A polypeptide-rich feed and its preparation method

Abstract

The present invention belongs to the technical field of bioengineering, and particularly relates to a polypeptide-rich feed and a preparation method thereof. By using non-starch polysaccharide degrading enzymes such as cellulase and α-galactosidase to pre-treat soybean meal, the viscosity of the feed liquid is significantly reduced and the fluidity is increased; inoculating mixed strains after enzymatic hydrolysis in an aqueous solution of soy protein concentrate not only helps to shorten the fermentation time but also improves the fermentation efficiency; by limiting the preparation of the polypeptide-rich feed under stirring conditions, it not only helps to improve the enzymatic hydrolysis and fermentation rates, but also avoids the blockage of pipelines and die holes caused by caking, greatly improving the fermentation efficiency. Therefore, the technical solution provided by the present invention has the characteristics of short time, high efficiency and high content of protein oligopeptides.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering technology, and particularly relates to a polypeptide-rich feed and a preparation method thereof. Background Art

[0002] As a by-product of soybean processing, soybean meal is inexpensive and rich in nutrients. For example, the protein content in soybean meal is 40% - 50%, the fat is 1% - 2%, and the carbohydrate is about 10% - 15%. It is currently the most widely used plant-based protein feed raw material. However, "antinutritional factors" in soybean meal, such as soybean antigens (glycinin and β-conglycinin), trypsin inhibitors, etc., affect the digestive ability of animals and increase the endogenous nitrogen loss of animals. Therefore, after directly feeding untreated soybean meal to animals, it is easy to cause problems such as diarrhea and intestinal allergy, seriously affecting the production performance of livestock and poultry and the feed utilization rate.

[0003] Currently, the methods for eliminating antinutritional factors in soybean meal mainly include: physical method, chemical method, microbial fermentation method, and enzyme preparation addition method. Although the microbial fermentation method is relatively safe and healthy, the fermentation time is long and the turnover is slow; although the enzymatic hydrolysis method can degrade non-starch polysaccharides and proteins in soybean meal, it will also increase the bitterness of the product and reduce the palatability of the feed. Summary of the Invention

[0004] The purpose of the present invention is to provide a polypeptide-rich feed and a preparation method thereof, which have the characteristics of short time, high efficiency, high content of protein small peptides, and good palatability.

[0005] The present invention provides a method for preparing a polypeptide-rich feed, comprising the following steps:

[0006] Mix an aqueous solution of soy protein concentrate, alkaline protease, and neutral protease for the first enzymatic hydrolysis, and inoculate a compound bacterium agent into the aqueous solution of soy protein concentrate after the first enzymatic hydrolysis for the first fermentation to obtain a fermented aqueous solution of soy protein concentrate after the first fermentation;

[0007] Mix an aqueous solution of soybean meal, cellulase, and α-galactosidase for the second enzymatic hydrolysis, and mix the enzymatic hydrolysate obtained from the second enzymatic hydrolysis, alkaline protease, and neutral protease for the third enzymatic hydrolysis to obtain an enzymatic hydrolysate of the aqueous solution of soybean meal;

[0008] Perform the second fermentation and drying on the fermented aqueous solution of soy protein concentrate after the first fermentation and the enzymatic hydrolysate of the aqueous solution of soybean meal to obtain the polypeptide-rich feed;

[0009] The compound bacterium agent includes yeast, lactic acid bacteria, and bacillus;

[0010] The content of crude protein in the soybean meal ≥ 46%;

[0011] The first enzymatic hydrolysis, the first fermentation, the second enzymatic hydrolysis, the third enzymatic hydrolysis and the second fermentation are all carried out under stirring conditions.

[0012] Preferably, the mass ratio of soy protein concentrate to water in the aqueous soy protein concentrate solution is 10-15:100;

[0013] During the first enzymatic hydrolysis, the addition amount of the alkaline protease is 0.1 wt.% - 0.3 wt.% of the soy protein concentrate; the addition amount of the neutral protease is 0.1 wt.% - 0.15 wt.% of the soy protein concentrate;

[0014] The conditions for the first enzymatic hydrolysis include: time is 2-6 h, and temperature is 50-55 °C.

[0015] Preferably, the inoculation amount of the compound bacterial agent is 0.1 wt.% - 0.2 wt.% of the soy protein concentrate in the aqueous soy protein concentrate solution;

[0016] The viable count of yeast in the compound bacterial agent ≥ 4×10 9 CFU / g, the viable count of lactic acid bacteria ≥ 6×10 9 CFU / g, the viable count of bacillus ≥ 6×10 9 CFU / g.

[0017] Preferably, the conditions for the first fermentation include: temperature is 35-38 °C, and time is 8-16 h.

