Description:Technical field of the invention
[0002] The present invention relates to a process for preparation of coconut protein, and more particularly, the invention relates to a process of separating the coconut protein and fibers from the coconut spent to derive pure form of coconut protein.
Background of the invention
[0003] The transformation of food processing sector with a significant increase in producing enormous volumes of agro-industrial spent material, leads to billions of tons of food waste. The majority of this plant-based waste is either incinerated with other combustible materials or landfilled or rarely used as animal feed. Because of its nutritional profile and functional properties, this food industrial spent material can be further value-added for food/feed and functional ingredients.
[0004] Coconut is a high-quality agricultural product, whose various parts and components are converted into various products including dried coconut kernels, coconut fibre, coconut coir, coconut water and meat, coconut oil., and many more. The coconut meat contains 4-5% of protein, and the coconut can be processed into special foods with rich nutrition such as coconut juice, coconut sugar, coconut cake, coconut powder and the like besides the production of the coconut oil. Upon extraction of coconut oil, the remaining coconut spent material is considered as waste and is discarded. Coconut protein is predominantly found in coconut flesh, where various extraction techniques such as chemical, enzymatic, and physical methods are used to extract coconut protein.
[0005] Coconut protein is plant sourced protein, that is 100% vegan, containing all essential amino acids. Cysteine and methionine are two rate limiting amino acids that are rarely found in plant source of protein. However, coconut protein consists of cysteine and methionine. The amino acid analysis of coconut protein indicates that coconut protein consists of all 18 important amino acids, with protein digestibility amino acid score as 1. Hence, coconut protein is considered as a whole protein. The coconut protein further contains balanced ratio of proteins and fibers, facilitating increase in the bioavailability of the protein.
[0006] Coconut proteins have several applications, where the coconut protein has hypolipidemic effect attributable to L-arginine, a precursor of nitric oxide, acting as a vasodilator and antiplatelet agent. Coconut protein can help in maintaining healthy weight, reducing inflammation, and even lower the risk of heart disease. Several methods have been developed to extract coconut protein from the coconut flesh.
[0007] The Patent Application No. CN102356809A entitled “Preparation process of coconut protein active peptide” discloses a preparation process of coconut protein active peptide, where the preparation process comprises the steps of: with copra as the raw material, converting protein contained in a coconut into coconut protein active peptide by using a biologic fermentation method. The preparation method has the characteristics of stable production and reaction process, easiness for control, low impurity content in finished products, high amino acid content, good taste without foreign taste such as bitter, astringency and the like and great reduction of the production cost.
[0008] The Patent Application No. CN117625718A entitled “Preparation method of coconut protein oligopeptide” discloses a preparation method of coconut protein oligopeptide, which belongs to the technical field of oligopeptide preparation and mainly comprises the following steps: (1) raw material treatment; (2) preparing coconut protein liquid; (3) adjusting the enzymolysis pH of the coconut protein liquid, and adding alkaline protease for primary enzymolysis; (4) after the primary enzymolysis is finished, adding neutral protease and exopeptidase to carry out secondary enzymolysis, and after the enzymolysis is finished, carrying out enzyme deactivation and cooling to carry out solid-liquid separation; (5) filtering the separated enzymatic hydrolysate with a ceramic membrane, purifying with an ultrafiltration membrane, deodorizing and decoloring with a cellulose membrane, and desalting with a nanofiltration membrane; and (6) concentrating and drying the refined peptide liquid. The coconut protein oligopeptide product obtained by the method is reasonable in molecular weight distribution, strong in functionality, and good in solubility, acid resistance and heat resistance stability.
