Method for producing high-quality soy protein concentrate product and application of product

Abstract

The present invention provides a method for producing a high quality soy protein concentrate product, use of the soy protein concentrate product thus obtained in the preparation of a feed, and a method for preparing a fermented feed comprising the soy protein concentrate product. According to the method disclosed by the invention, the high-temperature meal concentrated protein obtained by alcohol method separation is subjected to low-moisture solid-state glycolysis by adopting a specific bacterium-enzyme combination, so that the clearance rate of antigen protein and oligosaccharide can be remarkably increased, the mildew-proof effect is remarkably improved, meanwhile, the production cost of concentrated protein enzymolysis is greatly reduced, and the application of the high-temperature meal concentrated protein in feed is favorably improved.

Description

Technical Field

[0001] The invention relates to the field of protein food processing, in particular to a method for producing a high-quality soybean protein concentrate product and uses of the product obtained thereby. Background Art

[0002] The wet meal obtained by solvent extraction of soybean oil can be desolventized at high temperature to obtain high-temperature defatted soybean meal (high-temperature meal). High-temperature meal is rich in protein and is widely used as a feed protein raw material. However, high-temperature meal often has problems such as high protein denaturation and high content of anti-nutritional factors, which not only destroys the nutritional value and utilization efficiency of the feed itself, but also has a certain degree of protein denaturation and high content of anti-nutritional factors. [1] The anti-nutritional factors such as antigenic proteins, oligosaccharide flatulence factors, trypsin inhibitors, lectins, etc. in high-temperature meal can cause digestive stress reactions such as intestinal allergies and diarrhea when used in large quantities in animal feed. [2,3] , which leads to a decrease in feed digestibility, and to a certain extent limits its application in the feed field. It is urgent to convert the macromolecular protein in the high-temperature meal into small molecular substances such as peptides and amino acids, and eliminate anti-nutritional factors at the same time, so as to improve the utilization rate of feed protein. The anti-nutritional factors of soybeans are mainly divided into three categories, namely conglobulins, oligosaccharides and trypsin inhibitors. Since most anti-nutritional factors are heat-sensitive substances, their content can be reduced by improving processing conditions, but there are also heat-stable anti-nutritional factors such as conglobulins and oligosaccharides. Therefore, it is necessary to develop different soy protein processing technologies to minimize anti-nutritional factors while ensuring that the utilization efficiency of protein is not lost.

[0003] The methods for modifying soybean protein and degrading antigenic factors mainly include physical method, chemical method, enzymatic method and microbial fermentation method. [4] . Among them, the yield of soy protein concentrate produced by physical methods is low and the quality is poor. Bio-enzymatic hydrolysis and microbial fermentation are two methods that are now widely used to process soy protein. Bio-enzymatic hydrolysis can be performed by different enzyme preparations for selective enzymatic hydrolysis, and is considered to be an effective method for breaking down large molecular proteins into small molecular substances, reducing or even completely eliminating the allergenicity of soy protein. The enzymatic hydrolysis method has the characteristics of substrate specificity, clear products, stability and high efficiency, but bitter substances are easily produced during the enzymatic hydrolysis process, which affects the palatability of feed products. The enzymatic hydrolysis of soy protein concentrate is mostly used to improve the functional properties of concentrated protein, and the enzymatic hydrolysis process is mainly based on high-water liquid enzymatic hydrolysis (the enzymatic hydrolysis efficiency of solid-state fermentation in the prior art is not high). At present, the research on enzymatic hydrolysis of concentrated protein is mostly concentrated in the field of food processing, and the water content of the enzymatic hydrolysis system is above 90%. There are problems such as high energy consumption for product concentration and drying, high water consumption, and high labor costs. [5-9]Yang Ning et al. tried to enzymatically hydrolyze concentrated protein for feed and found that when the water content of the system was less than 50%, the addition of protease A at 3% resulted in only 7.4% acid-soluble protein; when the water content of the enzymatic system was greater than 50% and less than 70%, the addition of protease B at 0.8% resulted in a maximum acid-soluble protein content of 39.7%. Therefore, the current enzymatic efficiency and cost need to be further improved.

[10] . Studies have found that the enzymatic hydrolysis effect of the biological enzymatic hydrolysis method is affected by many factors such as the type of enzyme and the enzymatic hydrolysis conditions. Due to the structural problems of the polypeptide itself, enzymatic protein will inevitably lead to the presence of bitterness, which greatly affects the palatability of the product. The microbial fermentation method works through the protease produced by fermentation. In addition, a large amount of organic acids and flavor substances will be produced during the fermentation process, which has positive significance for improving the palatability of feed and regulating animal intestinal health. However, the single microbial fermentation method has a low amount of protease and cannot meet actual production needs.

[0004] At present, the bacterial enzyme synergistic fermentation process has been developed in the field of fermented feed, that is, a certain amount of probiotics such as lactic acid bacteria, yeast, spores, and even molds are added to the fermentation process under the treatment of enzymatic hydrolysis.

[11] These probiotics will produce a variety of aromatic substances during the fermentation process, which can regulate the bitterness of feed products.

[12] , and it can also overcome the problem of insufficient enzyme production by using microbial fermentation alone. However, the current bacterial-enzyme synergistic fermentation technology is not ideal in removing antigenic proteins and oligosaccharides and solving the problem of contaminating bacteria and mildew. Summary of the invention

[0005] In order to produce high-quality soybean protein concentrate based on high-temperature defatted soybean meal, the applicant has found through research that the quality of the prepared soybean protein concentrate can be significantly improved by using the alcohol method to separate the high-temperature meal protein concentrate and then performing bacterial enzyme synergistic fermentation. In the method of the present invention, by using a specific bacterial enzyme combination to ferment the high-temperature meal protein concentrate, the clearance rate of antigenic proteins and oligosaccharides can be significantly improved, and the anti-mildew effect can be significantly improved. Moreover, the method of the present invention can significantly reduce the production cost of concentrated protein hydrolysis through low-moisture solid-state fermentation under bacterial enzyme synergistic conditions, which helps to improve its application in feed; in addition, while controlling the amount of water added, the method of the present invention also achieves the purpose of increasing the acid-soluble protein content and significantly degrading antigenic factors.

