Process of production of edible protein from chlorella sorokiniana

Abstract

The present disclosure provides a process for production of protein (CP) from Chlorella sorokiniana (SVMBIOEN3) (NCIM 5561) with significant nutritional benefits. C. sorokiniana is cultivated in a growth medium containing essential macro and micronutrients, using sodium acetate as a carbon source and sodium nitrate as a nitrogen source. This cultivation method results in a substantial biomass with a high protein content. The C. sorokiniana protein produced is rich in protein, comprising 54.4 % of its composition, and contains eight essential amino acids (EAA) at 26.36 % and nine non-essential amino acids (NEAA) at 73.36 %.

Description

PROCESS OF PRODUCTION OF EDIBLE PROTEIN FROM CHLORELLA SOROKINIANAFIELD OF INVENTION

[0001] The present invention broadly relates to the field of protein production from Chlorella sorokinianci (SVMBIOEN3) (NCIM 5561). Particularly the present invention pertains to the production of protein (CP) from Chlorella sorokiniana (SVMBIOEN3) (NCIM 5561) wherein the growth medium contains essential macro and micronutrients, using sodium acetate as a carbon source and sodium nitrate as a nitrogen source. More particularly, the present invention provides a method of production of C. sorokiniana protein wherein the protein comprises eight essential amino acids (EAA) at 26.36 % and nine non-essential amino acids (NEAA) at 73.36 %. Further, the Protein Efficiency Ratio (PER) surpasses that of soy isolates, with a value >2, whereas soy isolates PER ranges between 1.1 to 1.7.BACKGROUND OF THE INVENTION

[0002] Chlorella, particularly Chlorella sp., stood out as a prominent microalgae genus for large-scale cultivation, with several species within this genus recognized for their potential to produce food-grade, high-quality protein for human consumption. Chlorella' s distinct characteristics includes rapid growth and its reputation as a nutrient-rich superfood, with high concentrations of valuable components, such as protein, essential amino acids, chlorophyll, lutein, and essential micronutrients. Chlorella demonstrated remarkable adaptability to various environmental and nutritional conditions, displaying significant resilience against external stressors, resulting in higher biomass and nutritional yields.

[0003] Unlike traditional crop plants, which utilize only about 1 % of solar energy for photosynthesis, Chlorella sp. enhanced this efficiency by up to eight-fold. The harnessing of Chlorella biomass as an alternative protein source for both human and animal nutrition was of primary importance due to its impressive intracellular protein content, which typically ranged from 45 % to 60 % of the algal dry weight, coupled with a well-balanced profile of essential amino acids, closely resembling that of animal proteins.

[0004] The market offered an array of Chlorella-based products, encompassing powdered and liquid Chlorella extracts. Liquid Chlorella extracts were recognized as significant nutritional supplements with wide-ranging commercial applications. Chlorella' s extract is considered a valuable resource, consisting of nucleic acids, polysaccharides, glycoproteins, amino acids,vitamins, and minerals, all essential for supporting human and animal immunity and overall growth. The extract's nutritive value plays a crucial role in regulating cellular regeneration, repairing damaged cells, and mitigating the aging process. It also demonstrated antiviral properties, enhanced the body's defense mechanisms against diseases, and limited cancer proliferation.

[0005] Reports also indicated that Chlorella protein (CP) contributes to lowering blood pressure, enhancing liver function, regulating blood parameters, managing blood sugar levels, rejuvenating cells, expediting the healing of skin conditions, including ulcers and anemia, alleviating constipation and headaches, warding off common colds, and rejuvenating overall vitality. Dietary CP has been shown to enhance immunological function, foster growth and recovery, and maintain body weight and blood lipid levels. CP inclusion in poultry diets has led to improved egg quality, notably with higher levels of lutein in the yolk.OBJECTIVE OF THE INVENTION

[0006] The main objective of the invention is to provide high-protein Chlorella sorokiniana with exceptional nutritional benefits.

[0007] Another objective of the present invention is to introduce an active C. sorokiniana (SVMBIOEN3) culture into a bioreactor to utilize a culturing medium with sodium acetate as the carbon (C) source and sodium nitrate as the nitrogen (N) source, maintaining a C / N ratio of 2: l.

[0008] Yet another objective of the present invention is to continuously monitor the biomass culture phase and assess C and N concentrations which are known to facilitate the estimation of C. sorokiniana C and N consumption rates.