[0018] Preferably, the mass ratio of soybean meal to water in the aqueous soybean meal solution is 25-30:100;

[0019] During the second enzymatic hydrolysis, the addition amounts of the cellulase and α-galactosidase are respectively 0.05 wt.% - 0.1 wt.% of the soybean meal in the aqueous soybean meal solution;

[0020] The conditions for the second enzymatic hydrolysis include: temperature is 50-55 °C, and time is 1-2 h.

[0021] Preferably, during the third enzymatic hydrolysis, the addition amounts of the alkaline protease and neutral protease are respectively 0.1 wt.% - 0.3 wt.% of the soybean meal in the enzymatic hydrolysate obtained from the second enzymatic hydrolysis;

[0022] The conditions for the third enzymatic hydrolysis include: time is 3-5 h, and temperature is 50-55 °C.

[0023] Preferably, during the second fermentation, the volume ratio of the aqueous soy protein concentrate solution after the first fermentation to the aqueous soybean meal solution after enzymatic hydrolysis is 1:5;

[0024] The conditions for the second fermentation include: temperature is 35-38 °C, and time is 2-4 h.

[0025] Preferably, the stirring speed is 20 - 40 rpm.

[0026] Preferably, the drying conditions include: inlet air temperature of 170 - 180 °C, outlet air temperature of 80 - 85 °C, and time of 7 - 10 s; during drying, the frequency of the atomizer frequency converter is 42 - 48 Hz.

[0027] The present invention provides a polypeptide-rich feed prepared by the method described in the above technical solution, characterized in that the acid-soluble protein content in the polypeptide-rich feed is ≥ 56%, the crude protein content is ≥ 50%, and the pH ≤ 5.0.

[0028] Beneficial effects:

[0029] The present invention provides a method for preparing a polypeptide-rich feed, comprising the following steps: mixing a soy protein concentrate aqueous solution, an alkaline protease, and a neutral protease for first enzymatic hydrolysis, and inoculating a compound bacterium agent into the soy protein concentrate aqueous solution after the first enzymatic hydrolysis for fermentation to obtain a first fermented soy protein concentrate aqueous solution; mixing a soybean meal aqueous solution, a cellulase, and an α-galactosidase for second enzymatic hydrolysis, mixing the enzymatic hydrolysate obtained from the second enzymatic hydrolysis, an alkaline protease, and a neutral protease for third enzymatic hydrolysis to obtain an enzymatic hydrolyzed soybean meal aqueous solution; performing second fermentation and drying on the first fermented soy protein concentrate aqueous solution and the enzymatic hydrolyzed soybean meal aqueous solution to obtain the polypeptide-rich feed; the compound bacterium agent includes yeast, lactic acid bacteria, and bacillus; the crude protein content of the soybean meal is ≥ 46%; the first enzymatic hydrolysis, fermentation, second enzymatic hydrolysis, third enzymatic hydrolysis, and second fermentation are all carried out under stirring conditions. By using non-starch polysaccharide degrading enzymes such as cellulase and α-galactosidase to pre-treat the soybean meal, the present invention significantly reduces the viscosity of the feed liquid and increases the fluidity; by inoculating a mixed strain after enzymatic hydrolysis in the soy protein concentrate aqueous solution, it not only helps to shorten the fermentation time but also improves the fermentation efficiency; by limiting the preparation of the polypeptide-rich feed under stirring conditions, it not only helps to improve the enzymatic hydrolysis and fermentation speed but also avoids blockage of pipelines and die holes caused by caking, greatly improving the fermentation efficiency.

[0030] Based on the above technical advantages, the present invention also provides a polypeptide-rich feed prepared by the above technical solution, wherein the acid-soluble protein in the polypeptide-rich feed is ≥ 56%, the crude protein content is ≥ 50%, and the pH ≤ 5.0. Experiments prove that by adopting the technical solution provided by the present invention, soybean protein antigens and anti-nutritional factors are completely eliminated. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0032] Figure 1This is the gel electrophoresis antigen detection diagram of the obtained finished product in Example 9. Detailed implementation method

[0033] The present invention provides a method for preparing a polypeptide-rich feed, which includes the following steps: mixing a soy protein concentrate aqueous solution, an alkaline protease, and a neutral protease for the first enzymatic hydrolysis, and inoculating a compound bacterium agent into the soy protein concentrate aqueous solution after the first enzymatic hydrolysis for fermentation to obtain a first fermented soy protein concentrate aqueous solution; mixing a soybean meal aqueous solution, a cellulase, and an α-galactosidase for the second enzymatic hydrolysis, mixing the enzymatic hydrolysate obtained from the second enzymatic hydrolysis, an alkaline protease, and a neutral protease for the third enzymatic hydrolysis to obtain an enzymatic hydrolyzed soybean meal aqueous solution; performing a second fermentation and drying on the first fermented soy protein concentrate aqueous solution and the enzymatic hydrolyzed soybean meal aqueous solution to obtain the polypeptide-rich feed; the compound bacterium agent includes yeast, lactic acid bacteria, and bacillus; the content of crude protein in the soybean meal is ≥46%; the first enzymatic hydrolysis, the first fermentation, the second enzymatic hydrolysis, the third enzymatic hydrolysis, and the second fermentation are all carried out under stirring conditions.

