Low-temperature refrigeration method for preparing soybean protein peptide through enzymolysis and application of low-temperature refrigeration method

Abstract

The invention relates to the technical field of plant polypeptide, and discloses a low-temperature refrigeration method for preparing soybean protein peptide through enzymolysis and application of the low-temperature refrigeration method. Soybean protein is subjected to enzymolysis by simultaneously fixing alkaline protease and bromelain with sodium alginate, then enzymolysis is performed by respectively fixing pepsase and trypsin to prepare soybean protein peptide, and the soybean protein peptide can be added to pig feed. According to the low-temperature refrigeration method, active ingredients in soybeans can be effectively reserved, meanwhile, the inhibition effect of a soybean trypsin inhibitor on trypsin is prevented, and meanwhile generation of bitter peptides is reduced; the obtained soybean protein peptide has the effect of inhibiting swine pathogenic bacteria such as streptococcus suis and clostridium perfringens. The compound can be used as a medicine for treating bacterial infection of pigs, replaces traditional antibiotics, avoids the problems of drug resistance and food safety, and realizes healthy breeding and benefit improvement.

Description

Technical Field

[0001] This application belongs to the field of plant polypeptide technology, and in particular relates to a low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis and its application. Background Technology

[0002] Currently, the industry mainly uses two methods to process enzymatic hydrolysis products: ① Room temperature storage: This is only suitable for products with high doses of preservatives (such as sodium benzoate and potassium sorbate), and the shelf life is less than 7 days, and there are also food safety controversies; ② Freezing (below -18℃): Although it can effectively inhibit microbial and oxidation reactions, the ice crystals formed during freezing will destroy the spatial structure of peptide molecules, resulting in decreased solubility of the product after thawing (solubility decreases by 5%-12%) and stratification precipitation. Moreover, the energy consumption and storage cost of freezing are 3-5 times that of low-temperature refrigeration, which is not suitable for large-scale continuous production scenarios.

[0003] Existing low-temperature refrigeration methods (4-8℃) are not optimized for the characteristics of soybean protein peptides and have three main shortcomings: First, the temperature range design is unreasonable: it does not consider the minimum growth temperature of psychrophilic microorganisms (some Pseudomonas bacteria can still reproduce at 2℃), and temperatures close to the freezing point (0℃) easily lead to localized freezing, compromising product stability. Second, pretreatment processes are lacking: the enzymatic hydrolysate is not pretreated by filtration to remove enzymes, desalination (removing free salt ions generated by hydrolysis to reduce microbial osmotic pressure adaptation), or the addition of natural antioxidants (such as vitamin E and tea polyphenols), and direct refrigeration will accelerate deterioration. Third, storage conditions are not adequately controlled: oxygen concentration (e.g., vacuum packaging or nitrogen-filled packaging) and light exposure (ultraviolet light promotes peptide bond breakage) are not controlled during refrigeration, further exacerbating activity loss and flavor deterioration.

[0004] In summary, given the nutritional characteristics and stability deficiencies of enzymatically hydrolyzed soybean protein peptides, there is an urgent need to develop an optimized low-temperature refrigeration method. Summary of the Invention

[0005] This application aims to provide a low-temperature refrigeration method for the enzymatic hydrolysis preparation of soybean protein peptides and its application. Soybean protein is enzymatically hydrolyzed by simultaneously immobilizing alkaline protease and bromelain with sodium alginate, and then further hydrolyzed by immobilized pepsin and trypsin to prepare soybean protein peptides. These peptides can be used as an additive in pig feed. This method effectively retains the active ingredients in soybeans while preventing the inhibitory effect of soybean trypsin inhibitors on trypsin. The resulting soybean protein peptides have the effect of inhibiting pathogenic swine bacteria such as Streptococcus suis and Clostridium perfringens.

[0006] To achieve the above objectives, this application provides a low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis, comprising the following steps:

[0007] S1. Take raw soybean meal, crush it, add n-hexane, stir to defatt it, and recover the solvent by vacuum distillation to obtain defatted soybean meal;

[0008] S2. Immobilized composite enzyme gel beads were prepared using sodium alginate solution, alkaline protease, and bromelain; immobilized pepsin gel beads were prepared using sodium alginate solution and pepsin; immobilized trypsin gel beads were prepared using sodium alginate solution and trypsin.