[0009] The Patent Application No: CN107502645A entitled “Method for preparing coconut polypeptides” discloses a method for preparing coconut polypeptides, comprising the steps of preparing coconut proteins; secondly, uniformly mixing the coconut proteins and distilled water with one another and carrying out heating and pressure treatment to obtain moist and hot coconut protein serous fluid; thirdly, regulating the pH (potential of hydrogen) of the moist and hot coconut protein serous fluid, adding primary complex enzymes into the moist and hot coconut protein serous fluid and carrying out ultrasonic-assisted enzymatic hydrolysis to obtain primary enzymatic hydrolysate after primary enzymatic hydrolysis reaction is completely carried out; fourthly, regulating the pH of the primary enzymatic hydrolysate, adding secondary complex enzymes into the primary enzymatic hydrolysate, and carrying out ultrasonic-assisted enzymatic hydrolysis to obtain secondary enzymatic hydrolysate after secondary enzymatic hydrolysis reaction is completely carried out; fifthly, carrying out microwave enzyme deactivation treatment on the secondary enzymatic hydrolysate to obtain enzyme-deactivated enzymatic hydrolysate, sequentially cooling and centrifuging the enzyme-deactivated enzymatic hydrolysate to obtain supernatant, carrying out ultra-filtration to obtain filter liquor and carrying out freeze drying to obtain the coconut polypeptides which are finished products. The method for preparing the coconut polypeptides has the advantages that the method includes safe processes, and the coconut polypeptides can be conveniently and quickly prepared by the aid of the method and are high in purity.
[0010] The existing methods facilitate extraction of coconut protein from the coconut meat, and the coconut that is used for producing coconut oil and other products is directly wasted, leading to disposal of large amount of coconut spent.
[0011] Therefore, there is a need for a process for extraction of coconut protein from coconut spent or coconut kernel cake devoid of fat or oil content, wherein the proteins and fibers are separated, and the extracted coconut protein is used for various applications.
Summary of the invention
[0012] The present invention overcomes the drawbacks of the existing processes in the prior arts to provide a novel process for extraction of coconut protein from coconut kernel cake or coconut spent, wherein the process facilitates separation of coconut proteins and coconut fibre from the coconut spent, thus reducing the wastage of coconut after oil extraction.
[0013] According to the invention, the process for the extraction of coconut protein from coconut spent includes subjecting the raw material to a water extraction process in the extractor equipment, wherein the coconut kernel cake is mixed with water in a ratio of 1:10, and the resultant is extracted at 65°C ± 5°C for 3 hours. Subsequently, the mass is filtered with 5-micron filter cloth.
[0014] Further, the liquid filtrate is subjected to membrane separation, wherein the desired protein size is selected as 20 kDa. The membrane separation process yielded two types of filtrates including retention filtrate and the permeate filtrate, wherein the retention filtrate and permeate filtrate are further concentrated in a reactor to achieve 30% of total dissolved solids (TDS) separately and the pH is adjusted between 6 to 7 using potassium hydroxide solution. Further, the concentrated mass is dried in a spray drier, where the inlet temperature is set at 160°C ±10°C and outlet temperature is set at 98°C ±10°C. The retention filtrate is free of sugar content and contains high protein, whereas the permeate filtrate contains less sugar content and is rich in minerals but had a lower protein content.
[0015] Additionally, the retention filtrate was dissolved in water at the ratio of 1: 5 and maintained at the temperature of about 50°C and the pH is adjusted to between 8 to 9 using ammonia solution. Upon stabilizing the pH, the retention filtrate was subjected to enzyme hydrolysis, wherein 1.5% of digestive enzyme derived from plants is used for the hydrolysis at a temperature of about 50°C for 4 hours, with stirring. Further, the resultant was subjected to Thin Layer Chromatography (TLC) analysis to identify the free amino acids present, with the help of ninhydrin reagent.
[0016] Further, upon confirmation of presence of free amino acids in the retention filtrate, the reaction mass was cooled to room temperature and was further subjected to extraction using ethanol in a 1:1 ratio and allowed for precipitate separation. Subsequently, the resulting mixture was filtered through a 5-micron filter cloth, and the filtrate was concentrated and dried at a temperature of 60°C ± 3°C. The resultant dried material was milled to 0.5mm and sifted through 40# yielding coconut protein hydrolysate. The retention filtrate is composed of 45% protein and 35% fibre, and the permeate filtrate is composed of sugars, minerals, fiber and 20% protein.