[0006] In a first aspect of the present invention, a method for producing a high-quality soybean protein concentrate product is provided, the method comprising: adding an enzyme preparation, a composite strain, a supplementary powder and a sorbate to a fermentation raw material to obtain a fermentation matrix, and fermenting the fermentation matrix to obtain a soybean protein concentrate product;

[0007] The fermentation matrix comprises soybean high-temperature meal concentrated protein and water, the enzyme preparation comprises neutral protease and papain; the composite bacteria comprises Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis, and the sorbate is one or more selected from potassium sorbate, sodium sorbate and calcium sorbate.

[0008] In another aspect of the present invention, provided is use of a soybean protein concentrate product obtained by the method described in the first aspect of the present invention in preparing feed.

[0009] In another aspect of the present invention, a method for preparing a fermented feed comprising the above-mentioned soybean protein concentrate product is provided.

[0010] Beneficial Effects

[0011] (1) The method of the present invention uses a specific composite enzyme (neutral protease and papain) + composite bacteria (Bacillus subtilis ZL1 + Lactobacillus plantarum ZM3 + Saccharomyces cerevisiae ZY1) to ferment soybean high-temperature meal concentrated protein, which can effectively increase the acid-soluble protein content and significantly improve the degradation rate of antigenic proteins and oligosaccharides.

[0012] (2) The method described in the present invention can solve the problem of bacterial contamination and mildew of protein products, and further improve the quality of the obtained soybean protein concentrate product.

[0013] (3) The method of the present invention utilizes low-moisture solid fermentation (moisture content does not exceed 55wt%, for example, using a breathing bag method) to greatly reduce costs. In comparison, the industry currently generally uses liquid fermentation to process feed protein, which has high drying costs and relatively low profits. This study is the first to use a bacterial enzyme-coordinated solid fermentation process under low-moisture conditions to process soybean high-temperature meal concentrated protein, and has achieved the effects of increasing the acid-soluble protein content, significantly degrading antigenic proteins and oligosaccharides, improving feed palatability, increasing feed nutritional value, and improving digestion and absorption rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The acid-soluble protein content and antigenic protein content in the samples of soybean high-temperature meal concentrated protein fermented by different Bacillus in the fermentation strain screening test are shown.

[0015] Figure 2 The acid-soluble protein content and antigen protein content in the samples of soybean high-temperature meal concentrated protein fermented by different lactobacilli (lactic acid bacteria) in the fermentation strain screening test are shown.

[0016] Figure 3 The acid-soluble protein and crude protein content of soybean high-temperature meal concentrated protein after hydrolysis with different complex enzymes under respective pH conditions are shown.

[0017] Figure 4 It is a graph showing the effect of bacterial inoculation amount on the quality change of soybean protein concentrate products.

[0018] Figure 5 It is a graph showing the effect of the amount of compound enzyme added on the quality change of soybean protein concentrate products.

[0019] Figure 6 It is a graph showing the effect of fermentation temperature on the quality change of soy protein concentrate products.

[0020] Figure 7 It is a graph showing the effect of fermentation time on the quality change of soy protein concentrate products.

[0021] Figure 8 It is a graph showing the effect of water content on the quality change of soy protein concentrate products. DETAILED DESCRIPTION

[0022] Next, exemplary embodiments of the present invention are described, but the protective scope of the present invention is not limited thereto.

[0023] In this document, unless otherwise specified, "high-temperature soybean meal protein concentrate" refers to the product obtained by alcohol extraction of high-temperature defatted soybean meal.

[0024] In one aspect, the present invention provides a method for producing a high-quality soybean protein concentrate product, the method comprising: adding an enzyme preparation, a composite strain, a supplementary powder and a sorbate to a fermentation raw material to obtain a fermentation matrix, and fermenting the fermentation matrix to obtain a soybean protein concentrate product;

[0025] The fermentation matrix comprises soybean high temperature meal protein concentrate and water, the enzyme preparation comprises neutral protease and papain, the composite bacteria comprises plant lactobacillus, saccharomyces cerevisiae and bacillus subtilis, and the sorbate is one or more selected from potassium sorbate, sodium sorbate and calcium sorbate. Due to the antibacterial effect of sorbate itself, the use of sorbate, especially potassium sorbate, may inhibit the fermentation bacteria added in the present invention, but the inventors unexpectedly found in the research process that in the method of the present invention, the addition of sorbate does not affect the fermentation ability of the composite bacteria itself, and can also effectively prevent and control mold.

[0026] In some embodiments, the method further comprises: adding C1-C3 alcohol (preferably methanol, ethanol) to the high-temperature defatted soybean meal for extraction, and then centrifuging and drying to obtain the high-temperature soybean meal concentrated protein.

[0027] In some embodiments, the water content of the fermentation substrate is 30 wt%-60 wt%, preferably 40 wt%-55 wt%, more preferably 45 wt%-55 wt%, such as 47 wt% or 54 wt%.

[0028] In some embodiments, the amount of sorbate added to the fermentation matrix is ​​0.01wt%-0.2wt%, preferably 0.05wt%-0.1wt%; the amount of added powder in the fermentation matrix is ​​0.1wt%-2.0wt%, preferably 0.5wt%-1.5wt%, for example 0.9wt%-1.0wt%.

[0029] In some embodiments, the method further comprises adding an additive to the fermentation raw material. In the present invention, additive refers to any auxiliary substance added during fermentation that helps fermentation (e.g., promotes the growth of fermentation strains, prevents the growth of miscellaneous bacteria, stabilizes pH). In some embodiments, the additive comprises glucose. In some embodiments, the addition amount of glucose in the fermentation substrate is 0wt%-2.0wt%, preferably 0.5wt%-1.0wt%.

[0030] In some embodiments, the mass ratio of neutral protease to papain in the enzyme preparation is (1.0-4.0):(1.0-4.0), preferably 1:1.

[0031] In some embodiments, the enzyme preparation is added in an amount greater than 500 U / g fermentation substrate, preferably 1000 U / g fermentation substrate to 3000 U / g fermentation substrate, such as 2000 U / g fermentation substrate.

[0032] In some embodiments, the composite strain is composed of Lactobacillus plantarum ZM3 (CICC R21790), Saccharomyces cerevisiae ZY1 (CGMCC 2.1638) and Bacillus subtilis ZL1 (CICC G20030). In some preferred embodiments, the mass ratio of Lactobacillus plantarum ZM3 (CICC R21790), Saccharomyces cerevisiae ZY1 (CGMCC 2.1638) and Bacillus subtilis ZL1 (CICCG20030) is (1.0-10.0): (1.0-6.0): (1.0-20.0), preferably 10:1.0:20.0.