[0009] Yet another objective of the present invention is to quantify the produced protein of the biomass, to evaluate the efficiency of the innovative approach and subject the nutrient-rich C. sorokiniana biomass obtained at the end of cultivation to non-chemical autolysis, a selfdigestion process to purify the targeted macromolecule (protein) for further analysis.

[0010] Still another objective of the present invention is to conduct amino acid profiling on the resulting autolysed product, called Chlorella protein (CP) and assess CP nutritional quality based on its amino acid composition and compare the nutritional attributes of CP with those of commercial protein sources.SUMMARY OF THE INVENTION

[0011] In an aspect of the present disclosure, there is provided a process for production of edible Chlorella protein from Chlorella sorokinianci (SVMBI0EN3), wherein the process comprises the steps: i. initiating biomass culture by introducing an active C. sorokiniana culture into a bioreactor, with culture medium having a carbon source and a nitrogen source, in a ratio of 2: 1, wherein both the nitrogen and carbon sources are introduced into the culture medium, and fermenter before inoculation, and the pH is maintained at 6.8 - 7 for a period of 20 days for biomass growth phase, and a photoperiod of 16:8 h (light: dark), under a light intensity of 100 pmol m’2s’1, with temperature in the range of 25 ± 2 °C at 120 rpm; ii. continuous monitoring of C. sorokinicma cells in step (i) over time during the biomass growth phase for a time-dependent increase in biomass cultivation resulting in biomass of 4.63 g L’1, with the nutrient uptake of 72.6 % carbon and 94.8 % nitrogen; iii. subjecting the C. sorokiniana biomass obtained in step (ii) to non-chemical autolysis at 50 °C for 24 hours, to purify 1.2 g of Chlorella protein from 4.63 g L’1of C. sorokiniana,' and iv. separating the cells in step (iii) by centrifugation at 10,000 rpm for 10 minutes and drying the resulting supernatant by a rotary evaporator to produce a powder of Chlorella protein, with an intracellular protein content of 54.4 %, followed by nutritional quality assessment and amino acid profding of Chlorella protein (CP).

[0012] In another embodiment of the present invention, the nitrogen source in culture medium is sodium nitrate.

[0013] In still another embodiment of the present invention, the carbon source in culture medium is sodium acetate.

[0014] In still another embodiment of the present invention, said protein contains 26.4 % of eight essential amino acid and 73 % of nine non-essential amino acids.

[0015] In yet another embodiment of the present invention, the total amino acid content in C. sorokiniana amounts to 51.9 g / 100 g, effectively meeting the human requirement of 5-20 g of amino acids.

[0016] In yet another embodiment of the present invention, said protein is superior quantitatively and qualitatively to other commercial protein sources, with an essential amino acid content of 13.7 g / 100 g which exceeds that of soy meal (12.5 g / 100 g).

[0017] In yet another embodiment of the present invention, the protein efficiency ratio of said protein (CP) is in the range of 2.8 to 3.2, exceeding that of soy isolates which range from 1.1 to 1.7.

[0018] In yet another embodiment of the present invention, said protein has an EAAI score of 92.83, and nutritional index value of 50.50.

[0019] In yet another embodiment of the present invention, said protein has a biological value of 89.4 %, exceeding that of soybean meal (84.2 %).

[0020] In yet another embodiment of the present invention, the protein solubility for Chlorella protein is 88.76 %, which is higher compared to commercial protein soybean meal with 85 % solubility.

[0021] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.NOVELTY OF THE INVENTION

[0022] The present invention proves to be a highly efficient method for the cultivation of Chlorella sorokiniana (SVMBIOEN3) (NCIM 5561), resulting in an elevated protein yield that stands as a viable alternative to traditional plant proteins. The innovation lies in the optimization of the culture medium composition, encompassing the selection of carbon (C) source, nitrogen (N) source, along with macro and micronutrients. This tailored composition catalyzes biomass growth and a substantial increase in protein content. Notably, this optimized approach not only achieves heightened protein production but also demonstrates a remarkable reduction in operational expenses, thereby enhancing overall production efficiency. This inventive cultivation strategy marks a significant rise in sustainable and cost-effective protein production, holding considerable promise for diverse applications in various industries.LIST OF ABBREVIATIONSBCAA - Branched chain amino acidsBV - Biological valueC - Carbon sourceC / N - Carbon to Nitrogen source ratioCP - Chlorella proteinDCW - Dry cell weightEAA - Essential amino acidsEAAI - Essential amino acid index g - Gram g L"1- Gram per litre mg g"1- Milligram per litreN - Nitrogen sourceNCIM - National collection of industrial microorganismsNEAA - Non-essential amino acidsPER - Protein efficiency ratioRPM - Revolutions per minuteE - SumDETAILED DESCRIPTION OF THE INVENTION

