A process for producing fermentation product through yeast and a nutrient composition

Abstract

The present disclosure relates to a simple, economical and energy efficient process for production of fermentation product. The process of the present disclosure employs nutrient composition obtained from cell biomass of fermentation process. The process of the present disclosure provides for efficient production of fermentation product when compared to conventional process. The present disclosure also relates to a nutrient composition extracted from cell biomass obtained from fermentation process.

Description

[0001] “A PROCESS FOR PRODUCING FERMENTATION PRODUCT THROUGH YEAST AND A NUTRIENT COMPOSITION”

[0002] TECHNICAE FIELD

[0003] 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.

[0004] BACKGROUND OF THE DISCLOSURE

[0005] 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. However, despite a widespread application, fermentation processes often encounter challenges related to low yield efficiencies and environmental sustainability.

[0006] Primary challenge in industrial fermentation is suboptimal utilization of nutrients by microorganisms, leading to lower product yield. Further, accumulation of metabolic byproducts and unused nutrients results in generation of nutrient-rich effluents, which pose environmental concerns when discharged into water bodies or soil.

[0007] Efforts to improve fermentation processes have traditionally focused on optimizing fermentation conditions, such as pH, temperature, oxygen levels, and substrate concentrations. While these approaches have led to incremental improvements in yield and productivity, they often fail to address the broader issue of nutrient utilization, efficiency and effluent management.

[0008] Thus, there is a need for a process for improved production of fermentation product by effectively utilizing the nutrients from the cell biomass. 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. SUMMARY OF THE DISCLOSURE

[0009] 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.

[0010] In one embodiment, 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. As a result, the cell biomass from each batch of the fermentation is effectively utilized for extracting nutrient composition for using in the subsequent fermentation process, thereby reducing burden on effluent treatment. The extracted nutrient composition from the cell biomass leads to efficient production of fermentation product due to rich nutritional value of the nutrient composition. As a result, making the fermentation process economical, efficient and leads to higher yield of fermentation product.

[0011] Accordingly, in one embodiment, the present disclosure relates to a process for producing fermentation product, said process comprising-

[0012] - extracting nutrient composition from cell biomass;

[0013] - adding the extracted nutrient composition to a culture medium comprising microorganism; and maintaining optimum condition for growth of the microorganism, thereby producing the fermentation product.

[0014] 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.

[0015] DETAILED DESCRIPTION OF THE DISCLOSURE

[0016] Unless otherwise defined, all terms used in the disclosure, including technical and scientific terms, have meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. By means of further guidance, term definitions are included for better understanding of the present disclosure.

[0017] As used herein, the singular forms ‘a’, ‘an’ and ‘the’ include both singular and plural referents unless the context clearly dictates otherwise.

[0018] The term ‘comprising’, ‘comprises’ or ‘comprised of as used herein are synonymous with ‘including’, ‘includes’, ‘containing’ or ‘contains’ and are inclusive or open-ended and do not exclude additional, non-recited members, elements, or method steps.

[0019] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0020] The term ‘about’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, is meant to encompass variations of ±10% or less, preferably ±5% or less, more preferably ±1% or less and still more preferably ±0.1% or less of and from the specified value, insofar such variations are appropriate to perform the present disclosure. It is to be understood that the value to which the modifier ‘about’ refers is itself also specifically and preferably disclosed.

[0021] The term ‘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.

[0022] Reference throughout this specification to ‘some embodiments’, ‘one embodiment’ or ‘an embodiment’ means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases ‘in some embodiments’, ‘in one embodiment’ or ‘in an embodiment’ in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

[0023] As used herein, the term ‘extracted nutrients’ or ‘nutrient extract’ are used interchangeable and refers to nutrient composition extracted from cell biomass of the fermentation process.

[0024] As used herein, the term ‘cell biomass’ or ‘microorganism biomass’ can be used interchangeably and refers to biomass obtained after each batch of fermentation process. In the present disclosure, the cell biomass is employed for extracting nutrient composition for use in subsequent fermentation process for efficient production of fermentation product.

[0025] 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.

[0026] In one embodiment, 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.

[0027] 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. The inventors have also noted that 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. In an embodiment, the fermentation product is selected from a group comprising cellulase enzyme, insulin, insulin analogues, egg proteins and milk proteins.