[0033] In some embodiments, the composite strain may be activated before inoculation using a method known in the art for culturing the strain. The composite strain may be activated in a solid culture medium and / or a liquid culture medium; preferably, the strain may be first activated on a solid culture medium and then transferred to a liquid culture medium for culturing to obtain a seed solution of the composite strain for inoculation.

[0034] In some embodiments, the inoculation amount of the composite bacteria is 1.0wt%-4.0wt% relative to the mass of the fermentation substrate. In a preferred embodiment, the inoculation amount of the composite bacteria is 1.5wt%-3.0wt%, 2.0wt%-3.0wt%.

[0035] In some embodiments, the viable bacteria count in the seed solution of the composite strain is 10 7 Pieces / mL-10 9 pcs / mL, e.g. 10 8 Pieces / mL.

[0036] In some embodiments, the fermentation is carried out at 30°C-50°C for 24h-72h. In a preferred embodiment, the fermentation is carried out at 35°C-45°C (eg, 35°C, 40°C or 45°C) for 48-72h.

[0037] In a specific embodiment, the water content of the fermentation substrate is 54 wt %, the added amount of the enzyme preparation is 2000 U / g fermentation substrate, the inoculation amount of the composite bacteria relative to the fermentation substrate is 2.25 wt %, and the fermentation is carried out at 35° C. for 60 h.

[0038] In another aspect of the present invention, there is provided use of the soybean protein concentrate product obtained by the method described above in preparing feed.

[0039] In another aspect of the present invention, a method for preparing a fermented feed comprising the above-mentioned soybean protein concentrate product is provided.

[0040] In some preferred embodiments, the method comprises adding enzyme preparation, composite strain, added powder and sorbate to the feed raw material containing soybean high temperature meal concentrated protein for fermentation, or mixing the soybean concentrated protein product obtained above with the fermented feed available in the prior art, wherein the enzyme preparation comprises neutral protease and papain; the composite strain comprises plant lactobacillus, saccharomyces cerevisiae and bacillus subtilis, and the sorbate is one or more selected from potassium sorbate, sodium sorbate and calcium sorbate. In some embodiments, the feed raw material may further include grain and oil processing byproducts or meal resources (such as soybean, peanut, rapeseed, sunflower and other cake and meal resources) other than soybean high temperature meal concentrated protein.

[0041] Example

[0042] Next, the present invention will be described in further detail by way of examples, but the present invention is not limited to these examples.

[0043] The experimental consumables, experimental equipment, strain activation method, and data analysis method used in the following examples are as follows:

[0044] (1) Test consumables

[0045] Raw materials: high-temperature defatted soybean meal (GB / T 19541-2017, protein 43 wt%, purchased from COFCO Oils & Fats (Jiujiang) Co., Ltd., crushed through a 30-mesh sieve).

[0046] Soybean high-temperature meal concentrated protein: The above-mentioned high-temperature defatted soybean meal is used as a raw material and is prepared by the following method: accurately weigh 1 kg of high-temperature defatted soybean meal and put it into an extraction container, prepare a 55 vol% ethanol solution, add the ethanol solution into the extraction container at a solid-liquid ratio of 1:6 (weight to volume ratio, kg:L), mix well and place in a constant temperature shaking water bath (or circulating extraction column) at 55°C, ensure that the temperature is relatively constant during the extraction process, after 90 minutes of extraction, centrifuge the extracted meal to remove the solvent, and dry to obtain the high-temperature meal soybean concentrated protein. The crude protein content of the soybean high-temperature meal concentrated protein prepared in this way reaches 65wt%, and soybean globulin, β-globulin, trypsin inhibitor, and lectin anti-nutritional factors are greatly eliminated, reaching the concentrated protein standard.

[0047] Bacterial strains (purchased from China Industrial Microorganism Culture Collection Center and China General Microorganism Culture Collection Center): Bacillus subtilis ZL1 (CICC G20030), Bacillus licheniformis ZL2 (CICC 20446), Bacillus subtilis ZL3 (CICC 10073), Bacillus amyloliquefaciens ZL4 (CICC 10160), Bacillus subtilis ZL5 (CICC 10210); Lactobacillus acidophilus ZM1 (CGMCC 1.1854), Lactobacillus plantarum ZM2 (CICC 21791), Lactobacillus plantarum ZM3 (CICC R21790) and Lactobacillus plantarum ZM4 (CGMCC R1.12934); Saccharomyces cerevisiae ZY1 (CGMCC 2.1638).

[0048] Culture medium: MRS medium (Beijing Luqiao Technology Co., Ltd.), YPD medium (Qingdao Haibo Biotechnology Co., Ltd.), LB broth medium (Beijing Luqiao Technology Co., Ltd.).

[0049] Enzyme preparations: papain (200,000 U / g, optimum pH = 6.0), neutral protease (200,000 U / g, optimum pH = 6.5), alkaline protease (200,000 U / g, optimum pH = 10.5), all purchased from Beijing Solebow Technology Co., Ltd.

[0050] Others: soybean globulin, β-conglycinin, trypsin inhibitor, lectin detection kit (Beijing Longke Ark Bioengineering Technology Co., Ltd.); stachyose, raffinose standards (China Food and Drug Biological Products Control Institute); Jiayi powder (Weifang Guanxiang Jiayi Powder Factory).

[0051] (2) Test equipment

[0052] Constant temperature incubator MIR-154-PC incubator (Panasonic, Japan), constant temperature shaker YZYF-113 (Thermo Fisher Scientific, USA), S-433(D) fully automatic amino acid analyzer (Sykam, Germany), Synergy HT multifunctional microplate reader (Biotek, USA), Kjeltec-TM-9 Kjeldahl nitrogen analyzer (FOSS, Denmark), 1260-Infinity-II high pressure liquid chromatography (Agilent, USA), SDS-III monogastric animal bionic digestion system (Hunan Zhongben Intelligent Technology Development Co., Ltd.).

[0053] (3) Bacterial Activation

[0054] First, inoculate one loop of each preserved bacterial species onto the corresponding slant solid culture medium, and place it in a 37°C constant temperature incubator for 24 hours; secondly, use an inoculation loop to inoculate two loops of bacterial species from the above fresh slant into a 100mL conical flask filled with 50mL liquid culture medium, where Bacillus and yeast are cultured in a shaking table at 35°C and 130rpm for 24 hours, and Lactobacillus is placed in a 37°C constant temperature incubator for static culture for 24 hours; finally, inoculate 2wt% of the above cultured bacterial solution into a 250mL conical flask filled with 100mL liquid culture medium and continue to expand the culture under the same conditions as above, so that the number of viable bacteria reaches 10 8 / mL, which is the fermentation seed liquid.