[0023] Those skilled in the art will be aware that the present disclosure is subject to variations and modifications other than those specifically described. It is to be understood that the present disclosure includes all such variations and modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features.Definitions

[0024] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0025] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article.

[0026] The terms “comprise” and “comprising” are used in the inclusive, open sense, meaning that additional elements may be included. It is not intended to be construed as “consists of only”.

[0027] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements or steps but not the exclusion of any other element or step or group of elements or steps.

[0028] The term “including” is used to mean “including but not limited to”. “Including” and “including but not limited to” are used interchangeably.

[0029] Ratios, concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or subranges encompassed within that range as if each numerical value and sub-range is explicitly recited.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosure, the preferred methods, and materials are now described. All publications mentioned herein are incorporated herein by reference.

[0031] The present invention focuses on the specialized cultivation of Chlorella sorokinianci (SVMBIOEN3) to achieve a high-yield protein output. This is accomplished through precise culture conditions, employing sodium acetate as the carbon source, and sodium nitrate as the nitrogen source. The biomass culture phase involves the introduction of an active C. sorokinianci culture into a bioreactor, where it is cultivated with continuous monitoring of biomass growth and protein content, while simultaneously tracking the consumption of carbon and nitrogen. The nutrient-rich biomass of C. sorokiniana was harvested at the end of the cultivation period and processed through autolysis, resulting in Chlorella protein (CP). Autolysis is an eco-friendly purification method, encompassing a multi-step process from cell disruption to fine extract product. It enables the yield of Chlorella components without the use of harsh chemicals, providing concentrated, purified compounds with applications in food and pharmaceuticals, contributing towards sustainability. The purified product from autolysis called CP exhibited a remarkable nutritional level. The quantification process yielded 1.2 g of CP from 4.63 g L"1DCW of C. sorokiniana, with 54.4 % protein. It is composed of a well- balanced amino acid content containing eight essential amino acids (EAA) and nine non- essential amino acids (NEAA). Notably, CP is rich in amino acids like lysine and methionine, which are typically in low concentration in soy products. The presence of sodium acetate ascarbon source, and sodium nitrate as nitrogen source in the cultivation medium of C. sorokiniana played a vital role in the accumulation of amino acids leading to increased protein synthesis. In C. sorokiniana, sodium acetate is converted into Acetyl-CoA, leading to oxaloacetic acid production, a precursor for amino acid synthesis, while sodium nitrate is converted to ammonium, initiating the synthesis of glutamic acid and subsequently other amino acids. CP proved to be nutritionally superior to other protein sources, addressing lysine deficiencies and offering adequate branched-chain amino acids (BCAA). CP showcases remarkable nutritional qualities that positions it as a superior protein source compared to conventional options. With an ample content of essential amino acids (EAA) at 13.7 g per 100 g, surpassing soy meal at 12.5 g per 100 g, CP offers both quantitative and qualitative advantages. Its protein efficiency ratio (PER) values of PERI (3), PER2 (3.2), and PER3 (2.8) categorize it as a high-quality protein, outperforming soy isolates having PER typically ranging from 1.1 to 1.7. The essential amino acid index (EAAI) score of CP is 92.83, surpassing most other protein sources. Further enhancing its nutritional profile, CP achieves a biological value (BV) of 89.4 %, surpassing soybean meal at 84.2 %. Additionally, with a protein solubility of 88.76 %, CP outperforms commercial soybean meal, which stands at 85 % solubility. These attributes collectively establish CP as a highly nutritious and versatile protein source with potential applications in diverse food, feed, and healthcare industries.

[0032] The current invention offers an innovative approach for cultivating Chlorella sorokiniana (SVMBIOEN3) (NCIM 5561) with enhanced protein yield, providing an alternative protein source to conventional plant proteins. The optimized culture medium composition, including sodium acetate as carbon (C) source, sodium nitrate as nitrogen (N) source, macro, and micronutrients, promotes biomass growth and increased protein content, significantly reducing operational expenses while enhancing production efficiency.

