism includes but not limited to basal salt medium, FM22 medium, complex medium and modified basal medium.
  • the extraction of nutrient composition comprises- cultivating microorganism including but not limited to Pichia pastoris in a culture medium including but not limited to basal salt medium; subjecting cell biomass to heat treatment at a temperature ranging from about 60 °C to 100 °C for a duration ranging from about 30 minutes to 60 minutes; subjecting the heat treated cell biomass to enzyme treatment by employing enzymes selected from a group comprising cellulase, protease, hemi-cellulase, amylase and combinations thereof, at a temperature ranging from about 50 °C to 60 °C and at pH ranging from about 5 to 7, for a duration ranging from about 60 to 120 minutes; and
  • the inventors have particularly identified that heating the cell biomass to a temperature ranging from about 60 °C to 100 °C for a duration ranging from about 50 minutes to 60 minutes, followed by enzyme treatment by employing enzymes selected from a group comprising cellulase, protease, hemi-cellulase, amylase and combinations thereof at a temperature ranging from about 50 °C to 60 °C and at pH ranging from about 5 to 7, for a duration ranging from about 60 to 120 minutes, ensures selective liberation of nutrient composition comprising components selected from a group comprising minerals, amino acids, vitamins, micronutrients and combinations thereof while preserving the integrity of sensitive nutrients and bioactive compounds.
  • the disclosed process of extracting nutrient composition from the cell biomass is scalable and suitable for industrial-scale applications, offering a cost- effective solution for the nutrient composition extraction from cell biomass including but not limited to yeast biomass.
  • the optimum condition for growth of the microorganism is- temperature in the range of about 20 °C to 30 °C, pH in the range of about 5 to 6, dissolved oxygen content of at least 25% and duration in the range of about 3 days to 8 days.
  • the temperature is about 20 °C, about 21 °C, about 22 °C, about 23 °C, about 24 °C, about 25 °C, about 26 °C, about 27 °C, about 28 °C, about 29 °C or about 30 °C.
  • the pH is about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7 a about 5.8, about 5.9 or about 6.0.
  • the duration is about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or about 8 days.
  • the dissolved oxygen content is maintained in a range of about 10% to 60%. In another embodiment, the dissolved oxygen content is maintained at about 10%, about 20%, about 30%, about 40%, about 50% or about 60%.
  • the process for producing fermentation product comprises-
  • the process of producing the fermentation product is an integrated process involving extraction of specific combination of nutrient in the form of nutrient composition, following by adding the nutrient composition, optionally along with treated effluent of cell biomass to subsequent batch of fermentation to produce said fermentation product at an improved rate or yield.
  • the process for producing fermentation product such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules comprises-
  • - cultivating microorganism in a culture medium at a temperature ranging from about 22 to 30°C, for a duration ranging from about 3 to 8 days; subjecting cell biomass to heat treatment at a temperature ranging from about 60 °C to 100 °C, for a duration ranging from about 30 minutes to 60 minutes; subjecting the heat treated cell biomass to enzyme treatment by employing enzymes selected from a group comprising cellulase, protease, hemi-cellulase, amylase and combinations thereof, at a temperature ranging from about 50 °C to 60 °C and at pH ranging from about 5 to 7, for a duration ranging from about 60 to 120 minutes;
  • the process for producing fermentation product such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules comprises-
  • - cultivating microorganism in a culture medium at a temperature ranging from about 22 to 30°C, for a duration ranging from about 3 to 8 days; subjecting cell biomass to heat treatment at a temperature ranging from about 60 °C to 100 °C, for a duration ranging from about 30 minutes to 60 minutes; subjecting the heat treated cell biomass to enzyme treatment by employing enzymes selected from a group comprising cellulase, protease, hemi-cellulase, amylase and combinations thereof, at a temperature ranging from about 50 °C to 60 °C and at pH ranging from about 5 to 7, for a duration ranging from about 60 to 120 minutes;
  • about 100 litres to 200 litres of the harvested volume of the nutrient composition is added to the culture medium for producing fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules in each batch.
  • the process of the present disclosure increases yield of the fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules by at least 22% when compared to conventional fermentation process not including addition of said nutrient composition as described in the present disclosure.
  • the nutrient composition obtained from the cell biomass of the microorganism are reintroduced into the culture medium, thereby enriching the culture medium with essential specific combination of nutrients, as a result improving the yield of the fermentation product by the cultured microorganism including but not limited to yeast, such as Pichia pastoris.
  • the process of the present disclosure uses the nutrient composition from the cell biomass of the previous fermentation batch rather than discarding the cell biomass for increasing nutritional value of the culture medium for efficient production of the fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules. Thereby, reduces burden on the effluent treatment and improves production of fermentation product.