[0055] (4) Measurement method

[0056] Crude protein content: Determined according to GB / T 6432-2018 "Determination of crude protein in feed, Kjeldahl method". Weigh 1g soybean meal sample, 0.2g CuSO4, and 6g K2SO4, mix them evenly and put them into the digestion tube, add 10mL H2SO4, put the digestion tube on the digestion device for digestion (420℃, 2h), and measure the digested sample with a Kjeldahl nitrogen analyzer.

[0057] Acid-soluble protein content: Determined by the Kjeldahl nitrogen method in NY / T 3801-2020 "Determination of Acid-soluble Protein in Feed Raw Materials".

[0058] Potassium hydroxide protein solubility: The potassium hydroxide protein solubility in Appendix A of GB / T 19541-2017 "Feed Ingredient Soybean Meal" is used for determination.

[0059] Moisture content: Determined using the moisture determination method in GB / T 6435-2014 “Determination of moisture in feeds”.

[0060] Amino acid content: Cysteine ​​and methionine contents were determined by the sulfur-containing amino acid determination method in GB / T 15399-2018 "Determination of sulfur-containing amino acids in feed by ion exchange chromatography", tryptophan content was determined by the high performance liquid chromatography determination method in GB / T 15400-2018 "Determination of tryptophan in feed", and other amino acids were determined by the conventional acid hydrolysis determination method in GB / T18246-2019 "Determination of amino acids in feed". Samples (such as soybean high temperature meal concentrated protein, fermentation enzymatic concentrated protein, soybean concentrated protein products, etc. of the present invention) were determined using a Sekam S-433 (D) fully automatic amino acid analyzer.

[0061] Glycinin, β-conglycinin, trypsin inhibitor, lectin: The test kits were purchased from Beijing Longke Ark Bioengineering Technology Co., Ltd., and the samples (such as soybean high temperature meal concentrated protein, fermentation enzymatic concentrated protein, soybean concentrated protein products, etc.) were measured using the Biotech Synergy HT multifunctional microplate reader. The test and determination were carried out according to the instructions of the kit.

[0062] Stachyose and raffinose content: Detected by referring to the method of raffinose and stachyose in GB / T 22491-2008 "Soybean oligosaccharides". Samples (such as soybean high temperature meal concentrated protein, fermentation enzymatic concentrated protein, soybean concentrated protein products, etc.) were determined using Agilent 1260-Infinity-II high pressure liquid chromatography.

[0063] In vitro digestibility of protein: This experiment was performed with reference to the SDS-III bionic digestion operation manual of the State Key Laboratory of Animal Nutrition. The enzymatic hydrolysis products of soybean high-temperature meal concentrated protein (i.e., crude protein) were determined using the porcine crude protein bionic digestion program. Each sample was loaded on the machine in 10 parallels, 5 of which were used to determine the amino acid digestibility, and 5 were used to determine the crude protein digestibility. The in vitro digestibility of soybean high-temperature meal concentrated protein after enzymatic hydrolysis was calculated according to the following formula: D / % = (m1-m2) / m1×100, where: D, in vitro digestibility of protein, %; m1, mass of crude protein without enzymatic hydrolysis, g; m2 mass of crude protein after enzymatic hydrolysis, g. (Refer to Li Qingxiao et al.

[13] method)

[0064] (5) Data analysis

[0065] SPSS 22.0 statistical software was used to process the data, and one-way analysis of variance was used for comparison between multiple groups. P < 0.05 indicated a significant difference between the two groups. The test results were expressed as "mean ± standard deviation".

[0066] Example 1 Fermentation strain screening

[0067] 1 Test method

[0068] The above-mentioned high temperature meal protein concentrate is used as raw material, and the conditions for the screening of breathing bag fermentation are as follows: the high temperature meal protein concentrate charge is 100g (57.75wt%), water is 69.2g (39.96wt%), alkaline protease is 0.362g (0.21wt%), single strain inoculation amount is 2.17mL (the bacterial solution addition amount of each of Lactobacillus ZM1-ZM4, Saccharomyces cerevisiae ZY1 or Bacillus ZL1-ZL5 accounts for 1.25wt%), glucose addition is 0.72g (0.42wt%), and the addition amount of Jiayi powder is 0.72g (0.42wt%), wherein, Lactobacillus is fermented anaerobically, yeast and Bacillus are fermented aerobically, fermentation temperature is 40°C, and fermentation time is 60h. The sample after the fermentation of high temperature meal protein concentrate is dried at 55°C, crushed, sieved through a 40-mesh sieve, and used.

[0069] 2 Test results

[0070] After soybean high temperature meal concentrated protein was fermented by different Bacillus, the acid soluble protein content and antigen protein content in the samples were shown in Figure 2. Figure 1 . Specifically, after fermentation with different Bacillus (ZL1-ZL5), the acid-soluble protein content in the high-temperature meal concentrated protein fermentation samples of the ZL1 group was 341.1 mg / g, and the acid-soluble protein content in the high-temperature meal concentrated protein fermentation samples of the ZL2 group was 361.6 mg / g. Compared with other fermentation groups, the acid-soluble protein content in the high-temperature meal concentrated protein fermentation samples of the ZL1 and ZL2 groups was at a higher level; in addition, compared with the other groups (ZL2-ZL5 groups), the antigenic proteins (soybean globulin and β-conglycinin) of the ZL1 group were at the lowest level. In general, choosing Bacillus ZL1 to ferment high-temperature meal concentrated protein can achieve the results of high acid-soluble protein content and the lowest antigenic protein content, and the overall fermentation effect is the best.

[0071] The acid-soluble protein content and antigen protein content of soybean high-temperature meal concentrate after fermentation by different lactobacilli Figure 2As shown. After fermentation with different lactobacilli, compared with other fermentation groups (ZM1, ZM2 and ZM4), the acid-soluble protein content in the fermentation sample of high-temperature meal concentrated protein in the ZM3 group was the highest (32.9 mg / g), and the soybean globulin content in the fermentation sample of the ZM3 group was the lowest (10.65 mg / g), and the β-conglycinin content was also the lowest (9.38 mg / g). Based on the decomposition of acid-soluble protein and antigen protein, ZM3 was selected as the fermentation lactic acid strain.