[0009] S3. Take defatted soybean meal, add distilled water and mix well. Adjust the pH to 8.5-9.5 with NaOH. Add immobilized complex enzyme gel beads and perform enzymatic hydrolysis by stirring at a constant temperature for the first time. Filter and remove the gel beads to obtain the first filtrate.

[0010] S4. Adjust the pH of the first filtrate to 2.0-4.0 with hydrochloric acid, add immobilized pepsin gel beads, stir and hydrolyze at a constant temperature for the second time, filter and remove the gel beads to obtain the second filtrate;

[0011] S5. Adjust the pH of the second filtrate to 8.0-9.0 with NaOH, add immobilized trypsin gel beads, and perform a third enzymatic hydrolysis by stirring at a constant temperature. Filter to remove the gel beads. Centrifuge the hydrolysate to remove waste residue, filter the supernatant through an ultrafiltration membrane, and then desalt the filtrate using a desalting column to obtain soybean protein peptides.

[0012] Preferably, in step S1, the soybean meal is pulverized to pass through an 80-mesh sieve; the ratio of soybean meal to n-hexane is 1 g: (4-6) mL; the stirring degreasing temperature is 25-35℃, and the time is 1.5-2.5 h; the soybean meal is pulverized to pass through an 80-mesh sieve; the material-to-liquid ratio of n-hexane is 1: (4-6); the stirring degreasing temperature is 25-35℃, and the time is 1.5-2.5 h.

[0013] Preferably, in step S2, the ratio of sodium alginate to water in the sodium alginate solution is (2.5–4.0) g: 100 mL; the ratio of alkaline protease to sodium alginate solution is (4 × 10⁻⁶) g: 100 mL. 5 -6×10 5 U: 500mL; the ratio of bromelain to sodium alginate solution is (1.5 × 10⁻⁶). 5 -2.5×10 5 U: 500mL; the ratio of pepsin to sodium alginate solution is (1.5 × 10⁻⁶). 4 -2.5×10 4 U: 500mL; the ratio of trypsin to sodium alginate solution is (5 × 10⁻⁶). 4 -7×10 4 U: 500mL.

[0014] Preferably, in step S3, the ratio of defatted soybean meal to distilled water is 1g:(7-9)mL; the mass ratio of immobilized composite enzyme gel beads to defatted soybean meal is (0.8-1.2):1; the first constant temperature stirring enzymatic hydrolysis temperature is 40-50℃, the stirring speed is 80-120rpm, and the enzymatic hydrolysis time is 1.5-2.5h.

[0015] Preferably, in step S4, the mass ratio of the immobilized pepsin gel beads to defatted soybean meal is (0.8-1.2):1; the second isothermal stirring enzymatic hydrolysis temperature is 35-45℃, the stirring speed is 80-120rpm, and the enzymatic hydrolysis time is 3-4h.

[0016] Preferably, in step S5, the mass ratio of the immobilized trypsin gel beads to defatted soybean meal is (0.8-1.2):1; the third isothermal stirring enzymatic hydrolysis temperature is 40-55℃, the stirring speed is 80-120rpm, and the enzymatic hydrolysis time is 1-2h; the ultrafiltration membrane has a molecular weight cutoff of 5kDa.

[0017] On the other hand, this application provides the application of soybean protein peptides prepared by a low-temperature refrigeration method for enzymatic hydrolysis in pig feed additives.

[0018] Compared with the prior art, the beneficial effects of this application are reflected in:

[0019] (1) Alkaline protease and bromelain were co-immobilized in sodium alginate gel beads in a certain ratio. Alkaline protease hydrolyzed the carboxyl terminus of basic amino acids, opening the protein structure. Bromelain cleaved the internal hydrophobic region, reducing the production of bitter peptides and further reducing the peptide chain length. The cleavage of the carboxyl terminus of basic amino acids by alkaline protease can destroy the active site of KTI and the P1 active site of BBI. The cleavage of hydrophobic amino acids by bromelain can cleave the P2 active site of BBI. This design disrupts the activity of soybean trypsin inhibitors, prevents interference with subsequent trypsin degradation, and also eliminates the impact of soybean trypsin inhibitors on intestinal digestion after entering the intestine.