[0033] The Chlorella sorokiniana (SVMBIOEN3) (NCIM 5561) deployed in the present invention, was isolated from the Peddacheruvu water body, (17.42°N 78.55°E, Nacharam, Ram Reddy Colony, Secunderabad, Telangana-500076) at Bioengineering and Environmental Sciences Lab, CSIR-Indian Institute of Chemical Technology (CSIR-IICT), Hyderabad. The isolated strain was deposited in the National Collection of Industrial Microorganisms (NCIM) under accession number NCIM 5561.

[0034] The biomass culture was initiated by introducing an active C. sorokiniana culture into a bioreactor. The culturing medium featured sodium acetate as the carbon source and sodium nitrate as the nitrogen source, maintaining a C / N ratio of 2: 1 .

[0035] Continuous monitoring of the biomass culture phase included regular assessment of C and N concentrations, facilitating the estimation of C. sorokinianci C andN consumption rates.

[0036] This phase encompassed simultaneous biomass culture and protein enhancement, followed by the quantification of the produced protein.

[0037] The nutrient-rich C. sorokinianci biomass obtained at the end of cultivation was subjected to non-chemical autolysis, a self-digestion process, to purify the targeted macromolecule (protein).

[0038] The resulting autolysed product, known as Chlorella protein (CP), underwent amino acid profiling. Nutritional quality assessment of CP, based on amino acid composition, was conducted and compared with commercial protein sources.

[0039] The present invention provides a process for production of edible Chlorella protein from Chlorella Sorokiniana (SVMBIOEN3), wherein the process comprises the steps: i. initiating biomass culture by introducing an active C. sorokiniana culture into a bioreactor, with culture medium having a carbon source and a nitrogen source, in a ratio of 2: 1, wherein both the nitrogen and carbon sources are introduced into the culture medium and fermenter before inoculation, and the pH is maintained at 6.8 - 7 for a period of 20 days for the biomass growth phase, and a photoperiod of 16:8 hours (light: dark), under a light intensity of 100 pmol m’2s’1, with temperature in the range of 25 ± 2 °C at 120 rpm; ii. continuous monitoring of C. sorokiniana cells in step (i) over time during the biomass growth phase for a time-dependent increase in biomass cultivation resulting in biomass of 4.63 g L’1, with the nutrient uptake of 72.6 % carbon and 94.8 % nitrogen; iii. subjecting the C. sorokiniana biomass obtained in step (ii) to non-chemical autolysis at 50 °C for 24 hours, to purify 1.2 g of Chlorella protein from 4.63 g L’1of C. sorokiniana,' and iv. separating the cells in step (iii) by centrifugation at 10,000 rpm for 10 minutes and drying the resulting supernatant by a rotary evaporator to produce a powder of Chlorella protein, with an intracellular protein content of 54.4 %, followed by nutritional quality assessment and amino acid profding of Chlorella protein.

[0040] The process for production of edible Chlorella utilizes nitrogen source and carbon source in culture medium and the said nitrogen source is sodium nitrate and the carbon source in culture media is sodium acetate.

[0041] The edible Chlorella protein contains 26.4 % of eight essential amino acid; 73% of nine non-essential amino acids; and the total amino acid content in C. sorokinianci amounts to 51.9 g / 100 g, effectively meeting the human requirement of 5-20 g of amino acids.

[0042] The edible Chlorella protein is superior quantitatively and qualitatively to other commercial protein sources, wherein the essential amino acid content of said Chlorella protein is 13.7 g / 100 g, which exceeds than soy meal (12.5 g / 100 g).

[0043] The edible Chlorella protein has a protein efficiency ratio in the range of 2.8 to 3.2, exceeding that of soy isolates which range from 1.1 to 1.7.

[0044] The edible Chlorella protein has an EAAI score of 92.83, and a nutritional index value of 50.50.

[0045] The edible Chlorella protein has a biological value of 89.4 %, exceeding that of soybean meal (84.2 %) and the protein solubility for Chlorella protein is 88.76 %, which is higher compared to commercial protein soybean meal with 85 % solubility.

