A method for purifying chickpea polypeptides

Abstract

The present application belongs to the technical field of chickpea polypeptide purification, and particularly relates to a chickpea polypeptide purification method. The chickpea polypeptide purification method comprises the following steps: (1) preparing a chickpea powder mixture; (2) sequentially using alkaline protease, neutral protease and bromelain to enzymatically digest the chickpea powder mixture to obtain an enzymatic hydrolysate; (3) adding complex bacteria to the enzymatic hydrolysate to perform fermentation, sterilization and obtain a fermentation liquor; (4) after centrifugation of the fermentation liquor, the supernatant is obtained, and the supernatant is subjected to ultrafiltration using an ultrafiltration membrane to obtain a chickpea polypeptide solution; and (5) freeze-drying the chickpea polypeptide solution to obtain purified chickpea polypeptide powder. The purified chickpea polypeptide powder prepared by the present application has high nutritional value and good storage stability when used for preparing yogurt.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of chickpea polypeptide purification, and specifically relates to a chickpea polypeptide purification method. BACKGROUND

[0002] Chickpea polypeptide not only has potential biological activity, but also is rich in various nutrients, which makes it widely used in the food industry, especially in fermented dairy products such as yogurt. Chickpea polypeptide can increase the protein content of yogurt, provide more essential amino acids, and enhance its nutritional value, which is very beneficial to people who need to supplement protein (such as athletes, the elderly, etc.). At the same time, chickpea polypeptide contains a certain amount of dietary fiber, which can increase the satiety of yogurt, promote intestinal health, and improve digestive function.

[0003] Currently, when chickpea polypeptide on the market is added to milk containing lactobacillus to prepare yogurt, due to the low purity of polypeptide, it may cause a series of problems, affecting the quality, flavor, texture and functionality of yogurt. 1. Unstable fermentation process: inhibition or promotion of lactobacillus growth: impurities in chickpea polypeptide (such as incompletely hydrolyzed proteins, polysaccharides, phenolic compounds, etc.) may inhibit or promote the growth of lactobacillus. Some impurities may compete with lactobacillus for nutrients, or release metabolic products that inhibit bacterial growth, resulting in slower or uneven fermentation. On the contrary, some impurities may provide additional carbon or nitrogen sources, accelerating the fermentation process, resulting in premature yogurt coagulation or excessive acidity. 2. Abnormal flavor and aroma: impurities in chickpea polypeptide may produce undesirable flavors during fermentation. For example, incompletely hydrolyzed proteins may decompose into bitter or polypeptides, affecting the overall taste of yogurt; some impurities may adsorb or consume volatile aroma components in yogurt, resulting in reduced or lost aroma, affecting its flavor quality. 3. Stability problems: some impurities may continue to catalyze reactions, causing changes in yogurt texture and flavor, affecting its stability and shelf life. 4. Decreased nutritional value: if chickpea polypeptide contains too many impurities, it may reduce the content of polypeptide with biological activity, weakening its antioxidant, anti-inflammatory, immune regulation and other functions. This will directly affect the functional value of yogurt, especially for consumers who hope to obtain health benefits by consuming yogurt. At the same time, the presence of impurities may interfere with the digestion and absorption of polypeptide, reducing its bioavailability. For example, incompletely hydrolyzed proteins may be difficult to be effectively absorbed in the intestine, resulting in a decrease in their nutritional value.

[0004] Currently, chickpea polypeptide is mainly purified by ultrafiltration membrane. The pore size of the ultrafiltration membrane is relatively large, and it cannot effectively remove small molecular impurities (such as organic acids, phenolic compounds, minerals, etc.). These small molecular substances will still affect the growth of lactobacillus, leading to unstable fermentation process, or affecting the flavor and aroma of yogurt.

[0005] Therefore, there is an urgent need for a chickpea polypeptide purification method. SUMMARY

[0006] The purpose of the present application is to provide a chickpea polypeptide purification method.