[0028] In some embodiments, 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.

[0029] In some embodiments of the present disclosure, the process for producing fermentation product comprises-

[0030] - extracting nutrient composition from cell biomass;

[0031] - adding the extracted nutrient composition to a culture medium comprising microorganism; and maintaining optimum condition for growth of the microorganism, thereby producing the fermentation product.

[0032] In some embodiments of the present disclosure, in the process of producing the fermentation product, extraction of nutrient composition comprises-

[0033] - lysing cell biomass / biomass of the microorganism;

[0034] - extracting nutrients from the lysed cell biomass of the microorganism; and

[0035] - processing / treating the extracted nutrients to obtain nutrient composition.

[0036] In some embodiments of the present disclosure, in the process of producing fermentation product, extraction of nutrient composition comprises-

[0037] - cultivating microorganism in a culture medium;

[0038] - lysing cell biomass / biomass of the microorganism; - extracting nutrients from the lysed cell biomass / biomass; and

[0039] - processing / treating the extracted nutrients to obtain nutrient composition.

[0040] In some embodiments of the present disclosure, microorganism includes but not limited to yeast. In an embodiment, the microorganism includes but not limited to Pichia pastoris, Saccharomyces cerevisiae and Hansenulci polymorphci .

[0041] In some embodiments of the present disclosure, the lysing the cell biomass is carried out by techniques including but not limited to heat treatment and enzyme treatment.

[0042] In an embodiment of the present disclosure, 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.

[0043] In an embodiment, 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. In an embodiment, 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.

[0044] In another embodiment, 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.

[0045] In an embodiment, 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.

[0046] In some embodiments, 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. In some embodiments, the enzyme treatment of the cell biomass is carried out by enzyme cocktail comprising cellulase, hemi -cellulase, protease, and amylase.

[0047] In an embodiment, 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.

[0048] In an embodiment, 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. In an embodiment, 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. In an embodiment, 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.

[0049] In an embodiment, 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.

[0050] In some embodiments, 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.

[0051] In an embodiment of the present disclosure, 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.

[0052] In an embodiment, 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. In an embodiment, 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.

[0053] In some embodiments, the minerals present in the nutrient composition includes but not limited to potassium, calcium, magnesium, copper, sodium, manganese, boron, cobalt, zinc and iron. In an embodiment, in the nutrient composition, 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; and the iron is in an amount ranging from about 1000 to 2500 ppb.

[0054] In an exemplary embodiment, in the nutrient composition, 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, and iron is in an amount of about 1547 ppb. In an embodiment, 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 on and so forth, up until 75 g / lOOg.

[0055] In an exemplary embodiment, the nutrient composition comprises total amino acid content of about 57. 17 g / 100 g of proteins.

[0056] In some embodiments, 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. In an embodiment, 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, the 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 in an amount ranging from about 2 to 10 g / lOOg of protein, the tyrosine is in an amount ranging from about 1 to 5 g / lOOg of protein, the phenylalanine is in an amount ranging from about 1 to 5 g / lOOg of protein, the histidine is in an amount ranging from about 0.5 to 2.5 g / lOOg of protein, the lysine is in an amount ranging from about 2 to 10 g / lOOg of protein, arginine is in an amount ranging from about 1 to 5 g / lOOg of protein, the proline is in an amount ranging from about 2 to 10 g / lOOg of protein and the tryptophan is in an amount ranging from about 0.2 to 2 g / lOOg of protein.

[0057] In an exemplary embodiment, 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 2.74 g / lOOg of protein, histidine in an amount of about 1.36 g / lOOg of protein, lysine in an amount of about 4.14 g / lOOg of protein, arginine in an amount of about 2.73 g / lOOg of protein, proline in an amount of about 3.79 g / lOOg of protein and tryptophan in an amount of about 0.78 g / lOOg of protein.

[0058] In some embodiments, the vitamin in the nutrient composition includes but not limited to riboflavin, calciferol, niacin and thiamine.

[0059] In some embodiments, the micronutrient in the nutrient composition includes but not limited to iodine and molybdenum.

[0060] In an embodiment, 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.

[0061] In an embodiment, the culture medium employed for cultivation of microorganism includes but not limited to basal salt medium, FM22 medium, complex medium and modified basal medium.