[0034] In the present invention, unless otherwise specified, the corresponding raw materials used are all purchased conventionally.

[0035] The present invention preferably mixes soy protein concentrate with water. In the present invention, the mass ratio of soy protein concentrate to water during the mixing is preferably 10-15:100, more preferably 12:100; the protein content of the soy protein concentrate is preferably ≥65%. By adding soy protein concentrate, it is beneficial to make the solution rich and comprehensive in nutrition after enzymatic hydrolysis and achieve rapid propagation. The temperature of the water during the mixing in the present invention is preferably 50-60°C, more preferably 55°C; there are no special requirements for the mixing method and equipment, and the well-known methods and equipment in the art can be used.

[0036] After the mixing, the present invention mixes the soy protein concentrate aqueous solution, an alkaline protease, and a neutral protease for the first enzymatic hydrolysis. During the first enzymatic hydrolysis, the addition amount of the alkaline protease in the present invention is preferably 0.1 wt.% - 0.3 wt.% of the soy protein concentrate, more preferably 0.25 wt.%; the enzyme activity of the alkaline protease is preferably 200,000 - 250,000 U / g, more preferably 250,000 U / g; the addition amount of the neutral protease is preferably 0.1 wt.% - 0.15 wt.% of the soy protein concentrate, more preferably 0.15 wt.%; the enzyme activity of the neutral protease is preferably 40,000 - 50,000 U / g, more preferably 50,000 U / g. By adding the alkaline protease and the neutral protease, it is beneficial to the efficiency of protein decomposition into polypeptides.

[0037] The first enzymatic hydrolysis of the present invention is carried out under stirring conditions. The stirring speed is preferably 20-40 rpm, more preferably 32 rpm; the time of the first enzymatic hydrolysis is preferably 2-6 h, more preferably 4 h, and the temperature is preferably 50-60 °C, more preferably 55 °C; the pH value of the solution during the first enzymatic hydrolysis is preferably 6.5-7.0, more preferably 7.0. Reasonably regulating the conditions of the first enzymatic hydrolysis is beneficial to the uniform mixing of materials and more sufficient enzymatic hydrolysis reaction.

[0038] After the first enzymatic hydrolysis, the present invention inoculates a compound bacterium agent into the aqueous solution of soy protein concentrate after the first enzymatic hydrolysis for the first fermentation; the compound bacterium agent includes yeast, lactic acid bacteria and bacillus. The present invention preferably inoculates when the temperature of the aqueous solution of soy protein concentrate after the first enzymatic hydrolysis is 35-38 °C, more preferably when the temperature is 38 °C; the inoculation amount of the compound bacterium agent is preferably 0.1 wt.% - 0.2 wt.% of the soy protein concentrate in the aqueous solution of soy protein concentrate after the first enzymatic hydrolysis, more preferably 0.2 wt.%; the viable count of yeast in the compound bacterium agent is preferably ≥4×10 9 CFU / g, the viable count of lactic acid bacteria is preferably ≥6×10 9 CFU / g, and the viable count of bacillus is preferably ≥6×10 9 CFU / g. Adding yeast, lactic acid bacteria and bacillus is beneficial to increasing the aromatic flavor of the product, improving the taste of the product, increasing the palatability, and significantly degrading anti-nutritional factors such as oligosaccharides in soybean meal.

[0039] The first fermentation of the present invention is carried out under stirring conditions. The stirring speed is preferably 20-40 rpm, more preferably 32 rpm; the temperature of the first fermentation is preferably 35-38 °C, more preferably 38 °C, and the time is preferably 8-16 h, more preferably 14 h. Through the first fermentation, an aqueous solution of soy protein concentrate after the first fermentation can be prepared, which has a high concentration and a large number of proliferated microbial flora, and is beneficial to quickly reaching the fermentation end point during the second fermentation.

[0040] While preparing the aqueous solution of soy protein concentrate after the first fermentation, the present invention preferably mixes the crushed soybean meal with water. In the present invention, the crude protein content of the soybean meal ≥46%; the particle size of the crushed soybean meal is preferably 60-80 mesh, more preferably 80 mesh; the mass ratio of the crushed soybean meal to water is preferably 25-30:100, more preferably 25:100; the temperature of the water during the mixing is preferably 50-60 °C, more preferably 55 °C; the stirring speed of the mixing is preferably 20-40 rpm, more preferably 32 rpm; there is no special requirement for the stirring time, and it is subject to uniform stirring.