[0007] To achieve the above-mentioned purpose, the present application provides the following technical solutions:

[0008] A chickpea polypeptide purification method, comprising the following steps:

[0009] (1) Dry and crush chickpea beans to obtain chickpea powder; mix the chickpea powder with water, adjust the pH to 8.5-8.7, and prepare a chickpea powder mixture;

[0010] (2) Add alkaline protease to the chickpea powder mixture, and carry out enzymolysis at 45-48°C for 60-70 min; inactivate the enzyme, adjust the pH to neutral, add neutral protease, and carry out enzymolysis at 45-48°C for 30-50 min; inactivate the enzyme, continue to add bromelain, and carry out enzymolysis at 45-48°C for 30-40 min; inactivate the enzyme to obtain an enzymolysis liquid;

[0011] (3) Ferment the enzymolysis liquid with compound bacteria, sterilize, and obtain a fermentation liquid;

[0012] (4) After centrifugation of the fermentation liquid, take the supernatant, and perform ultrafiltration with an ultrafiltration membrane to prepare a chickpea polypeptide liquid.

[0013] (5) Perform low-temperature freeze-drying treatment on the chickpea polypeptide liquid to prepare a purified chickpea polypeptide powder.

[0014] Further, in step (1), the chickpea beans are dried and crushed to less than 100 mesh to obtain the chickpea powder.

[0015] Further, in step (1), the chickpea powder and water are mixed at a weight ratio of 1:(10-12).

[0016] Further, in step (1), the pH is adjusted to 8.5-8.7 with a 5-7wt% NaOH solution.

[0017] Further, in step (2), the pH is adjusted to neutral with a 7-9wt% citric acid.

[0018] Further, in step (2), the amount of alkaline protease is 600-800u / mL of the chickpea powder mixture; the amount of neutral protease is 1000-1200u / mL of the chickpea powder mixture; and the amount of bromelain is 800-900u / mL of the chickpea powder mixture.

[0019] The present application can improve the purity of chickpea polypeptide and the small intestine propulsion rate of chickpea polypeptide by using enzymes of specific types and amounts to perform gradient fermentation on chickpea. By selecting multiple proteases for combination use, the chickpea proteins can be hydrolyzed in multiple levels at different stages. First, endoprotease is used to decompose the proteins into larger polypeptide fragments, and then exoprotease is used to further decompose the polypeptide fragments into smaller peptide segments or amino acids. This multi-enzyme combination method can ensure that the proteins are fully hydrolyzed, reduce the residues of incompletely hydrolyzed proteins, and thus improve the purity of the polypeptides. Some short-chain polypeptides in the chickpea polypeptides have the effect of prebiotics, which can promote the growth of beneficial bacteria in the intestinal tract and inhibit the reproduction of harmful bacteria. The increase of beneficial bacteria can improve the intestinal microecological balance, promote intestinal health, and thus improve the small intestine propulsion rate.

[0020] Further, the fermentation condition in step (3) is 35-40℃ for 15-20h.

[0021] Further, the complex bacteria in step (3) include Lactobacillus casei, Bifidobacterium bifidum and Bifidobacterium longum.

[0022] Further, the amount of Lactobacillus casei in step (3) is 10 7 -10 8 CFU / mL of enzyme solution; the amount of Bifidobacterium bifidum is 10 9 -10 10 CFU / mL of enzyme solution; the amount of Bifidobacterium longum is 10 8 -10 9 CFU / mL of enzyme solution.

[0023] The present application uses specific complex bacteria for fermentation after enzymolysis, and the prepared polypeptide can improve the sensory effect of yogurt and improve the stability of yogurt. By selecting specific complex bacteria, the synergistic generation of multiple metabolites can be achieved during the fermentation process, enhancing the flavor, texture and functionality of yogurt. The active components produced during the fermentation of complex bacteria can adjust the pH value of yogurt and inhibit the growth of harmful microorganisms. In addition, the substances produced by complex bacteria can further inhibit the reproduction of harmful microorganisms, ensuring the safety and stability of yogurt.

[0024] Further, the step (4) uses a 3000-4000 Da ultrafiltration membrane for ultrafiltration.

[0025] Compared with the prior art, the present application has the following advantages and beneficial effects:

[0026] 1、The present application can improve the purity of chickpea polypeptide and the small intestine propulsion rate of chickpea polypeptide by using enzymes of specific types and amounts to perform gradient fermentation on chickpea.

[0027] 2、The polypeptide prepared by the method has good sensory effect and stability. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0029] The raw materials used in the following embodiments of the present application are all commercially available:

[0030] Lactobacillus brevis, No. SHBCCD14346, purchased from Shanghai Collection Center of Microorganisms.