[0072] After the soybean high temperature meal concentrated protein was fermented by Saccharomyces cerevisiae ZY1, the acid-soluble protein content in the sample was 173.40 mg / g, the glycinin content was 13.73 mg / g, and the β-conglycinin content was 6.74 mg / g.

[0073] Example 2 Composite enzyme screening

[0074] Test methods

[0075] The soybean high temperature meal concentrated protein was crushed and passed through a 40 mesh sieve, and then enzymolysis was performed. The enzymolysis conditions were as follows: the high temperature meal concentrated protein charge was 100 g, 400 mL of distilled water was added at a ratio of 1:4 between the concentrated protein and distilled water, 0.5 g of each group of composite proteases (composite enzyme M1: 50 wt% of neutral protease and papain; composite enzyme M2: 50 wt% of neutral protease and alkaline protease; composite enzyme M3: 50 wt% of papain and alkaline protease) accounting for 0.5% of the mass of the high temperature meal concentrated protein was added to each triangular flask, mixed evenly, and the pH of each group of enzymolysis system was adjusted with 1 mol / L NaOH and HCl solution. Specifically, the pH of the compound enzyme M1 group was first adjusted to 6.5 and enzymolyzed at 45°C for 30 minutes, and then the pH was adjusted to 6.0 and continued to enzymolyze at 45°C for 30 minutes, the pH of the compound enzyme M2 group was first adjusted to 10.5 and enzymolyzed at 45°C for 30 minutes, and then the pH was adjusted to 6.5 and continued to enzymolyze at 45°C for 30 minutes, and the pH of the compound enzyme M3 group was first adjusted to 10.5 and enzymolyzed at 45°C for 30 minutes, and then the pH was adjusted to 6.0 and continued to enzymolyze at 45°C for 30 minutes. At the same time, a control group M4 without adding enzyme was set up. Except for not adding enzyme, other conditions were the same as those of the M1 group. After heating the enzymolysis sample in a boiling water bath for 10 minutes, the sample was dried. Crushed through a 40-mesh sieve and stored in a cool and dry place for measurement.

[0076] After enzymatic hydrolysis, the moisture, total protein, acid-soluble protein, protein solubility and anti-nutritional factors were tested.

[0077] Test results

[0078] After enzymolysis with different complex enzymes as above, the acid-soluble protein and crude protein content in the obtained samples are shown in Figure 3 After the high-temperature meal concentrated protein was treated with compound enzymes, the acid-soluble protein content was significantly increased, much higher than that of the control group (non-enzyme treatment). Among them, the acid-soluble protein content was the highest after treatment with compound enzyme M1 (neutral protease and papain), reaching 262.1 mg / g, which was 371% higher than that of the control group. Therefore, compound enzyme M1 was selected for subsequent bacterial enzyme synergistic fermentation.

[0079] Compared with the control group, the mass concentration of soybean high-temperature meal concentrated protein in each complex enzyme group decreased significantly, but the protein mass concentration in the complex enzyme M1 treatment group was the highest. This may be because the enzymatic hydrolysis caused the system stability of the high-temperature meal concentrated protein to change, generating small molecular weight, low-boiling point water-soluble substances, and losing some dry matter during the drying and heating process.

[0080] Example 3 Bacterial enzyme synergistic fermentation

[0081] Test methods

[0082] (1) Research on the basic process of concentrated protein bacterial enzyme synergistic fermentation

[0083] Based on a large number of previous experimental studies, a study on bacterial-enzyme synergistic fermentation was conducted using a breathing bag under the following conditions: the above-mentioned soybean high-temperature meal concentrated protein was used as the raw material, the high-temperature meal concentrated protein charge was 100g (63.07wt%), water was 49.76g (31.38wt%), complex enzyme was 0.796g (0.5wt%; neutral protease and papain were half each), the bacterial inoculum was 4.81mL (3.0wt%, Lactobacillus plantarum ZM3: Saccharomyces cerevisiae Z Y1: Bacillus subtilis ZL1 = 10: 1: 20, wherein plant lactobacillus 1.55mL, saccharomyces cerevisiae 0.16mL, Bacillus subtilis 3.1mL), glucose addition 1.60g (1.0wt%), addition of benefit powder 1.52g (0.95wt%), addition of potassium sorbate 0.08g (0.05wt%), the breathing bag containing the fermentation substrate thus obtained is sealed and fermented, the fermentation temperature is 40°C, and the fermentation time is 60h. The sample after the bacterial enzyme cooperative fermentation is dried at 55°C, crushed, and passed through a 40-mesh sieve to obtain fermentation enzymatic concentrated protein (i.e., a soybean concentrated protein product as a cooperative fermentation basic process product).

[0084] Through determination, the content of acid-soluble protein in the fermentation enzymatic concentrated protein obtained was 271.10 mg / g, the content of soybean globulin was 1.43 mg / g, the content of β-conglycinin was 7.84 mg / g, the content of trypsin inhibitor was 0.42 mg / g, the content of lectin was 0.04 mg / g, and the contents of stachyose and raffinose were not detected.

[0085] Next, the factors that may affect the bacterial-enzyme synergistic fermentation were analyzed to obtain better bacterial-enzyme synergistic fermentation conditions.

[0086] (2) Effect of bacterial inoculation amount on the quality of soybean protein concentrate products

[0087] Based on the experiment of (1) above, the water content was set to 40wt%, and the inoculation amount of the strains was adjusted (the ratio between the strains remained unchanged) so that their contents in the fermentation matrix were 1.0wt%, 1.5wt%, 2.0wt%, 2.5wt%, and 3.0wt%, respectively, in order to explore the effect of different inoculation amounts of strains on the quality of soy protein concentrate products.

[0088] (3) Effect of enzyme addition on the quality of soybean protein concentrate products

[0089] On the basis of the experiment in (1) above, the water content was set to 40wt% and the bacterial inoculation amount was set to 3.0wt%, and the amount of composite enzyme added was set to 100U / g (0.0796g; 0.05wt%; the amount of papain and neutral protease each accounted for half), 300U / g (0.2388g; 0.15wt%; the amount of papain and neutral protease each accounted for half), 500U / g (0.398g; 0.25wt%; the amount of papain and neutral protease each accounted for half), 1000U / g (0.796g; 0.5wt%; the amount of papain and neutral protease each accounted for half), 2000U / g (1.592g; 1.0wt%; the amount of papain and neutral protease each accounted for half) to explore the effect of different enzyme addition amounts on the quality of soy protein concentrate products.