[0017] The primary advantage of the present invention is that the method for extraction of coconut protein yields coconut protein containing balanced ratio of protein and fibre, thereby increases the bioavailability of protein. The retention filtrate has several applications, specifically for diabetics and in adults’ nutrition, whereas the permeate filtrate has applications in kids’ nutrition. Further, the extracted coconut protein does not contain any traces of sodium, and it exhibits antioxidant, anti-obesity, anti-inflammatory, and prebiotic activity, since the coconut protein contains a combination of protein and dietary fibre. Additionally, coconut protein facilitates endurance, weight management and muscle building.
Brief description of the drawings
[0018] The foregoing and other features of embodiments will become more apparent from the following detailed description of embodiments when read in conjunction with the accompanying drawings. In the drawings, like reference numerals refer to like elements.
[0019] FIG 1 illustrates the flowchart disclosing the process of extraction of coconut protein from coconut spent.
[0020] FIG 2 illustrates the flowchart disclosing the process of production of coconut protein extract A (low sugar) and coconut protein extract B (free of sugar).
[0021] FIG 3 illustrates the flowchart disclosing the process of producing coconut protein hydrolysate.
[0022] FIG 4 illustrates the graphical representation of the COX-2 inhibitor assay disclosing the anti-inflammatory activity of the coconut protein.
[0023] FIG 5 illustrates the graphical representation of (2,2-diphenyl-1-picrylhydrazyl) (DPPH) radical scavenging activity of the coconut protein.
[0024] FIG 6A-6C illustrates the graphical representation of the growth of Lactobacillus plantarum in De Man–Rogosa–Sharpe (MRS) media with different compositions of the coconut protein.
[0025] FIG 7A-7C illustrates the graphical representation of the growth of Lactobacillus rhamnosus in De Man–Rogosa–Sharpe (MRS) media with different compositions of the coconut protein.
[0026] FIG 8A-8C illustrates the graphical representation of the growth of E Coli in De Man–Rogosa–Sharpe (MRS) media with different compositions of the coconut protein.
Detailed Description of the Invention
[0027] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.
[0028] The present invention discloses a process for extraction of coconut protein from coconut spent, wherein the process facilitates separation of protein and fibre from the coconut spent, thus reducing the wastage of coconut upon oil extraction.
[0029] FIG 1 illustrates the flowchart disclosing the process (100) of extraction of coconut protein from coconut spent, wherein the process (100) comprises the steps of: subjecting the coconut spent to aqueous extraction process using the extractor equipment in the step (101), wherein the coconut spent is mixed with water in a ratio of 1:10, and the resultant is extracted at 65°C ± 5°C for 3 hours. Further, in the step (102) the extracted mass is subjected to filtration, wherein the mass is filtered with 5-micron filter cloth. Upon filtration, the solid waste produced is separated in the step (103) and the liquid filtrate is considered for further processing.
[0030] Additionally, in the step (103) the liquid filtrate is subjected to membrane separation, wherein a membrane with 20 kDa size is used to separate the liquid filtrate into two types of filtrates including retention filtrate and the permeate filtrate. Further, the retention filtrate and the permeate filtrate collected after membrane separation is subjected to spray drying in the step (104), wherein both the retention filtrate and permeate filtrate are further concentrated in a reactor to achieve 30% of total dissolved solids (TDS) separately, with the pH adjusted between 6-7 using potassium hydroxide solution. Further, the concentrated mass is dried in a spray drier, where the inlet temperature is set at 160°C ±10°C and outlet temperature is set at 98°C ±10°C. Additionally, the spray dried mixture is further processed in step (105), where the resultant dried material was milled to 0.5mm and sifted to produce the coconut protein extract.
[0031] FIG 2 illustrates the flowchart disclosing the process of production of coconut protein extract A (free of sugar) and coconut protein extract B (low sugar). With reference to FIG 2, the process of producing coconut protein extract A from the retention filtrate comprises the steps of, subjecting the retention filtrate obtained by membrane filtration in step (103) to spray drying in step (104), wherein the concentrated retention filtrate is dried in the spray drier at the inlet temperature of 160°C ±10°C and outlet temperature of 98°C ±10°C. Further, the spray dried mixture is subjected to further processing in step (105), where the resultant dried material was milled to 0.5mm and sifted to remove the unwanted impurities. The sifted material is then blended and screened through the metal detector to detect presence of any unwanted impurities to obtain the purified coconut protein extract A. The coconut protein extract A is free of sugar and is composed of 45% protein and 35% fibres.