[0031] Lactobacillus plantarum, No. CCTCC AB2010210, purchased from China Center for Type Culture Collection.

[0032] Bifidobacterium animalis, No. SHBCCD24407AS1.1852, purchased from China Center for Type Culture Collection.

[0033] Lactobacillus casei, No. SHBCCD24737, purchased from Shanghai Collection Center of Microorganisms.

[0034] Bifidobacterium bifidum, No. SHBCCD24408 ATCC35914, purchased from Shanghai Collection Center of Microorganisms.

[0035] Bifidobacterium longum, purchased from Shanghai Collection Center of Microorganisms, No. SHBCCD24310.

[0036] The enzymes used in the present application are all purchased from Shanghai Yuan Ye Biotechnology Co., Ltd. Alkaline protease, item No. S10154. Neutral protease, item No. S10013.

[0037] Embodiment 1

[0038] The present embodiment provides a method for purifying chickpea polypeptide, comprising the following steps:

[0039] (1) Dry the chickpea beans, crush them to less than 100 mesh to obtain chickpea powder; mix 1:11 by weight of the chickpea powder with water, and adjust the pH to 8.6 with 7wt% NaOH solution to prepare a chickpea powder mixture;

[0040] (2) adding alkaline protease to the chickpea powder mixed solution, and carrying out enzymolysis at 46℃ for 65 min; inactivating the enzyme, adjusting the pH value to neutral with 9wt% citric acid, adding neutral protease, and carrying out enzymolysis at 47℃ for 40 min; inactivating the enzyme, continuously adding bromelain, and carrying out enzymolysis at 46℃ for 35 min; inactivating the enzyme to obtain an enzymolysis solution;

[0041] The amount of alkaline protease in step (2) is 700 u / mL of the chickpea powder mixed solution; the amount of neutral protease is 1100 u / mL of the chickpea powder mixed solution; and the amount of bromelain is 850 u / mL of the chickpea powder mixed solution.

[0042] (3) adding compound bacteria to the enzymolysis solution, and carrying out fermentation at 38℃ for 16 h to obtain a fermentation solution; the compound bacteria include Lactobacillus casei, Bifidobacterium bifidum and Bifidobacterium longum;

[0043] The amount of Lactobacillus casei is 10 7 CFU / mL of the enzymolysis solution; the amount of Bifidobacterium bifidum is 10 10 CFU / mL of the enzymolysis solution; and the amount of Bifidobacterium longum is 10 8 CFU / mL of the enzymolysis solution.

[0044] (4) after centrifugation, the supernatant is obtained, and the supernatant is subjected to ultrafiltration with a 3000 Da ultrafiltration membrane to obtain a chickpea polypeptide solution.

[0045] (5) the chickpea polypeptide solution is subjected to low-temperature freeze-drying treatment to obtain a purified chickpea polypeptide powder.

[0046] Example 2

[0047] The embodiment provides a chickpea polypeptide purification method, which comprises the following steps:

[0048] (1) drying chickpea beans, and crushing the chickpea beans to less than 100 mesh to obtain chickpea powder; mixing the chickpea powder with water at a weight ratio of 1:12, and adjusting the pH value to 8.7 with a 7wt% NaOH solution to obtain a chickpea powder mixed solution;

[0049] (2) adding alkaline protease to the chickpea powder mixed solution, and carrying out enzymolysis at 48℃ for 70 min; inactivating the enzyme, adjusting the pH value to neutral with 9wt% citric acid, adding neutral protease, and carrying out enzymolysis at 48℃ for 30 min; inactivating the enzyme, continuously adding bromelain, and carrying out enzymolysis at 48℃ for 30 min; inactivating the enzyme to obtain an enzymolysis solution;

[0050] The amount of alkaline protease in step (2) is 800 u / mL of the chickpea powder mixed solution; the amount of neutral protease is 1000 u / mL of the chickpea powder mixed solution; and the amount of bromelain is 900 u / mL of the chickpea powder mixed solution.