[0090] (4) Effect of fermentation temperature on the quality of soybean protein concentrate products

[0091] Based on the experiment of (1) above, the enzyme addition amount was set to 1000U / g (0.796g; 0.5wt%; the amount of papain and neutral protease was half each), the water content was 40wt%, and the bacterial inoculation amount was 3.0wt%, and the effect of fermentation temperature on the quality of soy protein concentrate products was investigated at 30°C, 35°C, 40°C, 45°C, and 50°C.

[0092] (5) Effect of fermentation time on bioprocessing protein concentration

[0093] Based on the experiment of (1) above, the enzyme addition amount was set to 1000U / g (0.796g; 0.5wt%; the amount of papain and neutral protease was half each), the water content was 40wt%, and the bacterial inoculation amount was 3.0wt%, and the effect of fermentation time of 24h, 36h, 48h, 60h, and 72h on the quality of soy protein concentrate products was investigated.

[0094] (6) Effect of water content on bioprocessed protein concentrate

[0095] Based on the experiment of (1) above, the enzyme addition amount was set to 1000U / g (0.796g; 0.5wt%; the amount of papain and neutral protease was half each), and the bacterial inoculation amount was 3.0wt% to explore the effect of the water content of 35wt%, 40wt%, 45wt%, 50wt% and 55wt% in the fermentation matrix on the quality of the soy concentrate protein product.

[0096] Test results

[0097] (1) Effect of inoculation rate on the quality of soybean protein concentrate products

[0098] Depend on Figure 4 It can be seen that the inoculation amount of the strain has an impact on the quality of the soybean concentrate protein product. When the inoculation amount is 1.5wt%-3.0wt%, the content of small peptides is at a higher level, and soybean globulin and β-conglycinin are at a low level. Therefore, the more optimal inoculation amount is determined to be 1.5wt%-3.0wt%.

[0099] (2) Effect of compound enzyme addition on the quality of soybean protein concentrate products

[0100] Depend on Figure 5 It can be seen that the amount of compound enzyme added has an impact on the quality of soybean protein concentrate products. With the increase of enzyme addition, the content of acid-soluble protein gradually increases; when the enzyme addition amount is greater than 500U / g (0.398g; 0.25wt%; the amount of papain and neutral protease each accounts for half), soybean globulin and β-conglycinin show a downward trend; considering the increase of acid-soluble protein content and the control of soybean globulin and β-conglycinin content to the lowest, the more preferred enzyme addition amount is 2000U / g (1.592g; 1.0wt%; the amount of papain and neutral protease each accounts for half).

[0101] (3) Effect of fermentation temperature on the quality of soybean protein concentrate products

[0102] Depend on Figure 6 It can be seen that the fermentation temperature has an impact on the quality of soy protein concentrate products. As the fermentation temperature increases, the acid-soluble protein content gradually increases. The acid-soluble protein content at a fermentation temperature of 50°C is the highest (192.0 mg / g), indicating that 50°C is suitable for protease hydrolysis, and relatively high temperatures can enhance protease activity; however, when the temperature exceeds 35°C, the antigen protein content gradually decreases with increasing temperature, and is at the lowest level at 50°C. At a fermentation temperature of 50°C, although the acid-soluble protein content is high and the antigen protein is at the lowest level, it is not conducive to probiotic fermentation, so the more preferred fermentation temperature is 35°C-45°C.

[0103] (4) Effect of fermentation time on the quality of soybean protein concentrate products

[0104] Depend on Figure 7 It can be seen that the fermentation time has an impact on the quality of soybean protein concentrate products. Within a certain fermentation time, the acid-soluble protein content increases with the increase of fermentation time. After 60 hours of fermentation, the acid-soluble protein content is the highest (181.70 mg / g). The antigen protein content decreases with the continuous increase of time. When the fermentation is 60 hours, the content of soybean globulin and β-conglycinin tends to be stable and at a low level. Therefore, according to the acid-soluble protein content and the antigen protein content, the more preferred processing time is determined to be 60 hours.

[0105] (5) Effect of water content on the quality of soy protein concentrate products

[0106] Depend on Figure 8 It can be seen that the water content has an impact on the quality of soybean protein concentrate products. Figure 8 It can be seen that when the water content is greater than 40wt%, the acid-soluble protein content is relatively at a high level, and glycinin and β-conglycinin are at a low level, thus determining that the more preferred water content is 45wt%-55wt%.

[0107] Example 4 Further Optimization of Fermentation Conditions

[0108] In order to screen the optimal fermentation process conditions for soybean concentrate protein products, the amount of complex enzyme added, fermentation temperature, initial moisture content of the fermentation matrix, and bacterial inoculation amount were used as variables, and the content of acid-soluble protein and antigenic protein (soybean globulin, β-conglycinin) in the obtained soybean concentrate protein product was used as the evaluation index, and the experiment was carried out according to the fermentation method of Example 3.

[0109] After research, it was found that when the acid-soluble protein content was used as the evaluation indicator, the influencing factors were water content > fermentation temperature > enzyme addition > inoculation amount, and the optimal conditions were: compound enzyme addition 2000U / g (1.592g; 1.0wt%; the amount of papain and neutral protease each accounted for half), fermentation temperature 40°C, water content 54wt%, inoculation amount 2.25wt%.

[0110] When soybean globulin content is used as the evaluation index, the influencing factors are as follows: compound enzyme addition amount > water content > fermentation temperature > inoculation amount. The optimal conditions are: compound enzyme addition amount 2000U / g (1.592g; 1.0wt%; the amount of papain and neutral protease each accounts for half), fermentation temperature 35℃, water content 40wt%, and inoculation amount 2.25wt%.

[0111] When β-conglycinin content was used as the evaluation index, the influencing factors were, in order, compound enzyme addition amount > inoculation amount > water content > fermentation temperature. The optimal conditions were: compound enzyme addition amount 2000U / g (1.592g; 1.0wt%; the amount of papain and neutral protease each accounted for half), fermentation temperature 45°C, water content 40wt%, and inoculation amount 2.25wt%.