[0041] After mixing, the present invention mixes the soybean meal aqueous solution, cellulase and α-galactosidase for the second enzymatic hydrolysis. During the second enzymatic hydrolysis, the addition amount of the cellulase of the present invention is preferably 0.05 wt.% to 0.1 wt.%, more preferably 0.8 wt.%, based on the soybean meal in the soybean meal aqueous solution; the cellulase activity is preferably 19,000 to 20,000 U / g, more preferably 20,000 U / g; the addition amount of the α-galactosidase is preferably 0.05 wt.% to 0.1 wt.%, more preferably 0.1 wt.%, based on the soybean meal in the soybean meal aqueous solution; the α-galactosidase activity is preferably 2000 to 3000 U / g, more preferably 3000 U / g. Adding cellulase and α-galactosidase is beneficial to degrade non-starch polysaccharides in soybean meal, reduce the effect of anti-nutritional factors, and at the same time can also reduce the viscosity of the feed liquid.

[0042] The second enzymatic hydrolysis of the present invention is carried out under stirring conditions. The stirring speed is preferably 20 to 40 rpm, more preferably 32 rpm; the time of the second enzymatic hydrolysis is preferably 1 to 2 h, more preferably 2 h, and the temperature is preferably 50 to 60 °C, more preferably 55 °C; the pH value of the solution during the second enzymatic hydrolysis is preferably 6.5 to 7.0, more preferably 7.0.

[0043] After the second enzymatic hydrolysis, the present invention mixes the enzymatic hydrolysate obtained from the second enzymatic hydrolysis, alkaline protease and neutral protease for the third enzymatic hydrolysis. During the third enzymatic hydrolysis, the addition amount of the alkaline protease of the present invention is preferably 0.1 wt.% to 0.3 wt.%, more preferably 0.25 wt.%, based on the soybean meal in the soybean meal aqueous solution; the alkaline protease activity is preferably 200,000 to 250,000 U / g, more preferably 250,000 U / g; the addition amount of the neutral protease is preferably 0.1 wt.% to 0.3 wt.%, more preferably 0.25 wt.%, based on the soybean meal in the soybean protein concentrate aqueous solution; the neutral protease activity is preferably 40,000 to 50,000 U / g, more preferably 50,000 U / g. After adding alkaline protease and neutral protease, it is beneficial to improve the enzymatic hydrolysis efficiency and increase the polypeptide content.

[0044] The third enzymatic hydrolysis of the present invention is carried out under stirring conditions. The stirring speed is preferably 20 to 40 rpm, more preferably 32 rpm; the time of the third enzymatic hydrolysis is preferably 3 to 5 h, more preferably 5 h, and the temperature is preferably 50 to 60 °C, more preferably 55 °C; the pH value of the solution during the third enzymatic hydrolysis is preferably 6.5 to 7.0, more preferably 7.0. After the third enzymatic hydrolysis, the enzymatically hydrolyzed soybean meal aqueous solution is prepared, which is beneficial to the rapid and large-scale reproduction of microorganisms and quickly reaches the fermentation end point.

[0045] After the third enzymatic hydrolysis, the present invention mixes the aqueous solution of fermented soy protein concentrate after the first fermentation with the aqueous solution of soybean meal after enzymatic hydrolysis for the second fermentation. When mixing, it is preferably carried out when the temperature of the aqueous solution of soybean meal after enzymatic hydrolysis is 35-38 °C, more preferably when the temperature of the aqueous solution of soybean meal is 38 °C; when mixing, the volume ratio of the aqueous solution of fermented soy protein concentrate after the first fermentation to the aqueous solution of soybean meal after enzymatic hydrolysis is preferably 1:3-5, more preferably 1:5.

[0046] The second fermentation of the present invention is carried out under stirring conditions. The stirring speed is preferably 20-40 rpm, more preferably 32 rpm; the time of the second fermentation is preferably 2-4 h, more preferably 3 h, and the temperature is preferably 35-38 °C, more preferably 38 °C. After the second fermentation, it is beneficial to thoroughly degrade anti-nutritional factors such as soybean antigens and oligosaccharides in soybean meal, increase the fermented fragrance and improve palatability.

[0047] The first enzymatic hydrolysis and the first fermentation of the present invention are preferably carried out in the same container; the second enzymatic hydrolysis, the third enzymatic hydrolysis and the second fermentation are preferably carried out in the same container for fermentation; the container is preferably a container with a breathing port. The present invention has no limitation on the source of the fermentation container, and it can be purchased conventionally.

[0048] After the second fermentation, the present invention preferably dries the liquid after the second fermentation. When drying, the inlet air temperature is preferably 170-180 °C, more preferably 180 °C, the outlet air temperature when drying is preferably 80-85 °C, the drying time of the material in the drying tower is preferably 7-10 s, and the frequency of the atomizer frequency converter when drying is preferably 42-48 Hz. By reasonably regulating the drying conditions, the biological activity of the material is maximally maintained, which is beneficial to maintaining its color, taste, fragrance and solubility.