[0046] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the art upon reference to the description of the subject matter. It is therefore contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.EXAMPLES

[0047] The disclosure will now be illustrated with following examples, which is intended to illustrate the working of disclosure and not intended to take restrictively to imply any limitations on the scope of the present disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, the exemplary methods, devices and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.EXAMPLE 1Production of biomass rich in protein

[0048] The growth parameters of C. sorokiniana cultivated in nutrient media with varied nutrient concentrations were estimated during the cultivation period. At the end of cultivation (20thday), the maximum biomass concentration achieved was 4.91 g L’1. The DCW indicated that the growth was significantly higher, reflecting the importance of mode of cultivation for maximized biomass growth. The results also inferred that the presence of both organic carbon and nitrogen is of high importance for the growth of C. sorokiniana.

[0049] Organic C and N uptake was evaluated, and the resultant nutrient consumption rates by the end of the cultivation period were observed to be 74.6 % and 96.2 %, respectively for C uptake of 158.7 mg L"1and N uptake of 161.8 mg L’1. The Chlorella ’s uptake of carbon enhances intracellular carbon metabolic flux, facilitated by acetyl-CoA synthetase, leading to acetate conversion to acetyl-CoA. This, in turn, contributes to the citric acid cycle and related pathways, augmenting macromolecule synthesis and ultimately promoting biomass growth.

[0050] Theoretically, the conversion of 1 mole of nitrate to 1 mole of amino acid requires 4 moles of ATP. The presence of carbon in the cultivation media supplies the necessary energy for nitrate absorption during metabolism. As a crucial component for protein, chlorophyll, and nucleic acid synthesis, nitrogen plays a vital role. The coexistence of carbon and nitrogen in Chlorella cultivation media is inferred to enhance nutrient uptake, leading to maximal biomass production.EXAMPLE 2Purification by autolysis

[0051] C. sorokiniana autolysis involves a multi-step process where the C. sorokiniana cells are allowed to self-digest and release their cellular components. This method is particularly valuable for producing specific compounds, such as proteins from biomass. The process typically includes the following steps: i. Harvesting: The first step is to harvest the C. sorokiniana biomass, which is obtained at the end of the cultivation. ii. Cell disruption: Before autolysis, it is often necessary to disrupt the C. sorokiniana cells to facilitate the release of intracellular components, which can be achieved by sonication. iii. Suspension in water: The disrupted C. sorokiniana biomass is suspended in water, typically with the addition of 40 mL of distilled water. This creates a homogeneous mixture that aids in the autolysis process. iv. Autolysis: The suspension is then subjected to autolysis, a controlled self-digestion of the C. sorokiniana cells. During autolysis, enzymes and other cellular components arereleased into the liquid phase. This process often takes place at an elevated temperature, typically around 50 °C, for 24 hours allowing for thorough digestion. v. Centrifugation: After the autolysis process is complete, the mixture is typically centrifuged at high speed of 10,000 rpm for 10 minutes to separate the cell debris and other solid components from the liquid phase. The supernatant, which contains the desired components like proteins, is collected and further processed. vi. Drying: To obtain a concentrated and purified form of the target component, the supernatant is dried using a rotary evaporator. Drying removes the water content, leaving behind a fine powder or concentrated product.

[0052] The advantage of this purification method is that it allows for the production of specific components from C. sorokinianci without the need for harsh chemicals, making it environmentally friendly. It also provides a concentrated and purified form of the desired proteins which can have applications in various industries, including food and pharmaceutical.

[0053] Additionally, this process is well-suited for the recovery of valuable components from Chlorella, contributing to sustainable and eco-friendly practices.EXAMPLE 3Quantification of Chlorella protein (CP)

[0054] The purification by autolysis process yielded 1.2 g of CP from 4.63 g L"1DCW of C. sorokinianci. CP exhibited an intracellular protein content of 544.1 mg g"1and was accompanied by a well-balanced amino acid profile containing eight essential amino acids (EAA; 26.36 %) and nine non-essential amino acids (NEAA; 73.36 %) (Table 1). EAA, namely threonine, methionine, phenylalanine, histidine, lysine, valine, isoleucine, and leucine were observed to be in adequate quantity. Among NEAA, glutamic acid (29.28 %) and alanine (17.39 %) were in higher concentration than others. CP contained a lower concentration (< 2 %) of sulfur- containing amino acids like methionine, cysteine, and serine. It is observed that the presence of threonine, methionine, histidine, lysine, valine, and isoleucine makes C. sorokiniana biomass a protein-rich source, which is nutritionally equivalent to eggs and soy products. Methionine and lysine are the two amino acids that are in low concentration in soy products, whereas they were observed to be abundant in CP. Lysine, often the first limiting amino acid in many plant protein sources, displayed a relatively high concentration in CP (8.5 %), surpassing other protein sources. This suggests that CP, when used as a nutritional supplement, can address dietary lysine insufficiencies.