[0032] Subsequently, the coconut protein extract B is obtained from the permeate filtrate comprises the steps of, subjecting the permeate filtrate obtained by membrane filtration in step (103) to spray drying in step (104), wherein the concentrated retention filtrate is dried in the spray drier at the inlet temperature of 160°C ±10°C and outlet temperature of 98°C ±10°C. Further, the spray dried mixture is subjected to milling and sifting in step (105), where the resultant dried material was milled to 0.5mm and sifted to remove the unwanted impurities. The sifted material is then blended and screened through the metal detector to detect presence of any unwanted impurities to obtain the purified coconut protein extract B. The coconut protein extract B contains less sugar and is composed of 20% protein, sugars, minerals and fibres.
[0033] FIG 3 illustrates the flowchart disclosing the process of producing coconut protein hydrolysate. With reference to FIG 3, the process of obtaining the coconut protein hydrolysate comprises the steps of, subjecting the spray dried coconut protein extract obtained in the retention filtrate in the step (104) to enzyme hydrolysis in step (201), wherein the coconut protein extract was dissolved in water at the ratio of 1: 5 and at the temperature about 50°C and the pH is adjusted between 8 to 9 using ammonia solution. Upon stabilizing the pH, the coconut protein extract was subjected to enzyme hydrolysis, wherein 1-2% of digestive enzyme derived from plants is used for the hydrolysis at a temperature of about 45-60°C for 4 hours, with continuous stirring. According to an embodiment of the invention, the digestive enzyme is Specialty Enzymes and Biotechnologies (SEB) digest enzyme. Subsequently, the resultant was subjected to Thin Layer Chromatography (TLC) analysis to identify the free amino acids present, with the help of ninhydrin reagent.
[0034] Further, in step (202), the reaction mass was subjected to extraction, wherein the reaction mass was cooled to room temperature and subjected to extraction using ethanol in a 1:1 ratio and allowed for precipitate separation. Subsequently, in step (203), the resultant mixture was subjected to filtration, wherein the resultant mixture was filtered through a 5-micron filter cloth, and the filtrate was further concentrated in step (204). The concentration mixture was spray dried at a temperature of 60°C ± 3°C, in step (205) and the resultant dried material was milled to 0.5mm and sifted through 40#, in step (206). Further, the milled and sifted material was blended in step (207) and screened through the metal detector in step (208) to detect any unwanted impurities. The purified material obtained through the process was coconut protein hydrolysate.
[0035] According to the invention, the hydrolysis of the coconut protein extract A using the digestive enzyme improves the nutrient absorption by breaking down the coconut protein extract into easily absorbable amino acids and peptides. The digestive enzyme further enhances the body's ability to absorb and utilize the nutrients from coconut protein extract. Further, enzyme hydrolysis of the coconut protein extract improves the availability of essential amino acids also facilitates increased amino acid absorption, aiding in muscle recovery and repair after exercise. Further, hydrolysis of the coconut protein extract with non-GMO, and gluten-free plant based digestive enzyme makes the process environment friendly, and caters to those with gluten intolerance, celiac disease, or the users who avoid genetically modified organisms (GMOs) for health or ethical reasons.
[0036] According to some embodiments of the invention, the coconut protein extract A and coconut protein extract B were assessed for anti-oxidant activity, anti-inflammatory activity and prebiotic activity.
Example 1: COX-2 Inhibitor Assay (Anti-inflammatory activity)
[0037] FIG 4 illustrates the graphical representation of the COX-2 inhibitor assay disclosing the anti-inflammatory activity. With reference to FIG 4, the anti-inflammatory activity of the coconut protein extract was determined and compared to Celecoxib as a control, where Celecoxib is a known nonsteroidal anti-inflammatory drug (NSAID). The COX-2 inhibitor assay revealed that the coconut protein extracts A and B showed significant anti-inflammatory activity with respect to positive control (Celecoxib). The anti-inflammatory activity of the coconut protein extract B was found to be 28% and of coconut protein extract A was found to be 71.9%.