[0049] The present invention provides a polypeptide-rich feed prepared by the method according to the above technical solution. In the polypeptide-rich feed, the acid-soluble protein ≥ 56%, the crude protein content ≥ 50%, and the pH ≤ 5.0. Experiments have proved that in the polypeptide-rich feed prepared by the present invention, soybean protein antigens and anti-nutritional factors are completely eliminated, which not only reduces the cost of the feed.

[0050] In order to further illustrate the present invention, the following describes in detail a polypeptide-rich feed and its preparation method provided by the present invention with reference to the drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Experimental Example 1

[0052] The influence of enzyme preparations on the enzymatic hydrolysis effect of soybean meal, the steps are as follows:

[0053] 1) Take 100 g of soybean meal passing through 80 mesh and dissolve it in 400 g of water to obtain a soybean meal aqueous solution. The physicochemical indexes of the soybean meal are as follows: the content of crude protein in the soybean meal ≥ 46%, crude ash ≤ 7%, moisture ≤ 13%, and the manufacturer is Henan Sunshine Oil Group Co., Ltd.;

[0054] 2) Add 1.74 g of Compound Protease No. 1 (the addition amount of Compound Protease No. 1 is 1.74 wt.% of the soybean meal in step 1)) to the soybean meal aqueous solution in step 1), and adjust the pH of the soybean meal aqueous solution to 8.0 with sodium hydroxide solution to obtain a soybean meal aqueous solution containing enzyme preparation; among them, the mass ratio of alkaline protease and neutral protease in Compound Protease No. 1 is 7:3, that is, it consists of 1.218 g of alkaline protease and 0.522 g of neutral protease; the enzyme activity of alkaline protease is 250,000 U / g, and the enzyme activity of neutral protease is 50,000 U / g; both alkaline protease and neutral protease are purchased from Nanning Pangbo Bioengineering Co., Ltd.;

[0055] 3) Place the soybean meal aqueous solution containing enzyme preparation in step 2) in a water bath at 55 °C, then stir with a stirrer, observe the enzymolysis rate, and after enzymolysis for 6 h, place it in a constant temperature drying oven at 65 °C and dry overnight, and then pulverize it to measure crude protein, ash, moisture, acid-soluble protein and protein molecular weight distribution.

[0056] Experimental Example 2

[0057] The difference from Example 1 is that in step 2), 1.74 g of Compound Protease No. 2 (the addition amount of Compound Protease No. 2 is 1.74 wt.% of the soybean meal in step 1)) is added, and the pH of the soybean meal aqueous solution is adjusted to 8.0 with sodium hydroxide solution to obtain a soybean meal aqueous solution containing enzyme preparation; among them, the mass ratio of alkaline protease and neutral protease in Compound Protease No. 2 is 7:3, that is, it consists of 1.218 g of alkaline protease and 0.522 g of neutral protease; the enzyme activity of alkaline protease is 250,000 U / g, and the enzyme activity of neutral protease is 50,000 U / g; both alkaline protease and neutral protease are purchased from Wuhan New Huayang Biological Co., Ltd.

[0058] Experimental Example 3

[0059] The difference from Example 1 is that in step 2), 1.74 g of hydrolase is added, and the pH of the soybean meal aqueous solution does not need to be adjusted to obtain a soybean meal aqueous solution containing enzyme preparation. The hydrolase is purchased from Tianjin Nuoao Technology Development Co., Ltd. The enzyme activity of alkaline protease is 250,000 U / g, and the enzyme activity of neutral protease is 50,000 U / g.

[0060] Experimental Example 4

[0061] The difference from Example 1 is that in step 2), 1.74 g of Compound Protease No. 3 is added (the addition amount of Compound Protease No. 3 is 1.74 wt.% of the soybean meal in step 1)), and it is not necessary to adjust the pH of the soybean meal aqueous solution to obtain a soybean meal aqueous solution containing the enzyme preparation; the mass ratio of alkaline protease to neutral protease in Compound Protease No. 3 is 1:1, that is, it consists of 0.87 g of alkaline protease and 0.87 g of neutral protease; the enzyme activity of alkaline protease is 250,000 U / g, and the enzyme activity of neutral protease is 50,000 U / g; both alkaline protease and neutral protease are purchased from Nanning Donghenghuadao Biotechnology Co., Ltd.