[0055] In the cellular context, glutamate dehydrogenase played a pivotal role in facilitating the reductive amination of a-ketoglutaric acid with NH4+to produce glutamic acid. This glutamic acid then contributed to the formation of aspartic acid under the guidance of aspartate aminotransferase activity. Additionally, leucine synthesis occurred through the interaction of a-ketoisocaproate and glutamic acid, catalyzed by leucine transferase. Glycine production was the result of L-serine synthesis, catalyzed by serine hydroxyl-methyltransferase, with glutamate playing a crucial role in the synthesis of 3-phosphoserine, a precursor of serine.

[0056] The accumulation of glutamic acid in CP could be attributed to the presence of both carbon and nitrogen sources in the cultivation media of C. sorokinianci. This facilitated the anabolism of other amino acids, leading to increased protein accumulation and synthesis.EXAMPLE 4Role of selected nutrient sources in CP production

[0057] Sodium acetate, readily available as a carbon source in the growth medium of C. sorokinianci, is converted into acetyl-CoA by acetyl-CoA synthetase. Acetyl-CoA then undergoes a series of conversions in a cycle to ultimately produce oxaloacetic acid. Oxaloacetic acid serves as a precursor for the synthesis of aspartic acid, which, in turn, is a precursor for the synthesis of other amino acids, such as serine, threonine, and isoleucine.

[0058] Sodium nitrate, the nitrogen source, is assimilated by first converting nitrate to nitrite and then to ammonium. This process leads to the synthesis of glutamic acid, which serves as the primary precursor for the synthesis of other amino acids, including proline, arginine, glutamine, and histidine.EXAMPLE 5Nutritional parameters that distinguish CP from commercially available protein sources

[0059] CP contained adequate levels of branched-chain amino acids (BCAA), including isoleucine, leucine, and valine, which are significant components of dietary supplements. The total content of amino acids in C. sorokiniana is 51.9 g / 100 g which is perfectly sufficient to meet a human need of nearly 5-20 g of amino acids. EAA content in CP is 13.7 g / 100 g which is slightly higher than the soy meal (12.5 g / 100 g), thus making CP superior both in terms of quantitative and qualitative EAA content compared to other commercial protein sources (Table 1).

[0060] The evaluation of CP nutritional properties extended to protein efficiency ratio (PER), an index of protein quality. CP achieved PER values of PERI (3), PERT (3.2), and PER3 (2.8),categorizing it as a high-quality protein. These values surpassed the PER values of soy isolates, which typically range from 1.1 to 1.7.

[0061] The essential amino acid index (EAAI) served as a more accurate measure of protein nutritional value, and CP resulted in a score of 92.83, surpassing most other protein sources. The nutritional index (NI), a measure of EAAI and protein content, reached 50.50.

[0062] Biological Value (BV), another vital nutritional factor, measures the integration of a protein into the human body's proteins. CP demonstrated a BV of 89.4 %, surpassing soybean meal (84.2 %).