[0062] In an exemplary embodiment, 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

[0063] - extracting nutrient composition by subjecting the enzyme treated microorganisms and its constituents to separation technique selected from a group comprising centrifugation and filtration to obtain the nutrient composition and residual solid biomass. The inventors of the present disclosure have identified that combination of heat treatment and enzyme treatment improves the efficiency of nutrient extraction from the cell biomass to obtain the nutrient composition when compared to conventional methods. 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.

[0064] In some embodiments of the present disclosure, in the process of producing the fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules, 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. In an embodiment, 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. In an embodiment, the pH is about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7aabout 5.8, about 5.9 or about 6.0. In an embodiment, the duration is about 3 days, about 4 days, about 5 days, about 6 days, about 7 days or about 8 days. In an embodiment, 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%.

[0065] In some embodiments of the present disclosure, the process for producing fermentation product comprises-

[0066] - extracting nutrient composition from cell biomass; - adding the nutrient composition and treated effluent of cell biomass to the culture medium comprising microorganism; and maintaining optimum condition for growth of the microorganism, thereby producing the fermentation product.

[0067] In some embodiments, the process of producing the fermentation product, such as proteins, enzymes, small organic molecules including but not limited to vitamins, antibiotics, APIs and building block molecules 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.

[0068] In some embodiments of the present disclosure, 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-

[0069] - 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;

[0070] - extracting nutrient composition by subjecting the enzyme treated cell biomass and its constituents to separation technique selected from a group comprising centrifugation and filtration to obtain nutrient composition and residual solid biomass;

[0071] - adding the nutrient composition to the culture medium comprising fresh batch of microorganism; and

[0072] - maintaining optimum condition for growth of the microorganism, thereby and producing the fermentation product. In some embodiments of the present disclosure, 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-

[0073] - 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;

[0074] - extracting nutrient composition by subjecting the enzyme treated cell biomass and its constituents to separation technique selected from a group comprising centrifugation and fdtration to obtain nutrient composition and residual solid biomass; subjecting the residual solid biomass to effluent treatment to obtain treated effluent of cell biomass;

[0075] - adding the nutrient composition and treated effluent of cell biomass to the culture medium comprising fresh batch of microorganism; and

[0076] - maintaining optimum condition for growth of the microorganism, thereby and producing the fermentation product.

[0077] In an embodiment, 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.

[0078] In another embodiment, about 50% to 100% of the harvest volume of the nutrient composition is added to the culture medium for producing fermentation product in each batch. In an exemplary embodiment, about 80 litres to 160 litres of the nutrient composition is added to the culture medium, depending on the volume of the fermentation batch. In some embodiments of the present disclosure, 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.

[0079] In one embodiment, according to the process of 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.

[0080] 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.

[0081] 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.

[0082] The process of the present disclosure addresses the problem of fermentation industries dealing with waste disposal in the form of cell biomass. In the present disclosure, 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. As a result, the quantity of effluent to be treated is significantly reduced and thus reducing the cost of effluent treatment. Further, there is an improvement in the production of said fermentation product.

[0083] The present disclosure further relates to nutrient composition obtained from the cell biomass of the fermentation process.

[0084] 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%.

[0085] In some embodiments, the nutrient composition comprises mineral, amino acid, vitamin, micronutrient and combinations thereof.

[0086] In an embodiment, the mineral in the nutrient composition is selected from a group comprising potassium, calcium, magnesium, copper, sodium, manganese, boron, cobalt, zinc and iron. In an embodiment, 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; and the iron is in an amount ranging from about 1000 to 2500 ppb.

[0087] In an embodiment, 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 on and so forth, up until 75 g / lOOg.

[0088] In an embodiment, 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. In an embodiment, 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, the 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 in an amount ranging from about 2 to 10 g / lOOg of protein, the tyrosine is in an amount ranging from about 1 to 5 g / lOOg of protein, the phenylalanine is in an amount ranging from about 1 to 5 g / lOOg of protein, the histidine is in an amount ranging from about 0.5 to 2.5 g / lOOg of protein, the lysine is in an amount ranging from about 2 to 10 g / lOOg of protein, arginine is in an amount ranging from about 1 to 5 g / lOOg of protein, the proline is in an amount ranging from about 2 to 10 g / lOOg of protein and the tryptophan is in an amount ranging from about 0.2 to 2 g / lOOg of protein.