[0062] Experimental Example 5

[0063] The difference from Example 1 is that in step 2), 1.74 g of Compound Protease No. 4 is added (the addition amount of Compound Protease No. 4 is 1.74 wt.% of the soybean meal in step 1)), and it is not necessary to adjust the pH of the soybean meal aqueous solution to obtain a soybean meal aqueous solution containing the enzyme preparation; the mass ratio of alkaline protease to neutral protease in Compound Protease No. 4 is 1:1, that is, it consists of 0.87 g of alkaline protease and 0.87 g of neutral protease; the enzyme activity of alkaline protease is 250,000 U / g, and the enzyme activity of neutral protease is 50,000 U / g; the alkaline protease is purchased from Henan Yangshao Biochemical Engineering Co., Ltd., and the neutral protease is purchased from Nanning Donghenghuadao Biotechnology Co., Ltd.

[0064] Results and Analysis

[0065] 1) Analyze the enzymatic hydrolysis fluidity of Experimental Examples 1 - 5. Use a calibrated viscometer to measure the viscosity of the material. The calibration method of the viscometer is to measure the viscosity of water at 25°C as 2 mPa·s. The results are shown in Table 1 (0 h in Table 1 refers to the moment when the enzyme preparation is added).

[0066] Table 1 Observation of Fluidity during Enzymatic Hydrolysis

[0067]

[0068] It can be seen from Table 1 that as the addition amount of alkaline protease in the formula increases, the viscosity of the feed liquid gradually becomes thinner; in the experiments with the same enzyme preparation formula, after adjusting the pH to 8.0 with sodium hydroxide, the viscosity of the feed liquid also slightly decreases, and the fluidity increases.

[0069] Measure the crude protein, crude ash, moisture, acid-soluble protein of the products after enzymatic hydrolysis and comminution in Experimental Examples 1 - 5, and the HPLC analysis refers to the standard NYT2218 - 2012. The specific results are shown in Table 2.

[0070] Table 2 Influence of Different Enzyme Preparations on Product Properties after Enzymatic Hydrolysis

[0071]

[0072] As can be seen from Table 2, the acid-soluble eggs in groups 1 and 2 adjusted to pH 8.0 are higher than those in the control groups 4 and 5. In the molecular weight distribution of HPLC, the corresponding molecular weight of the pH 8.0 group is smaller. It was also found that alkaline enzymes act quickly, and when the addition ratio of alkaline enzymes to neutral enzymes is close to 7:3, the enzymatic effect and speed can be significantly improved, but the differences between enzymes from different manufacturers are not obvious.

[0073] Example 1

[0074] A method for preparing polypeptide-rich feed, comprising the following steps:

[0075] 1) Preparation of strain propagation materials: Weigh 400 kg of soybean protein concentrate (protein content ≥ 65%, Shandong Yuwang Industrial Co., Ltd.), add 55°C water into a reaction tank to mix into a 15% liquid by mass, add 0.3wt.% alkaline protease (enzyme activity 250,000 units) and 0.15wt.% neutral protease (enzyme activity 50,000 units) of the soybean protein concentrate, stir and enzymolyze at 55°C and 32rpm for 2h (alkaline protease and neutral protease were purchased from Henan Yangshao Bioengineering Co., Ltd.);

[0076] 2) Inoculation and propagation: After the enzymatic hydrolysis is completed, the material obtained in step 1) is cooled to 38°C and inoculated with a composite bacterial agent composed of yeast, lactic acid bacteria and Bacillus. The inoculation amount of the composite bacterial agent is 0.1wt.% of the soybean protein concentrate in step 1); the number of live yeast cells in the composite bacterial agent is ≥4×10 9 CFU / g, the number of viable lactic acid bacteria ≥ 6×10 9 CFU / g, the number of viable Bacillus ≥ 6×10 9 CFU / g, yeast, lactic acid bacteria and bacillus were purchased from Qingdao Weilan Biotechnology Co., Ltd., and fermented at 32 rpm (fermentation temperature was 38 °C, time was 12 h), and the fermented soybean protein concentrate aqueous solution was obtained for use;

[0077] 3) Take 2000 kg of soybean meal (purchased from Jiangsu Huifu Protein Technology Co., Ltd.; soybean meal crude protein 46%) after crushing and sieving through a 60-mesh sieve, and mix it in a reaction tank with 55°C water to make a feed liquid with a mass ratio of soybean meal of 25%, and stir it evenly;

[0078] 4) Add 0.1wt.% of cellulase (enzyme activity 20,000 units) and 0.1wt.% of α-galactosidase (enzyme activity 250U / mg) of soybean meal to the liquid obtained in step 3), stir and enzymolyze at 55°C and 32rpm for 1h to obtain an enzymolyzed soybean meal aqueous solution; (cellulase and α-galactosidase were purchased from Jiangsu Yinong Biological Co., Ltd.)