Example 2: DPPH Radical scavenging activity assay
[0038] FIG 5 illustrates the graphical representation of DPPH radical scavenging activity. The antioxidant activity of the coconut protein extracts A and B was determined and compared to ascorbic acid as a control, where ascorbic acid is a natural antioxidant that regulates the level of reactive oxygen species. The antioxidant assay revealed that the coconut protein extracts A and B showed significant DPPH radical scavenging activity with respect to positive control (Ascorbic acid). The antioxidant activity of the coconut protein extract A was found to be around 386 µg/ml and of coconut protein extract B was found to be around 485 µg/ml.
[0039] Example 3: Biological analysis of prebiotics for the growth enhancement of probiotics.
[0040] In order to determine the prebiotic activity of the coconut protein extract, the Lactobacillus bacteria was used. The initial screening process was conducted to determine if Lactobacillus was appropriate for the prebiotic activity assay. For selection of Lactobacillus bacteria for the prebiotic assay, the growth curves of the bacterial growth in medium containing coconut protein extracts A and B were determined. The growth of the bacteria on basal MRS media (media without carbohydrates) containing 1 to 5 % prebiotics such as coconut protein extract A and coconut protein extract B was determined by growth curves using a spectrophotometer and by plating growth at 0 and 24 hours. The media without prebiotics and without sugar is taken as positive control and MRS Hi-media containing 2 % glucose was considered as an additional control. Further, the criteria for a probiotic bacterium to be selected were growth on the prebiotic that resembled the growth on MRS Hi media.
[0041] The procedure for prebiotic activity assay involves streaking different Lactobacilli cultures including Lactobacillus plantarum, Lactobacillus rhamnosus and Escherichia coli on MRS agar and incubating at 37°C for 48 hours in anaerobic conditions using an anaerobic chamber. Then, one colony from the MRS culture plates was transferred into 10 ml MRS broth and incubated at 37°C for 16 hours. Further, the overnight cultures were diluted by 1/10 using basal MRS with basal referring to preparation without carbohydrates. The test samples were prepared by taking 1 to 5 g of the sample prebiotic food and were prepared in duplicate for each of the three trials of the food product produced, for the probiotic samples.
[0042] Further, the control product was tested from one trial in duplicate, for the probiotic samples. Approximately 100-200 µl of the diluted overnight culture was added to the basal media containing 1 to 5 % prebiotic sample mixture. The samples were then diluted by taking 100 µl of the sample mixture and adding it to 900 µl 0.9% saline solution. Upon diluting the samples, 10 µl was placed on an MRS agar plate and spread. The final dilutions plated for the 0-hour time point were 10-4 and 10-5. The samples were then incubated at 37°C for 24 hours, and the plates were incubated at 37°C for 24 - 48 hours. After 24 hours, the samples were diluted and plated for final dilutions of 10-4 and 10-5. The plates were incubated at 37°C for 24 - 48 hours. The plates were counted in the dilution containing 25 - 250 colonies and recorded.
[0043] FIG 6A-6C illustrates the graphical representation of the growth of Lactobacillus plantarum in MRS media with different compositions of the coconut protein. The colony forming units per gram (cfu/g) were calculated and then applied to the prebiotic activity equation. The probiotic log cfu/g on the prebiotic at 24 hr − probiotic log cfu/g on the prebiotic at 0 hr were measured. Further, the probiotic log cfu/g on control at 24 hr − probiotic log cfu/g on control at 0 hr were measured. With reference to FIG 6A, FIG 6B and FIG 6C, the bacterial growth was measured in Hi-media (HM); MRS without Dextrose (NS); MRS with coconut protein extract B prebiotic (B); MRS with coconut protein extract A prebiotic (A). It was observed that the growth of probiotic bacteria was higher in HM, than MRS media supplemented with prebiotics coconut protein extract A and coconut protein extract B.
[0044] FIG 7A-7C illustrates the graphical representation of the growth of Lactobacillus rhamnosus in MRS media with different compositions of the coconut protein. The colony forming units per gram (cfu/g) were calculated and then applied to the prebiotic activity equation. The probiotic log cfu/g on the prebiotic at 24 hr − probiotic log cfu/g on the prebiotic at 0 hr were measured. Further, the probiotic log cfu/g on control at 24 hr − probiotic log cfu/g on control at 0 hr were measured. With reference to FIG 7A, FIG 7B and FIG 7C, the bacterial growth was measured in Hi-media (HM); MRS without Dextrose (NS); MRS with coconut protein extract B prebiotic (B); MRS with coconut protein extract A prebiotic (A). It was observed that the growth of probiotic bacteria was higher in HM, than MRS media supplemented with prebiotics coconut protein extract A and coconut protein extract B.