[0089] In an embodiment, the vitamin in the nutrient composition is selected from a group comprising riboflavin, calciferol, niacin and thiamine.

[0090] In an embodiment, the micronutrient in the nutrient composition is selected from a group comprising iodine and molybdenum.

[0091] While the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof have been shown by way of examples (and drawings) described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the appended claims. The present disclosure is therefore further described with reference to the following examples, which are only illustrative in nature and should not be construed to limit the scope of the present disclosure in any manner. YPG (Y east extract-Peptone-glycerol) medium was used as a seed medium for growing Pichia pastoris (from Biogrammatics, USA) by below composition. 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. b) Media Preparation and sterilization:

[0092] 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. Pichia trace elements solution (PTM) 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. c) Fermentation:

[0093] 5L of well grown inoculum of Pichia pastoris having optical density of 15 to 20 was transferred from Erlenmeyer flasks to Bio-Jenik fermenter aseptically.

[0094] Once the fermenter was inoculated, 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.

[0095] For the entire duration of fermentation, 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.

[0096] Example 2: Isolation of fermentation biomass (cell biomass)

[0097] Once the fermentation process (as described in Example 1c) attained the maximum productivity, 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.

[0098] 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. 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. To recover the maximum recombinant protein (fermentation product) from the cell broth, 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.

[0099] Example 3: Extraction of nutrient composition from cell biomass

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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:

[0104] Amino acids content

[0105] Table 2:

[0106] Example 4: Improving the yield of Fermentation product by addition of nutrient composition according to present disclosure

[0107] The fermentation product, cellulase enzyme was produced according to the process described herein.

[0108] About 120 litres of nutrient composition was added to subsequent batch of fermentation process. 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.

[0109] 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.

[0110] It was noted that the fermentation product- cellulase enzyme was expressed at 22.5 % higher than the previous batch.

[0111] Example 5: Improving the yield of Fermentation production by addition of nutrient composition according to present disclosure

[0112] The fermentation product, such as cellulase enzyme, insulin, insulin analogues, egg proteins or milk proteins was produced according to the process described herein.

[0113] About 120 litres of the nutrient composition of the cell biomass was added to subsequent batch of fermentation process. About 30 litres of basal salt medium was added to the fermenter to make up the volume up to about 160L.

[0114] 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.

[0115] It was noted that the fermentation products- cellulase enzyme, insulin, insulin analogues, egg proteins or milk proteins were expressed at 24.2 % higher than the previous batch. The present disclosure is further explained by the following numbered embodiments. These embodiments represent important aspects of the present disclosure, but should not be construed to limit its scope:

[0116] 1. A process for production of fermentation product in fermentation process, said process comprises-

[0117] - extracting nutrient composition from cell biomass;

[0118] - adding the extracted nutrients to culture medium comprising microorganism; and maintaining optimum condition for growth of the microorganism, thereby producing the fermentation product. The process as defined in embodiment 1, wherein the extraction of nutrients from cell biomass is carried out by-

[0119] - lysing cell biomass of microorganism;

[0120] - extracting nutrients from the lysed microorganism; and

[0121] - processing / treating the extracted nutrients to obtain nutrient composition. The process as defined in embodiment 2, wherein the lysing the cell biomass comprises subjecting the cell biomass to heat treatment and enzyme treatment. The process as defined in embodiment 3, wherein 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 process as defined in embodiment 3, wherein 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, wherein the enzyme treatment is carried out at a temperature ranging from about 50 °C to 60 °C at pH ranging from about 5 to 7, for a duration ranging from about 60 minutes to 120 minutes. 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 process as defined in embodiment 1 , wherein the nutrient composition comprises component selected from a group comprising minerals, amino acids, vitamins, micronutrients and combinations thereof. 9. The process as defined in embodiment 8, wherein the minerals is selected from a group comprising potassium, calcium, magnesium, copper, sodium, manganese, boron, cobalt, zinc, iron and combinations thereof.

[0122] 10. The process as defined in embodiment 8, wherein the 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.

[0123] 11. The process as defined in embodiment 8, wherein the extracted nutrient composition comprises total amino acid content ranging from about 40 g / 100 g to 75 g / 100 g.