  • the process of the present disclosure is a continuous fermentation process to produce fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules, wherein the nutrient composition from the cell biomass is effectively added to the culture medium for increasing the nutritional value of the culture medium and thereby improving the production of said fermentation product.
  • fermentation product such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules
  • the process of the present disclosure addresses the problem of fermentation industries dealing with waste disposal in the form of cell biomass.
  • the cell biomass is treated, and specific combination of nutrient is extracted in the form of nutrient composition to use in further fermentation batches for efficient production of fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules.
  • fermentation product such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules.
  • the present disclosure further relates to nutrient composition obtained from the cell biomass of the fermentation process.
  • the nutrient composition of the present disclosure aid in improving the growth the microorganism during fermentation, as a result enhances of the production of fermentation product when compared to a fermentation process not employing said nutrient composition.
  • the nutrient composition of the present disclosure aids in improving the yield of the fermentation product by at least 20%.
  • the nutrient composition comprises mineral, amino acid, vitamin, micronutrient and combinations thereof.
  • the mineral in the nutrient composition is selected from a group comprising potassium, calcium, magnesium, copper, sodium, manganese, boron, cobalt, zinc and iron.
  • the potassium is in an amount ranging from about 100,000 to 300,000 ppb;
  • the calcium is in an amount ranging from about 300 ppb to 900pb;
  • the magnesium is in an amount ranging from about 5000 to 10,000 ppb,
  • the copper is in an amount ranging from about 50 to 200 ppb;
  • the sodium is in an amount ranging from about 2000 to 4000 ppb;
  • the manganese is in an amount ranging from about 50 to 200 ppb,
  • the boron is in an amount ranging from about 20 to 100 ppb;
  • the cobalt is in an amount ranging from about 20 to 100;
  • the zinc is in an amount ranging from about 500 to 2000 ppb;
  • the iron is in an amount ranging from about 1000 to 2500 ppb.
  • the nutrient composition comprises total amino acid content ranging from about 40g/100 g to 75 g/100 g of proteins, including all the values in the range, for instance 41 g/100 g, 42 g/100 g, 43 g/100 g, 44 g/100 g and so forth, up until 75 g/lOOg.
  • the amino acid in the nutrient composition is selected from a group comprising aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucin, tyrosine, phenylalanine, histidine, lysine, arginine, proline and tryptophan.
  • the aspartic acid is in an amount ranging from about 2 to 10 g/lOOg of protein
  • threonine is in an amount ranging from about 1 to 5 g/lOOg of protein
  • the serine is in an amount ranging from about 1 to 5 g/lOOg of protein
  • the glutamic acid is in an amount ranging from about 5 to 15 g/lOOg of protein
  • the glycine is in an amount ranging from about 1 to 5
  • the alanine is in an amount ranging from about 2 to 10 g/lOOg of protein
  • cysteine is in an amount ranging from about 0.5 to 5 g/lOOg of protein
  • the valine is in an amount ranging from about 2 to 10 g/lOOg of protein
  • the methionine is in an amount ranging from about 0.5 to 5 g/lOOg of protein
  • the isoleucine is in an amount ranging from about 1 to 5 g/lOOg of protein
  • the leucin is
  • the vitamin in the nutrient composition is selected from a group comprising riboflavin, calciferol, niacin and thiamine.
  • the micronutrient in the nutrient composition is selected from a group comprising iodine and molybdenum.
  • Yeast extract 0.5%, Peptone 1.0%, and Glycerol 1.0% were autoclaved at 121 °C for 20 minutes.
  • a fresh recombinant culture of Pichia pastoris was removed from the deep freezer (-80°C) and inoculated into the sterile YPG medium and kept in shaker incubator at 200 rpm and 30 °C for 24 hours.
  • the seed culture was transferred to a bigger volume in the flask to match the volume required for inoculating 100L fermentation batch.
  • 5L inoculum was used to inoculate the 100L fermentation batch in 300 L fermenter.
  • Optical density, pH, and sterility were checked before transferring the inoculum from one stage to another. It was made sure the culture has attained the proper growth and is in the early logarithmic phase of the growth. Optical density (OD) of the culture was noted to be 15 to 20 at about 24 hours of incubation.
  • Optical density (OD) of the culture was noted to be 15 to
  • basal salt media Required amounts of individual basal salts components were weighed according to the formulation of the basal salt media (as provided in https://tools.thermofisher.com/content/sfs/manuals/pichiaferm_prot.pdf). Each component was added sequentially to a clean and sterile container containing a suitable volume of portable water, ensuring complete dissolution before adding the next component. The solution was gently stirred to facilitate dissolution and homogenization of the basal salts. Once all the basal salts were added and dissolved completely, the medium was transferred to the fermenter and the volume was made up to about 160 litres with the RO water. The medium was sterilized using the program given in the SCADA (Supervisory Control and Data Acquisition) computer.