[0051] (3) adding compound bacteria into the enzymatic hydrolysate, carrying out fermentation at 40℃ for 15h, sterilizing to obtain a fermentation liquor; the compound bacteria include Lactobacillus casei, Bifidobacterium bifidum and Bifidobacterium longum;

[0052] The amount of Lactobacillus casei is 10 7 CFU / mL of the enzymatic hydrolysate; the amount of Bifidobacterium bifidum is 10 9 CFU / mL of the enzymatic hydrolysate; the amount of Bifidobacterium longum is 10 8 CFU / mL of the enzymatic hydrolysate;

[0053] (4) centrifuging the fermentation liquor, taking supernatant, and carrying out ultrafiltration using a 4000Da ultrafiltration membrane to obtain chickpea polypeptide liquor.

[0054] (5) carrying out low-temperature freeze-drying treatment on the chickpea polypeptide liquor to obtain purified chickpea polypeptide powder.

[0055] Comparative Example 1

[0056] The difference between the present comparative example and Example 1 is that no fermentation is carried out.

[0057] A chickpea polypeptide purification method, comprising the following steps:

[0058] (1) drying chickpea beans, crushing to less than 100 mesh to obtain chickpea powder; mixing the chickpea powder with water at a weight ratio of 1:11, and adjusting pH to 8.6 using a 7wt% NaOH solution to obtain a chickpea powder mixture;

[0059] (2) adding alkaline protease to the chickpea powder mixture, carrying out enzymatic hydrolysis at 46℃ for 65min; inactivating the enzyme, adjusting pH to neutral using 9wt% citric acid, adding neutral protease, carrying out enzymatic hydrolysis at 47℃ for 40min; inactivating the enzyme, and continuously adding bromelain, carrying out enzymatic hydrolysis at 46℃ for 35min; inactivating the enzyme to obtain an enzymatic hydrolysate;

[0060] The amount of alkaline protease in step (2) is 700u / mL of the chickpea powder mixture; the amount of neutral protease is 1100u / mL of the chickpea powder mixture; and the amount of bromelain is 850u / mL of the chickpea powder mixture.

[0061] (3) centrifuging the enzymatic hydrolysate, taking supernatant, and carrying out ultrafiltration using a 3000Da ultrafiltration membrane to obtain chickpea polypeptide liquor.

[0062] (4) carrying out low-temperature freeze-drying treatment on the chickpea polypeptide liquor to obtain purified chickpea polypeptide powder.

[0063] Comparative Example 2

[0064] The difference between the present comparative example and Example 1 is that the neutral protease is replaced by ficin. The bromelain is replaced by pectinase.

[0065] A chickpea polypeptide purification method, comprising the following steps:

[0066] (1) Dry the chickpea beans, crush them to less than 100 mesh to obtain chickpea powder; mix the chickpea powder with water at a weight ratio of 1:11, adjust the pH to 8.6 with a 7wt% NaOH solution to prepare a chickpea powder mixture;

[0067] (2) Add alkaline protease to the chickpea powder mixture, and enzymatically hydrolyze at 46°C for 65 min; inactivate the enzyme, adjust the pH to neutral with 9wt% citric acid, add ficin, and enzymatically hydrolyze at 47°C for 40 min; inactivate the enzyme, continue to add pectinase, and enzymatically hydrolyze at 46°C for 35 min; inactivate the enzyme to obtain an enzymatic hydrolysate;

[0068] The amount of alkaline protease used in step (2) is 700u / mL of the chickpea powder mixture; the amount of ficin used is 1100u / mL of the chickpea powder mixture; and the amount of pectinase used is 850u / mL of the chickpea powder mixture.

[0069] (3) Add a complex bacteria to the enzymatic hydrolysate, ferment at 38°C for 16h, sterilize, and obtain a fermentation liquor; the complex bacteria include Lactobacillus casei, Bifidobacterium bifidum, and Bifidobacterium longum;

[0070] The amount of Lactobacillus casei used is 10 7 CFU / mL of the enzymatic hydrolysate; the amount of Bifidobacterium bifidum used is 10 10 CFU / mL of the enzymatic hydrolysate; and the amount of Bifidobacterium longum used is 10 8 CFU / mL of the enzymatic hydrolysate.

[0071] (4) After centrifugation of the fermentation liquor, take the supernatant, and perform ultrafiltration with a 3000Da ultrafiltration membrane to prepare a chickpea polypeptide solution.