[0124] 12. The process as defined in embodiment 1, wherein the process provides at least 22% increase in the yield of the fermentation product when compared to conventional process.

[0125] 13. A nutrient composition extracted from cell biomass of fermentation process, the nutrient composition comprises component selected from a group comprising mineral, amino acid, vitamin, micronutrients and combinations thereof.

[0126] Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known / conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.

[0127] The foregoing description of the specific embodiments reveal the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and / or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.

[0128] Throughout this specification, the term ‘combinations thereof or ‘any combination thereof or ‘any combinations thereof are used interchangeably and are intended to have the same meaning, as regularly known in the field of patents disclosures.

[0129] As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.

[0130] While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims

1. We Claim:

1. A process for producing fermentation product, said process comprises-- extracting nutrient composition from cell biomass obtained from fermentation;- adding the nutrient composition to a culture medium comprising microorganism; and maintaining optimum condition for growth of the microorganism, thereby producing the fermentation product.

2. The process as claimed in claim 1, wherein the extracting the nutrient composition from the cell biomass comprises-- lysing the cell biomass obtained from fermentation by subjecting the biomass to combination of heat treatment and enzyme treatment; and separating the nutrient composition from residual biomass solid to obtain the nutrient composition.

3. The process as claimed in claim 2, wherein the heat treatment is carried out at a temperature ranging from about 60 °C to 100 °C for a duration ranging from about 30 minutes to 60 minutes.

4. The process as claimed in claim 2, wherein the enzyme treatment is carried out at a temperature ranging from about 50 °C to 60 °C, at a pH ranging from about 5 to 7, for a duration ranging from about 60 minutes to 120 minutes.

5. The process as claimed in claim 2, wherein the enzyme treatment is carried out by employing enzyme selected from a group comprising cellulase, protease, hemicellulase, xylanase, carboxy methyl cellulase, arabinose and amylase and combinations thereof.

6. The process as claimed in claim 2, wherein the separating the nutrient composition is carried out by techniques selected from a group comprising centrifugation, fdtration and combination thereof.

7. The process as claimed in claim 2, wherein prior to separating the nutrient composition, the enzyme treated cell biomass is subjected to cooling to a temperature ranging from about 20 °C to 40 °C.

8. The process as claimed in claim 1, wherein the nutrient composition is added in an amount ranging from about 80 litres to 160 litres.

9. The process as claimed in claim 1, wherein the culture medium is selected from a group comprising basal salt medium, FM22 medium, complex medium and modified basal medium.

10. The process as claimed in claim 1, wherein the microorganism is selected from a group comprising Pichia pasioris. Saccharomyces cerevisiae. and Hansenulci polymorphci .

11. The process as claimed in claim 1, wherein the optimum condition for the growth of the microorganism comprises- temperature ranging from 20 °C to 30 °C, pH ranging from about 5 to 6, dissolved oxygen content ranging from about 10 to 60% and duration ranging from about 3 to 8 days.

12. The process as claimed in claim 2, wherein the residual biomass solid is subjected to effluent treatment to obtain treated effluent of cell biomass.

13. The process as claimed in claim 1, wherein the nutrient composition is optionally combined with treated effluent of cell biomass while adding to the culture medium.

14. The process as claimed in claim 1, wherein nutrient composition comprises components selected from a group comprising mineral, amino acid, vitamin, micronutrients and combinations thereof.

15. The process as claimed in claim 14, wherein the mineral is selected from a group comprising potassium in an amount ranging from about 100,000 ppb to 300,000 ppb, calcium in an amount ranging from about 300 ppb to 900pb, magnesium in an amount ranging from about 5000 to 10,000 ppb, copper in an amount ranging from about 50 to 200 ppb, sodium in an amount ranging from about 2000 to 4000 ppb, manganese in an amount ranging from about 50 to 200 ppb, boron in an amount ranging from about 20 to 100 ppb, cobalt in an amount ranging from about 20 to 100, zinc in an amount ranging from about 500 to 2000 ppb, iron in an amount ranging from about 1000 to 2500 ppb and combinations thereof.

16. The process as claimed in claim 14, wherein the nutrient composition comprises total amino acid content ranging from about 40 g to 75g per 100 of protein.