  • SCADA Supervisory Control and Data Acquisition
  • Pichia trace elements solution was prepared separately as per the protocol (as provided in https://tools.thermofisher.com/content/sfs/manuals/pichiaferm_prot.pdf) and filter sterilized using PES filter. PTM was added to the fermenter after adjusting the pH of the media to 5.00 with addition of ammonium hydroxide solution.
  • Fermentation was performed separately as per the protocol (as provided in https://tools.thermofisher.com/content/sfs/manuals/pichiaferm_prot.pdf) and filter sterilized using PES filter. PTM was added to the fermenter after adjusting the pH of the media to 5.00 with addition of ammonium hydroxide solution.
  • Fermentation was carried out in 3 phases- First one is batch phase where initial glycerol was consumed. Second one was glycerol fed batch to build the biomass where the cell OD was increased to 250 at 600 nm. Third one was induction phase where 100% methanol was fed to induce the recombinant protein expression, in this phase methanol acts as both inducer and carbon source. While increasing the methanol feed, accumulation of methanol was checked periodically by analysing the sample with GC (gas chromatography). If the accumulation of methanol was found, either the feed of methanol was stopped to let the culture consume the accumulated methanol or the feed rate of methanol was reduced to match the consumption.
  • GC gas chromatography
  • ammonium hydroxide was used to maintain the pH of about 5.5, temperature was maintained at about 25 °C and the fermentation process was carried out for about 6 days Dissolved oxygen was maintained at about 30% throughout the process. About 25% of autoclaved polypropylene glycol was manually added to the fermenter whenever the foam appears visually.
  • the fermentation batch was harvested by cooling it down to 10°C to prevent contamination and prevent protein degradation.
  • Volume of the fermenter at the end of fermentation was noted to be 220 litres because of glycerol and methanol feeding.
  • the wet weight of the total broth was 88 Kgs based on the packed cell volume calculations and the supernatant volume was 132 litres.
  • Harvested broth was then put through continuous centrifuge/fdter press for separating the cells from the supernatant which contains protein of interest (fermentation product), such as cellulase enzyme, insulin, insulin analogues, egg proteins or milk proteins.
  • protein of interest such as cellulase enzyme, insulin, insulin analogues, egg proteins or milk proteins.
  • Cell broth was continuously fed into the centrifuge through a feed inlet. Continuous centrifuge takes a feed at 25 litres per hour feed rate. As the broth entered to the centrifuge, it was subjected to highspeed rotation of 7500 rotations per minute. This force causes the denser components of the mixture to move outward towards the walls of centrifuge rotor while the lighter components remain closer to the centre. Inside the centrifuge, there was a separation zone where the denser components accumulate.
  • This zone was typically located near the walls of the centrifuge rotor.
  • the lighter components form an inner layer or core.
  • centrifugation was repeated till the protein recovery was >90%.
  • Final slurry which has nil to negligible amount of recombinant protein was collected separately.
  • the resultant supernatant volume was 264 litres after processing for 3 times and was further processed for extracting the protein of interest through various filtrations, chromatography techniques.
  • Example 3 Extraction of nutrient composition from cell biomass
  • Cell biomass left over after extraction of the fermentation product was transferred into a heat-resistant reactor and diluted to ease the heating process.
  • the cell biomass was heated at about 80°C for a predetermined duration of about 30 to 60 minutes, using a controlled heating system. The temperature was monitored throughout the process to ensure uniform heating and avoid overheating.
  • An enzyme cocktail was prepared containing a combination of cellulase, hemi-cellulase, protease, and amylase enzymes. This enzyme cocktail was added to the heated cell biomass at 5 ml/1 concentration, followed by thorough mixing. The biomass-enzyme mixture was incubated at an optimal temperature 50-60°C and pH was maintained at 5.0-7.0, respectively. After the enzymatic treatment, the mixture was cooled to room temperature and centrifuged or filtered to separate the liquid extract from residual biomass solids. The liquid extract contained combination of nutrients referred to as nutrient composition.
  • the residual biomass solids were sent for effluent treatment which is 15% of the original volume of the cell biomass before treatment which is a significant reduction of biomass in volume for effluent treatment.
  • the nutrient composition included amino acids, minerals, and vitamins, which were analysed using GC/Kjeldahl. Details of the minerals and amino acids in the nutrient composition is provided in Table 1 and Table 2. Table 1:
  • Example 4 Improving the yield of Fermentation product by addition of nutrient composition according to present disclosure
  • the fermentation product, cellulase enzyme was produced according to the process described herein.