[0063] Protein solubility, a key quality indicator, was observed at 88.76 % for CP, outperforming commercial soybean meal, which has 85 % solubility.Table 1: Amino acid (%) profiling of C. sorokiniana protein and other commercially available protein sourcesCPSoy product Whey Casein Corn Fish Meal (This invention)Essential Amino Acids (EAA)Threonine 2.3 5.4 2.6 1.8 4.5 2.3Methionine 0.3 1.8 1.6 1.1 2.8 0.9Phenylalanine 3.2 2.5 3.1 3.4 4.3 3Histidine 1.5 1.4 1.7 1.1 3.5 1Lysine 3.4 7.1 4.6 1.0 8.6 8.5Valine 2.2 3.5 3.0 2.1 5.4 2.1Isoleucine 1.9 3.8 2.3 1.7 4.5 0.8Leucine 5.0 8.6 5.8 8.8 7.8 7.8E of EAA 19.9 34.1 24.8 21.0 41.4 26.4Non - Essential Amino Acids (NEAA)Serine 3.4 4.0 3.4 2.9 3.68 3.3Glycine 2.7 1.5 1.2 1.6 6.89 5.3Glutamic acid 12.4 15.5 13.9 13.1 12.65 29.2Aspartic acid - - - - - 6.7Proline 3.3 4.8 6.5 5.2 4.70 2.8Cysteine 0.2 0.8 0.1 0.3 0.91 0.1Alanine 2.8 4.2 2.0 4.8 6.52 17.3Tyrosine 2.2 2.4 3.4 2.7 3.65 2.1Arginine 4.8 1.7 2.1 1.7 6.43 6.2L of NEAA 31.9 34.9 32.5 32.3 45.43 73Advantages of the present disclosure• Chlorella sp. offers an environmentally sustainable solution, because of their adaptability to cultivation methods and outstanding nutritional value.• The discoveries in this invention highlight the significant influence of sodium acetate as carbon source and sodium nitrate as nitrogen source on the macromolecular composition of C. sorokinianci.• Importantly, C. sorokiniana liquid extract obtained through autolysis demonstrates superior protein quality, nutritional content, and essential amino acids.• Given the amino acid profile of C. sorokinicma, there is a strong case for scaling up Chlorella cultivation using the mentioned nutrients to broaden the utilization in various food products and as protein supplements.

Claims

I / We claim:

1. A process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBI0EN3) (NCIM 5561), wherein the process comprises the steps: i. initiating biomass culture by introducing an active C. sorokiniana culture into a bioreactor, with culture medium having a carbon source and a nitrogen source, in a ratio of 2: 1, wherein both the nitrogen and carbon sources are introduced into the culture medium and fermenter before inoculation, and the pH is maintained at 6.8 - 7 for a period of 20 days for biomass growth phase, and a photoperiod of 16: 8h (light: dark), under a light intensity of 100 pmol m’2s’1, with temperature in the range of 25 ± 2 °C at 120 rpm; ii. continuous monitoring of C. sorokiniana cells in step (i) over time during the biomass growth phase for a time-dependent increase in biomass cultivation resulting in biomass of 4.63 g L’1, with the nutrient uptake of 72.6 % carbon and 94.8 % nitrogen; iii. subjecting the C. sorokiniana biomass obtained in step (ii) to non-chemical autolysis at 50 °C for 24 hours, to purify 1.2 g of Chlorella protein from 4.63 g L’1of C. sorokiniana,' and iv. separating the cells in step (iii) by centrifugation at 10,000 rpm for 10 minutes and drying the resulting supernatant by a rotary evaporator to produce a powder of Chlorella protein, with an intracellular protein content of 54.4 %, followed by nutritional quality assessment and amino acid profiling of Chlorella protein.

2. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBIOEN3) as claimed in claim 1, wherein the nitrogen source in culture medium is sodium nitrate.

3. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBIOEN3) as claimed in claim 1, wherein the carbon source in culture medium is sodium acetate.

4. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBIOEN3) as claimed in claim 1, wherein said protein contains 26.4 % of eight essential amino acids and 73 % of nine non-essential amino acids.

5. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBIOEN3) as claimed in claim 1, wherein the total amino acid content in C. sorokiniana amounts to 51.9 g / 100 g, effectively meeting the human requirement of 5-20 g of amino acids.

6. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBI0EN3) as claimed in claim 1, wherein said protein is superior quantitatively and qualitatively to other commercial protein sources, with an essential amino acid content of 13.7 g / 100 g, which exceeds that of soy meal (12.5 g / 100 g).

7. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBI0EN3) as claimed in claim 1, wherein the protein efficiency ratio of said protein is in the range of 2.8 to 3.2, exceeding that of soy isolates, which range from 1. 1 to 1.7.

8. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBI0EN3) as claimed in claim 1, wherein said protein has an EAAI score of 92.83, and nutritional index value of 50.50.

9. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBIOEN3) as claimed in claim 1, wherein said protein has a biological value of 89.4%, exceeding that of soybean meal (84.2 %).

10. The process for production of edible Chlorella protein from Chlorella sorokiniana (SVMBIOEN3) as claimed in claim 1, wherein the protein solubility for Chlorella protein is 88.76 %, which is higher compared to commercial protein soybean meal with 85 % solubility.