17. The process as claimed in claim 14, wherein the amino acid is selected from a group comprising aspartic acid in an amount ranging from about 2 to 10 g / lOOg of protein, threonine in an amount ranging from about 1 to 5 g / lOOg of protein, serine in an amount ranging from about 1 to 5 g / lOOg of protein, glutamic acid in an amount ranging from about 5 to 15 g / lOOg of protein, glycine in an amount ranging fromabout 1 to 5, alanine in an amount ranging from about 2 to 10 g / lOOg of protein, cysteine in an amount ranging from about 0.5 to 5 g / lOOg of protein, valine in an amount ranging from about 2 to 10 g / lOOg of protein, methionine in an amount ranging from about 0.5 to 5 g / lOOg of protein, isoleucine in an amount ranging from about 1 to 5 g / lOOg of protein, leucin in an amount ranging from about 2 to 10 g / lOOg of protein, tyrosine in an amount ranging from about 1 to 5 g / lOOg of protein, phenylalanine in an amount ranging from about 1 to 5 g / lOOg of protein, histidine in an amount ranging from about 0.5 to 2.5 g / lOOg of protein, lysine in an amount ranging from about 2 to 10 g / lOOg of protein, arginine in an amount ranging from about 1 to 5 g / lOOg of protein, proline in an amount ranging from about 2 to 10 g / lOOg of protein, tryptophan in an amount ranging from about 0.2 to 2 g / lOOg of protein, and combinations thereof.

18. The process as claimed in claim 14, wherein the vitamin is selected from a group comprising riboflavin, calciferol, niacin, thiamine and combination thereof.

19. The process as claimed in claim 14, wherein the micronutrient is selected from a comprising iodine, molybdenum and combination thereof.

20. A nutrient composition obtained from cell biomass of fermentation process, the nutrient composition comprises component selected from a group comprising mineral, amino acid, vitamin, micronutrients and combinations thereof.

21. The nutrient composition as claimed in claim 20, wherein- the mineral is selected from a group comprising potassium in an amount ranging from about 100,000 ppb to 300,000 ppb, calcium in an amount ranging from about 300 ppb to 900pb, magnesium in an amount ranging from about 5000 to 10,000 ppb, copper in an amount ranging from about 50 to 200 ppb, sodium in an amount ranging from about 2000 to 4000 ppb, manganese in an amount ranging from about 50 to 200 ppb, boron in an amount ranging from about 20 to 100 ppb, cobalt in an amount ranging from about 20 to 100, zinc in an amount ranging from about 500 to 2000 ppb, iron in an amount ranging from about 1000 to 2500 ppb and combinations thereof; the amino acid is selected from a group comprising aspartic acid in an amount ranging from about 2 to 10 g / lOOg of protein, threonine in an amount ranging from about 1 to 5 g / lOOg of protein, serine in an amount ranging from about 1 to 5 g / lOOg of protein, glutamic acid in an amount ranging from about 5 to 15 g / lOOg of protein,glycine in an amount ranging from about 1 to 5, alanine in an amount ranging from about 2 to 10 g / lOOg of protein, cysteine in an amount ranging from about 0.5 to 5 g / lOOg of protein, valine in an amount ranging from about 2 to 10 g / lOOg of protein, methionine in an amount ranging from about 0.5 to 5 g / lOOg of protein, isoleucine in an amount ranging from about 1 to 5 g / lOOg of protein, leucin in an amount ranging from about 2 to 10 g / lOOg of protein, tyrosine in an amount ranging from about 1 to 5 g / lOOg of protein, phenylalanine in an amount ranging from about 1 to 5 g / lOOg of protein, histidine in an amount ranging from about 0.5 to 2.5 g / lOOg of protein, lysine in an amount ranging from about 2 to 10 g / lOOg of protein, arginine in an amount ranging from about 1 to 5 g / lOOg of protein, proline in an amount ranging from about 2 to 10 g / 100g of protein, tryptophan in an amount ranging from about 0.2 to 2 g / lOOg of protein, and combinations thereof; the vitamin is selected from a group comprising riboflavin, calciferol, niacin, thiamine and combination thereof; and the micronutrient is selected from a group comprising iodine, molybdenum and combination thereof.

22. The nutrient composition as claimed in claim, wherein the wherein the nutrient composition comprises total amino acid content ranging from about 40 g to 75g per 100 of protein.

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