[0072] (5) Perform low-temperature freeze-drying treatment on the chickpea polypeptide solution to prepare a purified chickpea polypeptide powder.

[0073] Comparative Example 3

[0074] The difference between the present comparative example and Example 1 is that the amount of alkaline protease used in step (2) is 1100u / mL of the chickpea powder mixture; the amount of neutral protease used is 8500u / mL of the chickpea powder mixture; and the amount of bromelain used is 700u / mL of the chickpea powder mixture.

[0075] Comparative Example 4

[0076] The difference between the present comparative example and Example 1 is that the composite bacteria include Lactobacillus brevis, Lactobacillus plantarum and Bifidobacterium animalis. The amount of Lactobacillus brevis is 10 7 CFU / mL of the enzyme hydrolysate; the amount of Lactobacillus plantarum is 10 10 CFU / mL of the enzyme hydrolysate; and the amount of Bifidobacterium animalis is 10 8 CFU / mL of the enzyme hydrolysate.

[0077] Comparative Example 5

[0078] The difference between the present comparative example and Example 1 is that the amount of Lactobacillus casei is 10 10 CFU / mL of the enzyme hydrolysate; the amount of Bifidobacterium bifidum is 10 8 CFU / mL of the enzyme hydrolysate; and the amount of Bifidobacterium longum is 10 7 CFU / mL of the enzyme hydrolysate.

[0079] Performance test

[0080] 1. Yogurt was prepared using the chickpea polypeptide powder prepared in Examples 1-2 and Comparative Examples 1-5, and the specific steps were as follows:

[0081] (1) The raw materials were weighed according to the following weight percentages: chickpea polypeptide 9%, Lactobacillus 1%, Streptococcus thermophilus 1%, xylose 7%, whey protein 5%, and the rest was fresh milk.

[0082] (2) Half of the milk was warmed to 50°C, and the chickpea polypeptide, xylose and whey protein were added and stirred uniformly, and then the remaining milk was mixed to obtain a mixture.

[0083] (3) The mixture was preheated, degassed, homogenized, sterilized, cooled, inoculated with bacteria, and fermented at 45°C. When the acidity of the mixture reached 70°C, the fermentation was stopped, and pasteurization was performed to obtain the finished product of chickpea polypeptide yogurt.

[0084] 2. The performance of the above-prepared chickpea polypeptide yogurt was tested.

[0085] (1) Sensory test. 100 volunteers were selected to comprehensively score the taste, aroma and appearance color of the yogurt, with scores of 1-10 points, and the average value was calculated. The higher the average value, the better the quality of the yogurt.

[0086] Table 1 Performance test results

[0087] Sensory score Example 1 8.7 Example 2 8.5 Comparative Example 1 7.1 Comparative Example 2 7.4 Comparative Example 3 8.1 Comparative Example 4 7.6 Comparative Example 5 7.9

[0088] (2) The effect of the yogurt on the constipation model mice was evaluated: 8 BALB / c male mice per group. Gavage was performed at 9 am every day.

[0089] Each BALB / c male mouse was given physiological saline by gavage throughout as a normal control group (NC).

[0090] Each mouse was given 0.25 mL of physiological saline by gavage for 2 weeks and then given 5 mg / kg of loperamide by gavage for 3 days as a constipation model group (CM).

[0091] Each mouse was given 0.25 mL of phenolphthalein by gavage for 2 weeks and then given 5 mg / kg of loperamide by gavage for 3 days as a positive drug group (PC).

[0092] Each mouse was given 0.25 mL of phenolphthalein by gavage for 2 weeks and then given 5 mg / kg of loperamide by gavage for 3 days as a positive drug group (PC).

[0093] Determination of small intestine propulsion rate in mice: On the third night, the mouse food was taken out, the mice were fasted for 12 h, and free water was allowed. The next morning, the mice were given 0.4 mL of phenol red meal by gavage, and after 20 min, the mice were sacrificed by cervical dislocation. After immersion in 75% alcohol for disinfection, the abdominal cavity was opened, the stomach was found, and the small intestine segment was quickly separated, placed on filter paper, the excess blood water was absorbed, the small intestine was gently stretched, and carefully laid on white paper. The total length of the small intestine and the distance of the phenol red meal forward propulsion were measured, and the small intestine propulsion rate was calculated according to the following formula:

[0094] Small intestine propulsion rate (%) = distance of phenol red meal propulsion / total length of small intestine * 100%.