[0112] By analyzing the data of the above three, it was found that the fermentation temperature had a greater impact on the content of acid-soluble protein, and had a smaller impact on the content of soybean globulin and β-conglycinin. Further fermentation experiments found that when the fermentation temperature was 40°C, the content of acid-soluble protein was the highest, and when the fermentation temperature was 35°C, the content of acid-soluble protein was second, but both had a smaller impact on the content of soybean globulin and β-conglycinin. Considering the optimal fermentation temperature of yeast, the fermentation temperature is more preferably 35°C, which improves the nutritional value of the feed while having both enzymatic and fermentative hydrolysis capabilities.

[0113] When the water content is 54 wt %, it has a great influence on the acid-soluble protein content, but has a small influence on the glycinin and β-conglycinin contents, so the more preferred water content in the fermentation matrix is ​​54 wt %.

[0114] Taking into account factors such as crude protein (soybean high-temperature meal concentrated protein) content, acid-soluble protein content, KOH protein solubility, and antigen protein content, it was found that the parameters for achieving the optimal fermentation process included: compound enzyme addition of 2000U / g (1.592g; 1.0wt%; the amount of papain and neutral protease each accounted for half), fermentation temperature of 35°C, water content of 54wt%, compound bacteria inoculation amount of 2.25wt%, addition of enhanced powder of 0.95wt%, and addition of potassium sorbate of 0.05wt%. The optimal fermentation conditions for the corresponding bacterial enzyme synergy are as follows: the above-mentioned soybean high-temperature meal concentrated protein is used as the raw material, the high-temperature meal concentrated protein charge is 100g (47.38wt%), water is 100g (47.38wt%), composite protease is 2.10g (1.0wt%; the amount of neutral protease and papain is half each), the bacterial inoculation amount is 4.74mL (2.25wt%, Lactobacillus plantarum ZM3: Saccharomyces cerevisiae ZY1: Bacillus subtilis Z L1 = 10:1:20, wherein Lactobacillus plantarum 1.53mL, Saccharomyces cerevisiae 0.15mL, Bacillus subtilis 3.06mL), glucose addition 2.10g (1.0wt%), Jiayi powder addition 1.995g (0.95wt%), potassium sorbate addition 0.105g (0.05wt%), the breathing bag containing the fermentation substrate thus obtained is sealed and fermented, the fermentation temperature is 40°C, and the fermentation time is 60h. The sample after the bacterial enzyme cooperative fermentation is dried at 55°C, crushed, and passed through a 40-mesh sieve to obtain a soybean protein concentrate product for standby use.

[0115] Example 5 Fermentation Effect Evaluation

[0116] High-temperature defatted soybean meal from which high-temperature soybean meal concentrated protein is derived was used as the high-temperature soybean meal used as a control.

[0117] The high-temperature meal concentrated protein was prepared according to the preparation method of the high-temperature soybean meal concentrated protein in the examples.

[0118] Based on the basic process of bacterial-enzyme cooperative fermentation determined in Example 3, but without inoculating composite bacteria for enzymatic hydrolysis treatment, enzymatic hydrolysis concentrated protein was obtained.

[0119] Based on the basic process of bacterial-enzyme synergistic fermentation determined in Example 3, composite bacteria and composite enzymes were added according to the method described in Example 3 to perform bacterial-enzyme synergistic treatment to obtain fermentation enzymatic concentrated protein.

[0120] According to the optimal fermentation process determined in Example 4, the above-mentioned soybean high temperature meal concentrated protein is used as raw material, the addition amount is 100g (47.38wt%), the addition amount of glucose is 2.10g (1.0wt%), the addition amount of Jiayi powder is 1.995g (0.95wt%), and the addition amount of potassium sorbate is 0.105g (0.05wt%). After fermentation for 60h, the fermentation effect is evaluated. The soybean concentrated protein product is obtained by the above method.

[0121] Reference to existing literature

[14] The process is to treat with Lactobacillus plantarum ZM3, Saccharomyces cerevisiae ZY1, Bacillus subtilis ZL1 (composite strain addition amount 2.25wt%) + papain (enzyme addition amount 0.12wt%) selected by the present invention, specifically: 1) take 1000kg of high-temperature meal concentrated protein; 2) place the same composite strain as in Example 4 in about 5kg of 35°C warm water and stir for 5 minutes to activate the strain; the inoculation amount is 2.25wt% of the dry matter weight of the soybean meal raw material; 3) take papain (200,000 U / g, 1200g, the addition amount is 0.12wt% of the dry matter weight of the soybean meal raw material), place it in 2kg of 50°C warm water for enzyme activation; 4) place the activated bacterial solution and the activated enzyme solution together in 500kg After stirring evenly in 35℃ warm water, mix evenly with 1000kg soybean meal; 5) bag and place in a special breathing bag for fermenting soybean meal, 40kg per bag; 6) place at room temperature of about 30℃ for 72 hours for fermentation; 7) dry in a rotary dryer; 8) crush and pack. Comparative concentrated protein was obtained by the above method.

[0122] In addition, a similar concentrated protein product purchased from the market (its composition is shown in Table 1) was used as a control.

[0123] Fermentation effect evaluation

[0124] The results of various determinations of the concentrated proteins obtained by different treatment methods are shown in Tables 1 and 2 below. It can be seen from Tables 1 and 2 that after the high-temperature soybean meal is processed into soybean high-temperature meal concentrated protein and then treated by fermentation, enzymolysis, and bacterial enzyme synergistic process, the protein and digestion and absorption indicators are gradually improved, and the anti-nutritional factor indicators are gradually reduced. Among them, the soybean concentrated protein product obtained by the method of the present invention has the best improvement effect on various indicators (dry basis): the crude protein mass concentration is increased from 525.60 mg / g of high-temperature soybean meal to 633.30 mg / g, and the acid-soluble protein content is the highest ( reached 413.60mg / g); soybean globulin decreased from 148.80mg / g to 0.83mg / g, and β-conglycinin decreased from 100.72mg / g to 1.29mg / g. At the same time, the contents of trypsin inhibitor (not detected), lectin (0.02mg / g), stachyose (not detected), and raffinose (0.24mg / g) all dropped to lower levels; the in vitro digestibility of crude protein increased from the original 88.95% to 91.06%, and the digestibility of total amino acids increased from the original 90.52% to 91.67%.

[0125] Specifically, compared with high-temperature soybean meal, the degradation rates of the antigenic factors of the comparison concentrate protein are as follows: soybean globulin (98.49%), β-conglycinin (89.06%), stachyose (91.25%), raffinose (82.35%), trypsin inhibitor (77.94%), and lectin (52.94%). Compared with high-temperature meal concentrated protein, the degradation rates of the antigenic factors of the comparison concentrate protein are as follows: soybean globulin (85.45%), β-conglycinin (34.09%), stachyose (32.26%), raffinose (4.00%), trypsin inhibitor (90.20%), and lectin (0%).