  • Basal salt medium was prepared in reduced volume, i.e., about 30 litres and added to the fermenter to make up the volume to about 160 litres. In this fermentation process about75% of the volume has been carried forward from the previous batch along with the nutrient composition.
  • Fermentation was carried out according to the process described in Example 1c. In this fermentation process, growth of the microorganism was slightly faster than the previous batch (fermentation described in Example 1c) and the protein expression was 22.5% higher than the previous batch.
  • Example 5 Improving the yield of Fermentation production by addition of nutrient composition according to present disclosure
  • the fermentation product such as cellulase enzyme, insulin, insulin analogues, egg proteins or milk proteins was produced according to the process described herein.
  • Fermentation was carried out according to the process described in Example 1c. In this fermentation process, growth of the microorganism was slightly faster than the previous batch (fermentation described in Example 1c) and the protein expression was about 24.2 % higher than the previous batch.
  • a process for production of fermentation product in fermentation process said process comprises-
  • the lysing the cell biomass comprises subjecting the cell biomass to heat treatment and enzyme treatment.
  • the heat treatment is carried out for a duration ranging from about 60 °C to 100 °C, for a duration ranging from about 30 minutes to 60 minutes.
  • the enzyme treatment is carried out by adding enzyme selected from a group comprising cellulase, protease, hemicellulase, xylanase, carboxy methyl cellulase, arabinose and amylase and combinations thereof.
  • the process as defined in embodiment 3, post the enzyme treatment, the enzyme treated microorganism (cell biomass) and constituents are subjected to separation technique selected from a group comprising centrifugation and filtration to obtain liquid extract comprising nutrient composition and residual biomass solids.
  • the nutrient composition comprises component selected from a group comprising minerals, amino acids, vitamins, micronutrients and combinations thereof.
  • the minerals is selected from a group comprising potassium, calcium, magnesium, copper, sodium, manganese, boron, cobalt, zinc, iron and combinations thereof.
  • amino acid is selected from a group aspartic acid, threonine, serine, glutamic acid, glycine, alanine, cysteine, valine, methionine, isoleucine, leucin, tyrosine, phenylalanine, histidine, lysine, arginine, proline, tryptophan and combinations thereof.
  • a nutrient composition extracted from cell biomass of fermentation process comprises component selected from a group comprising mineral, amino acid, vitamin, micronutrients and combinations thereof.

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Abstract

La présente invention concerne un procédé simple, économique et économe en énergie pour la production de produit de fermentation. Le procédé de la présente invention utilise une composition nutritive obtenue à partir de biomasse cellulaire résultant d'un procédé de fermentation. Le procédé de la présente invention permet une production efficace de produit de fermentation par comparaison avec un procédé classique. La présente invention concerne également une composition nutritive extraite de la biomasse cellulaire obtenue à partir d'un procédé de fermentation.
PCT/IN2025/050807 2024-05-31 2025-05-29 Procédé de production d'un produit de fermentation par levure et composition nutritive Pending WO2025248558A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3961080A (en) * 1972-10-17 1976-06-01 Kikkoman Shoyu Co., Ltd. Process for autolysis of yeast
US6159724A (en) * 1994-05-27 2000-12-12 Agrano Ag Process for preparing culture mediums for culturing yeasts and lactic acid bacteria
US20100183767A1 (en) * 2007-07-10 2010-07-22 Bertus Noordam Yeast autolysates
US20130273555A1 (en) * 2010-05-05 2013-10-17 Mascoma Corporation Detoxification of Biomass Derived Acetate Via Metabolic Conversion to Ethanol, Acetone, Isopropanol, or Ethyl Acetate
WO2019175809A1 (fr) * 2018-03-13 2019-09-19 Lallemand Hungary Liquidity Management Llc Levure et produit de levure inactivés pour améliorer le rendement de fermentation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3961080A (en) * 1972-10-17 1976-06-01 Kikkoman Shoyu Co., Ltd. Process for autolysis of yeast
US6159724A (en) * 1994-05-27 2000-12-12 Agrano Ag Process for preparing culture mediums for culturing yeasts and lactic acid bacteria
US20100183767A1 (en) * 2007-07-10 2010-07-22 Bertus Noordam Yeast autolysates
US20130273555A1 (en) * 2010-05-05 2013-10-17 Mascoma Corporation Detoxification of Biomass Derived Acetate Via Metabolic Conversion to Ethanol, Acetone, Isopropanol, or Ethyl Acetate
WO2019175809A1 (fr) * 2018-03-13 2019-09-19 Lallemand Hungary Liquidity Management Llc Levure et produit de levure inactivés pour améliorer le rendement de fermentation

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