[0020] (2) This application uses four proteases to enzymatically hydrolyze soybean protein, resulting in soybean protein peptides with small molecular weights. Some of these soybean protein peptides have the effect of inhibiting swine pathogens such as Streptococcus suis and Clostridium perfringens. When added to pig feed, they can be used as drugs to treat bacterial infections in pigs, replacing traditional antibiotics, avoiding drug resistance and food safety issues, achieving healthy breeding and improved efficiency. Afterward, they can be stored at low temperatures to improve product stability. Attached Figure Description

[0021] Figure 1 Example 1: Electrophoretic detection of enzymatically digested soybean protein peptides;

[0022] Figure 2 A standard curve for the detection of soybean saponins;

[0023] Figure 3 This is a chromatogram of soybean protein peptide separation and purification in Example 1. Detailed Implementation

[0024] The following embodiments are only used to illustrate the technical solutions of this application more clearly, and should not be used to limit the scope of protection of this application.

[0025] The main compounds used in the examples and comparative examples were all commercially available products and were not subjected to any further purification treatment.

[0026] Example 1

[0027] A method for low-temperature refrigeration of soybean protein peptides prepared by enzymatic hydrolysis includes the following steps:

[0028] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 1L of n-hexane, stir at 30℃ to defatt it twice, 2 hours each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0029] S2. Weigh 16g of sodium alginate, dissolve it in 500mL of distilled water, and after dissolving at 50℃, add 5×10 5 U alkaline protease solution and 2×10 5 U bromelain solution was mixed, and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M Tris-HCl (pH=8.5) to obtain immobilized composite enzyme gel beads. 16g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 2×10⁻⁶ ppm was added. 4 U of pepsin solution was mixed, and then 0.1M CaCl2 solution was added dropwise. The mixture was allowed to stand for 1 hour, and then washed three times with 0.05M citrate buffer (pH=3.0) to obtain immobilized pepsin gel beads. 16g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 6×10⁻⁶ ppm was added. 4 U of trypsin solution was mixed and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M PBS buffer (pH=8.5) to obtain immobilized trypsin gel beads.

[0030] S3. Take 100g of defatted soybean meal, add 800mL of distilled water, adjust the pH to 9.0 with 1M NaOH, add 100g of immobilized complex enzyme gel beads, stir at 100rpm at 45℃ for 2h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0031] S4. Adjust the pH to 3.0 with 1M hydrochloric acid filtrate, add 100g of immobilized pepsin gel beads, stir at 100rpm at 40℃ for 3.5h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0032] S5. Adjust the pH of the filtrate to 8.5 with 1M NaOH, add 100g of immobilized trypsin gel beads, and hydrolyze at 45℃ with constant stirring at 100rpm for 1.5h. Filter and remove the gel beads. Adjust the pH of the hydrolysate to 7.0, centrifuge to remove waste residue, and use a 5kDa ultrafiltration membrane to remove large molecular weight proteins from the supernatant. Desalt the supernatant using a desalting column and freeze-dry to obtain soybean protein peptides.

[0033] S6. - The soybean protein peptides are stored at low temperature.

[0034] Example 2

[0035] A method for low-temperature refrigeration of soybean protein peptides prepared by enzymatic hydrolysis includes the following steps:

[0036] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 0.8L of n-hexane, stir at 25℃ to defatted twice, 2.5h each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0037] S2. Weigh 12.5g of sodium alginate, dissolve it in 500mL of distilled water, and after dissolving at 50℃, add 4×10 5 U alkaline protease solution and 1.5×10 5 U bromelain solution was mixed, and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M Tris-HCl (pH=8.5) to obtain immobilized composite enzyme gel beads. 12.5g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 1.5×10⁻⁶ ppm was added. 4 U of pepsin solution was mixed, and then 0.1M CaCl2 solution was added dropwise. The mixture was allowed to stand for 1 hour, and then washed three times with 0.05M citrate buffer (pH=3.0) to obtain immobilized pepsin gel beads. 12.5g of sodium alginate was weighed and dissolved in 500mL of distilled water. After dissolving at 50℃, 5×10⁻⁶ ppm of sodium alginate was added. 4 U of trypsin solution was mixed and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M PBS buffer (pH=8.5) to obtain immobilized trypsin gel beads.