[0079] 5) Add alkaline protease (enzyme activity: 250,000 units) at 0.3 wt.% of the soybean meal and neutral protease (enzyme activity: 50,000 units) at 0.15 wt.% of the soybean meal to the aqueous solution of soybean meal after enzymatic hydrolysis in step 4), and stir and hydrolyze at 55 °C and 32 rpm for 2 h to obtain the aqueous solution of soybean meal after enzymatic hydrolysis;

[0080] 6) Cool the aqueous solution of soybean meal in step 5) to 35 - 38 °C and set aside;

[0081] 7) Mix the aqueous solution of soybean meal obtained in step 6) and the aqueous solution of fermented soy protein concentrate obtained in step 2) at a volume ratio of 5:1, and stir and ferment at 32 rpm for 5 h, and the fermentation temperature is 38 °C;

[0082] 8) Spray-dry the product after fermentation in step 7) (specific conditions during drying: inlet air temperature is 170 - 180 °C, outlet air temperature is 80 - 85 °C, and the atomizer frequency is 44 Hz), pass through a magnetic separator and a 60-mesh sieve to remove impurities to obtain the finished product.

[0083] Example 2

[0084] The difference from Example 1 is that in step 5), the enzymatic hydrolysis is carried out for 4 h; in step 7), the fermentation is carried out for 3 h.

[0085] Example 3

[0086] The difference from Example 1 is that in step 5), the enzymatic hydrolysis is carried out for 5 h; in step 7), the fermentation is carried out for 4 h.

[0087] Comparative Example 1

[0088] The difference from Example 1 is that in step 2), only lactic acid bacteria are inoculated, and the inoculation amount is 50 g / ton of material; the viable count of lactic acid bacteria ≥ 6×10 9 CFU / g.

[0089] Comparative Example 2

[0090] The difference from Example 1 is that in step 2), only a compound bacterium agent composed of yeast and bacillus is inoculated, and the inoculation amount is 200 g / ton of material; the viable count of yeast in the compound bacterium agent ≥ 4×10 9 CFU / g, and the viable count of bacillus ≥ 6×10 9 CFU / g.

[0091] Comparative Example 3

[0092] The difference from Example 1 is that in step 7), the fermentation temperature is 36 °C.

[0093] Comparative Example 4

[0094] The difference from Example 1 is that in step 7), the fermentation temperature is 42 °C.

[0095] Comparative Example 5

[0096] It is different from Example 1 in that step 4) is omitted, that is, cellulase and α-galactosidase are not added.

[0097] Comparative Example 6

[0098] It is different from Example 1 in that the aqueous solution of soy protein concentrate obtained after fermentation of the aqueous solution of soybean meal in step 7) is in a volume ratio of 3:1

[0099] Comparative Example 7

[0100] It is different from Example 1 in that the process of stirring is not involved in steps 1) to 8).

[0101] Comparative Example 8

[0102] It is different from Example 1 in that this experiment starts from step 3). When reaching step 7), the propagated strains in step 2) are not added, but the strains are directly added according to the amount in step 1), and fermented for 12 hours.

[0103] Results and Analysis:

[0104] 1) The finished products prepared in Examples 1 to 3 and Comparative Examples 1 to 8 were tested, and the test indexes are shown in Table 3.

[0105] Table 3 Nutritional indexes of finished products prepared by different treatment methods

[0106]

[0107] As can be seen from Table 3, when only 1 to 2 strains were used in Comparative Examples 1 to 2, the soybean antigen was not completely eliminated; in Comparative Examples 3 to 4, the fermentation temperature was 36 to 42 °C, and the pH of the product was slightly higher than that of Example 3; in Comparative Examples 5 to 6, without cellulase and galactosidase, the acid-soluble protein of the product was significantly lower than that of Example 3. In the process of Example 1 with an enzymatic hydrolysis time of 2 h, even if the later fermentation process was extended, the acid-soluble protein of the final product was lower than that of Example 2 and Example 3, indicating insufficient hydrolysis degree; in Example 2 with an enzymatic hydrolysis time of 4 h and the fermentation time shortened to 3 h, the pH of the product was on the high side, indicating that the effect of the fermentation process was not achieved, and the electrophoresis test results also showed that the fermentation time needed to be extended; the acid-soluble protein of Example 3 exceeded 56% and the pH was lower than 5.0, which was the preferred scheme of the present invention.

[0108] 2) The stachyose and raffinose contents of the finished product prepared in Example 3 were inspected (the inspection was entrusted to Eurofins Analytical Technology Services (Suzhou) Co., Ltd.), and compared with the average contents of stachyose and raffinose in the known fermented soybean meal. The results are shown in Table 4. (Among them, the source of the average contents of stachyose and raffinose in the fermented soybean meal can be found in the well-known literature: Li Ying, Han Yunsheng, Zhao Qingyu, etc. Comparative Analysis of the Main Nutritional Components, Anti-nutritional Factors and in vitro Digestibility between Soybean Meal and Fermented Soybean Meal, DOI10.15906 / j.cnki.cn11-2975 / s.20192318).