[0095] Table 2 Determination results of small intestine propulsion rate

[0096]

[0097]

[0098] 3, The yogurt was placed in a 30°C environment for 3 months to observe whether water separation occurred, and the results are shown in Table 3.

[0099] Table 3 Stability of yogurt storage

[0100] Stability Example 1 No water separation Example 2 No water separation Comparative Example 1 Water separation Comparative Example 2 Water separation Comparative Example 3 Slight water separation Comparative Example 4 Water separation Comparative Example 5 Slight water separation

[0101] From the performance test results, it can be seen that the sensory quality of the chickpea polypeptide of Example 1-2 is high, the stability of the yogurt prepared therefrom is good, the intestinal propulsion rate is high, and it is of great significance to digestive health, nutrient absorption and overall intestinal function. In particular, the comprehensive performance of Example 1 is the most outstanding.

[0102] The comparative examples are obviously worse than the examples in the corresponding performance test because they do not adopt the necessary technical solutions. In the comparative example 1, the fermentation is not carried out, the sensory quality of the yogurt is reduced, and the storage stability is reduced. In the comparative examples 2-3, the types and amounts of enzymes used are different, the intestinal propulsion rate of the prepared yogurt is reduced. In the comparative examples 4-5, the types and amounts of the composite bacteria are changed, and the stability of the yogurt is reduced. The above is the preferred embodiment of the present application. It should be pointed out that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as the protection scope of the present application.

Claims

1. The application of purified chickpea polypeptide powder in the preparation of yogurt, characterized in that, The purification method for chickpea peptides includes the following steps: (1) Dry and crush chickpeas to obtain chickpea flour; mix chickpea flour with water and adjust the pH to 8.5-8.7 to obtain chickpea flour mixture; (2) Add alkaline protease to the chickpea flour mixture and hydrolyze at 45℃-48℃ for 60-70 min; inactivate the enzyme, adjust the pH to neutral, add neutral protease, and hydrolyze at 45℃-48℃ for 30-50 min; inactivate the enzyme, continue to add bromelain, and hydrolyze at 45℃-48℃ for 30-40 min; inactivate the enzyme to obtain the hydrolysate; The dosage of alkaline protease is 600-800 u / mL of chickpea flour mixture; the dosage of neutral protease is 1000-1200 u / mL of chickpea flour mixture; and the dosage of bromelain is 800-900 u / mL of chickpea flour mixture. (3) Add the compound bacteria to the enzymatic hydrolysate for fermentation, sterilize, and obtain the fermentation broth; the compound bacteria include Lactobacillus casei, Bifidobacterium bifidum, and Bifidobacterium longum; the amount of Lactobacillus casei is 10 7 -10 8 CFU / mL enzymatic hydrolysate; the amount of Bifidobacterium bifidum used was 10. 9 -10 10 CFU / mL enzymatic hydrolysate; the amount of Bifidobacterium longum used was 10. 8 -10 9 CFU / mL enzyme hydrolysate; (4) After centrifugation, the supernatant of the fermentation broth was taken and ultrafiltered using an ultrafiltration membrane to obtain chickpea polypeptide solution; (5) The chickpea polypeptide liquid was subjected to low-temperature freeze-drying to obtain purified chickpea polypeptide powder.

2. The application according to claim 1, characterized in that, In step (1), chickpeas are dried and pulverized to a fineness of less than 100 mesh to obtain chickpea flour.

3. The application according to claim 1, characterized in that, In step (1), chickpea flour and water are mixed in a weight ratio of 1:(10-12).

4. The application according to claim 1, characterized in that, In step (1), the pH is adjusted to 8.5-8.7 using a 5-7 wt% NaOH solution.

5. The application according to claim 1, characterized in that, In step (2), the pH value is adjusted to neutral using citric acid at a concentration of 7-9 wt%.

6. The application according to claim 1, characterized in that, The fermentation conditions in step (3) are: fermentation at 35-40℃ for 15-20 hours.

7. The application according to claim 1, characterized in that, In step (4), ultrafiltration is performed using a 3000-4000 Da ultrafiltration membrane.

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