[0126] Compared with high-temperature soybean meal, the degradation rates of various antigenic factors of the soybean concentrated protein product of the present invention are as follows: soybean globulin (99.44%), β-conglycinin (98.72%), stachyose (100%), raffinose (82.35%), trypsin inhibitor (100%), and lectin (88.24%). Compared with high-temperature meal concentrated protein, the degradation rates of various antigenic factors of the soybean concentrated protein product of the present invention are as follows: soybean globulin (94.61%), β-conglycinin (92.28%), stachyose (100%), raffinose (4%), trypsin inhibitor (100%), and lectin (66.67%).

[0127] In summary, the bacterial enzyme synergistic fermentation method of the present invention is superior to the fermentation and enzymatic hydrolysis method of concentrated protein in the prior art. The bacterial enzyme synergistic fermentation method of the present invention can not only effectively decompose soybean protein into acid-soluble protein, but also degrade a variety of anti-nutritional factors, enhance the digestibility of protein and amino acids, and thus improve the nutritional value of feed.

[0128]

[0129] Example 6 Effect of fermentation partner on fermentation effect

[0130] The method for preparing a soybean protein concentrate product according to Example 5 was carried out, except that no fermentation partner (potassium sorbate and Jiayi powder) was added, and the fermentation effect was evaluated after 60 hours of fermentation. The primary soybean protein concentrate product was obtained by the above method, and the results are shown in Table 1.

[0131] Compared with high-temperature soybean meal, the degradation rates of various antigenic factors of the primary product of soybean protein concentrate prepared without adding fermentation partner are as follows: glycinin (97.86%), β-conglycinin (90.06%), stachyose (100%), raffinose (100%), trypsin inhibitor (11.09%), and lectin (82.35%);

[0132] Compared with high-temperature meal concentrated protein, the degradation rates of various antigenic factors of the primary product of soybean concentrated protein prepared without adding fermentation partner are as follows: soybean globulin (79.34%), β-conglycinin (40.13%), stachyose (100%), raffinose (4.00%), trypsin inhibitor (95.08%), and lectin (50.00%).

[0133] Compared with the primary soy protein concentrate product prepared without adding fermentation partner, the degradation rates of various antigenic factors of the soy protein concentrate product are as follows: glycinin (73.90%), β-conglycinin (87.11%), stachyose (100%), trypsin inhibitor (100%), and lectin (33.33%). In addition, the content of acid-soluble protein was greatly increased from 230.21 mg / g to 413.60 mg / g.

[0134] In summary, adding fermentation partners (potassium sorbate and Jiayi powder) helps to break down soybean protein into acid-soluble protein, degrade various anti-nutritional factors, and enhance the digestibility of protein and amino acids, thereby improving the nutritional value of feed.

[0135] References

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[0143] [8] Ma Yuxiang, Yang Guolong, Zhou Ruibao, et al. Study on the hydrolysis of bromelain into alcohol-based soy protein concentrate [J]. Journal of Henan University of Technology, 2006(5):43-46.

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[0145]

[10] Yang Ning, Xuan Li, Liang Liping, et al. Study on the process of enzymatic hydrolysis of soy protein concentrate with low water content protease[J]. Chinese Fruit and Vegetables, 2021(3):13-18.

[0146]

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[0149]

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Claims

1. A method for producing a high-quality soybean protein concentrate product, the method comprising: Adding enzyme preparation, composite bacteria, added powder and sorbate to fermentation raw materials to obtain a fermentation matrix, and fermenting the fermentation matrix to obtain a soybean protein concentrate product; The fermentation matrix comprises soybean high-temperature meal concentrated protein and water, the enzyme preparation comprises neutral protease and papain; the composite bacteria comprises Lactobacillus plantarum, Saccharomyces cerevisiae and Bacillus subtilis, and the sorbate is one or more selected from potassium sorbate, sodium sorbate and calcium sorbate.

2. The method of claim 1, wherein: The method further comprises: adding C1-C3 alcohol to the high temperature defatted soybean meal for extraction, and then centrifuging and drying to obtain the high temperature soybean meal concentrated protein; Preferably, the water content in the fermentation substrate is 30 wt%-60 wt%.

3. The method according to claim 1 or 2, wherein: The amount of sorbate added to the fermentation substrate is 0.01wt%-0.2wt%; Preferably, the added amount of the enhanced powder in the fermentation matrix is ​​0.1wt%-2.0wt%.

4. The method according to any one of claims 1 to 3, wherein: The method further comprises adding glucose to the fermentation substrate; preferably, the amount of glucose added to the fermentation substrate is 0 wt%-2.0 wt%.

5. The method according to any one of claims 1 to 4, wherein: The mass ratio of neutral protease and papain in the enzyme preparation is (1.0-4.0):(1.0-4.0); Preferably, the added amount of the enzyme preparation is greater than 500 U / g fermentation substrate.

6. The method according to any one of claims 1 to 5, wherein: The composite bacteria are composed of Lactobacillus plantarum ZM3 (CICCR21790), Saccharomyces cerevisiae ZY1 (CGMCC 2.1638) and Bacillus subtilis ZL1 (CICC G20030); Preferably, the mass ratio of Lactobacillus plantarum ZM3, Saccharomyces cerevisiae ZY1 and Bacillus subtilis ZL1 is (1.0-10.0):(1.0-6.0):(1.0-20.0); In addition, preferably, the inoculation amount of the composite bacteria is 1.0 wt%-4.0 wt% relative to the mass of the fermentation substrate.

7. The method according to any one of claims 1 to 6, wherein: The fermentation is carried out at 30°C-50°C for 24h-72h.

8. The method according to any one of claims 1 to 7, wherein: The water content of the fermentation substrate was 54 wt %, the addition amount of the enzyme preparation was 2000 U / g fermentation substrate, the inoculation amount of the composite bacteria relative to the fermentation substrate was 2.25 wt %, and the fermentation was carried out at 35° C. for 60 h.

9. Use of the soybean protein concentrate product obtained by the method according to any one of claims 1 to 8 in preparing feed.

10. A method for preparing a fermented feed comprising a soybean protein concentrate product obtained by the method according to any one of claims 1 to 8.

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