[0109] Table 4 Determination of Stachyose and Raffinose Contents in Example 3

[0110]

[0111] As can be seen from Table 4, the enzymolysis fermentation protein has a very good degradation effect on stachyose and raffinose, reaching a level lower than the HPLC detection quantification limit.

[0112] Example 9

[0113] The technical solution of Example 3 was used to carry out enzymolysis fermentation on peeled soybean meal for a total of 5 batches (the numbers are 20200604, 20200605, 20200606, 20200607, and 20200608), and gel electrophoresis antigen detection was carried out on the finished products of the 5 batches (with the soybean meal raw material as the control). The results are shown in Figure 1 .

[0114] From Figure 1 it can be seen that compared with the unfermented soybean meal, the antigen protein band colors of each batch of products are very light, and only the outline can be vaguely distinguished. This shows that after adopting the technical solution provided by the invention, the inhibitory factors such as β-conglycinin, α-globulin and trypsin in the soybean meal have been completely degraded.

[0115] In summary, after enzymolysis fermentation of soybean meal by adopting the technical solution provided by the present invention, the contents of crude protein and acid-soluble protein in the product are both > 50%, and substances such as stachyose and raffinose are completely eliminated. Therefore, the polypeptide-rich feed prepared by the present invention is not only simple to operate but also has a high polypeptide content.

[0116] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments without creative work based on this embodiment, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A method for preparing a polypeptide-rich feed, characterized in that, The steps are as follows: Mix the aqueous solution of soy protein concentrate, alkaline protease and neutral protease for the first enzymatic hydrolysis, and inoculate a compound microbial agent into the aqueous solution of soy protein concentrate after the first enzymatic hydrolysis for the first fermentation to obtain the aqueous solution of soy protein concentrate after the first fermentation; the mass ratio of soy protein concentrate to water in the aqueous solution of soy protein concentrate is 10-15:100; the addition amount of the alkaline protease is 0.1wt.% - 0.3wt.% of the soy protein concentrate; the addition amount of the neutral protease is 0.1wt.% - 0.15wt.% of the soy protein concentrate; the time of the first enzymatic hydrolysis is 2-6h, and the temperature is 50-55°C; the inoculation amount of the compound microbial agent is 0.1wt.% - 0.2wt.% of the soy protein concentrate in the aqueous solution of soy protein concentrate; The composite bacterial agent is composed of yeast, lactic acid bacteria and bacillus; the viable count of yeast in the composite bacterial agent is ≥ 4×10 9 CFU / g, the viable count of lactic acid bacteria is ≥ 6×10 9 CFU / g, and the viable count of bacillus is ≥ 6×10 9 CFU / g; the temperature of the first fermentation is 35-38°C and the time is 8-16h; Mix the aqueous solution of soybean meal, cellulase and α-galactosidase for the second enzymatic hydrolysis, and mix the enzymatic hydrolysate obtained from the second enzymatic hydrolysis, alkaline protease and neutral protease for the third enzymatic hydrolysis to obtain the enzymatic hydrolysate of the aqueous solution of soybean meal; the mass ratio of soybean meal to water in the aqueous solution of soybean meal is 25-30:100; the addition amounts of the cellulase and α-galactosidase are respectively 0.05wt.% - 0.1wt.% of the soybean meal; the temperature of the second enzymatic hydrolysis is 50-55°C, and the time is 1-2h; during the third enzymatic hydrolysis, the addition amounts of the alkaline protease and neutral protease are respectively 0.1wt.% - 0.3wt.% of the soybean meal; the time of the third enzymatic hydrolysis is 3-5h, and the temperature is 50-55°C; Perform the second fermentation and drying on the aqueous solution of soy protein concentrate after the first fermentation and the enzymatic hydrolysate of the aqueous solution of soybean meal to obtain the polypeptide-rich feed; during the second fermentation, the volume ratio of the aqueous solution of soy protein concentrate after the first fermentation to the enzymatic hydrolysate of the aqueous solution of soybean meal is 1:3-5; The conditions of the second fermentation include: the temperature is 35-38°C, and the time is 2-4h; The crude protein content in the soybean meal is ≥46%; The first enzymatic hydrolysis, the first fermentation, the second enzymatic hydrolysis, the third enzymatic hydrolysis and the second fermentation are all carried out under stirring conditions.

2. The method according to claim 1, wherein The time of the third enzymatic hydrolysis is 4h; the time of the second fermentation is 3h.

3. The method according to claim 1, wherein The stirring speed is 20-40rpm.

4. The method according to claim 1, wherein The conditions of the drying include: the inlet air temperature is 170-180°C, the outlet air temperature is 80-85°C, and the time is 7-10s; during the drying, the frequency of the atomizer frequency converter is 42-48Hz.

5. The polypeptide-rich feed prepared by the method according to any one of claims 1 to 4, characterized in that, The acid-soluble protein content in the polypeptide-rich feed is ≥56%, and the crude protein content is ≥50%.

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