[0045] FIG 8A-8C illustrates the graphical representation of the growth of Escherichia coli in MRS media with different compositions of the coconut protein. The colony forming units per gram (cfu/g) were calculated and then applied to the prebiotic activity equation. The probiotic log cfu/g on the prebiotic at 24 hr − probiotic log cfu/g on the prebiotic at 0 hr were measured. Further, the probiotic log cfu/g on control at 24 hr − probiotic log cfu/g on control at 0 hr were measured. With reference to FIG 8A, FIG 8B and FIG 8C, the bacterial growth was measured in Hi-media (HM); MRS without Dextrose (NS); MRS with coconut protein extract B prebiotic (B); MRS with coconut protein extract A prebiotic (A). It was observed that the growth of probiotic bacteria was higher in HM, than MRS media supplemented with prebiotics coconut protein extract A and coconut protein extract B. However, the growth of probiotics in MRS media devoid of sugar was lower than all the other three medias.

[0046] There are several advantages of the present invention, where the process for extraction of coconut protein contains balanced ratio of protein and fibre, there by increases the bioavailability of protein. The coconut protein extract A is highly useful for diabetics and adults’ nutrition, and coconut protein extract B is useful for kids’ nutrition. Further, the coconut protein extracts A and B does not contain any traces of sodium, and it exhibits antioxidant, anti-obesity, anti-inflammatory, and pre-biotic activity, since the coconut protein contains a combination of protein and dietary fibre. Additionally, coconut protein extracts facilitate endurance, weight management and muscle building. , Claims:We Claim:
1. A process for extracting coconut protein from coconut spent, the process (100) comprising the steps of:
a. subjecting a coconut spent to an aqueous extraction in step (101) by mixing the coconut spent with water;
b. filtering a resultant mass through a 5-micron filter cloth in step (102) to separate as a solid waste and a liquid filtrate;
c. subjecting the liquid filtrate to membrane separation using a membrane in step (103) to obtain a retention filtrate and a permeate filtrate;
d. concentrating and drying the retention filtrate and the permeate filtrate in step (107) through a spray drying technique; and
e. milling the resultant dried material to 0.5mm and sifting to obtain the dried coconut protein extract A and coconut protein extract B.

2. The process (100) as claimed in claim 1 wherein, aqueous extraction involves extraction of the coconut protein with water as a solvent in the ratio of 1:10, with an operating temperature of 65°C ± 2°C for 4 hours.

3. The process (100) as claimed in claim 1 wherein, the membrane used in membrane filtration is a tubular and ceramic membrane with a pore size of 20 kDa facilitating filtration of the retention filtrate and the permeate filtrate.

4. The process (100) as claimed in claim 1 wherein, the retention filtrate and the permeate filtrate are spray dried at an inlet temperature of 160°C ±10°C and an outlet temperature of 98°C ±10°C.

5. The process (100) as claimed in claim 1 wherein, the coconut protein extract A derived from the retention filtrate is composed of 45% protein and 35% fibres.

6. The process (100) as claimed in claim 1 wherein, the coconut protein extract B derived from the permeate filtrate is composed of 20% protein, sugars, minerals, and fibers.

7. The process (100) as claimed in claim 1 wherein, the coconut protein extract A is hydrolyzed by enzyme hydrolysis through a plant derived, non-GMO, gluten-free digestive enzyme at a concentration of 1-2% at 45-60°C for 4 hours to obtain a coconut protein hydrolysate.

8. The process (100) as claimed in claim 7 wherein, the coconut protein hydrolysate is obtained by subjecting the enzyme hydrolyzed coconut protein extract A to ethanol extraction at a ratio of 1:1 and filtering through a 5-micron filter cloth before spray drying at 60°C ± 3°C.