[0038] S3. Take 100g of defatted soybean meal, add 700mL of distilled water, adjust the pH to 8.5 with 1M NaOH, add 80g of immobilized complex enzyme gel beads, stir at 80rpm at 40℃ for 1.5h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0039] S4. Adjust the pH of the filtrate to 2.0 with 1M hydrochloric acid, add 80g of immobilized pepsin gel beads, stir at 35℃ and 80rpm for 3h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0040] S5. Adjust the pH of the filtrate to 8.0 with 1M NaOH, add 80g of immobilized trypsin gel beads, and hydrolyze at 40℃ with constant stirring at 80rpm for 1h. Filter and remove the gel beads. Adjust the pH of the hydrolysate to 7.0, centrifuge to remove waste residue, and use a 5kDa ultrafiltration membrane to remove large molecular weight proteins from the supernatant. Desalt the supernatant using a desalting column and freeze-dry to obtain soybean protein peptides.

[0041] S6. - The soybean protein peptides are stored at low temperature.

[0042] Example 3

[0043] A method for low-temperature refrigeration of soybean protein peptides prepared by enzymatic hydrolysis includes the following steps:

[0044] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 1.2L of n-hexane, stir at 35℃ to defatted twice, 1.5h each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0045] S2. Weigh 20g of sodium alginate, dissolve it in 500mL of distilled water, and after dissolving at 50℃, add 6×10 5 U alkaline protease solution and 2.5×10 5 U bromelain solution was mixed, and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M Tris-HCl (pH=8.5) to obtain immobilized composite enzyme gel beads. 20g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 2.5×10⁻⁶ ppm was added. 4 U of pepsin solution was mixed, and then 0.1M CaCl2 solution was added dropwise. The mixture was allowed to stand for 1 hour, and then washed three times with 0.05M citrate buffer (pH=3.0) to obtain immobilized pepsin gel beads. 20g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 7×10⁻⁶ ppm was added. 4 U of trypsin solution was mixed and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M PBS buffer (pH=8.5) to obtain immobilized trypsin gel beads.

[0046] S3. Take 100g of defatted soybean meal, add 900mL of distilled water, adjust the pH to 9.5 with 1M NaOH, add 120g of immobilized complex enzyme gel beads, stir at 120rpm at 50℃ for 2.5h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0047] S4. Adjust the pH of the filtrate to 4.0 with 1M hydrochloric acid, add 120g of immobilized pepsin gel beads, stir at 120rpm at 45℃ for 4h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0048] S5. Adjust the pH of the filtrate to 9.0 with 1M NaOH, add 120g of immobilized trypsin gel beads, and hydrolyze at 55℃ with constant stirring at 120rpm for 2 hours. Filter and remove the gel beads. Adjust the pH of the hydrolysate to 7.0, centrifuge to remove waste residue, and use a 5kDa ultrafiltration membrane to remove large protein molecules from the supernatant. Desalt the supernatant using a desalting column and freeze-dry to obtain soybean protein peptides.

[0049] S6. - The soybean protein peptides are stored at low temperature.

[0050] Comparative Example 1

[0051] A low-temperature refrigeration method for preparing soybean protein peptides by co-enzymatic hydrolysis with immobilized alkaline protease, pepsin, and trypsin includes the following steps:

[0052] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 1L of n-hexane, stir at 30℃ to defatt it twice, 2 hours each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0053] S2. Weigh 16g of sodium alginate, dissolve it in 500mL of distilled water, and after dissolving at 50℃, add 5×10 5 U alkaline protease solution was mixed, and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M Tris-HCl (pH=8.5) to obtain alkaline protease gel beads. 16g of sodium alginate was weighed and dissolved in 500mL of distilled water. After dissolving at 50℃, 2×10⁻⁶ CaCl₂ solution was added. 4 U of pepsin solution was mixed, and then 0.1M CaCl2 solution was added dropwise. The mixture was allowed to stand for 1 hour, and then washed three times with 0.05M citrate buffer (pH=3.0) to obtain immobilized pepsin gel beads. 16g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 6×10⁻⁶ ppm was added. 4 U of trypsin solution was mixed and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M PBS buffer (pH=8.5) to obtain immobilized trypsin gel beads.

[0054] S3. Take 100g of defatted soybean meal, add 800mL of distilled water, adjust the pH to 9.0 with 1M NaOH, add 100g of fixed alkaline protease gel beads, stir at 100rpm at 45℃ for 2h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0055] S4. Adjust the pH of the filtrate to 3.0 with 1M hydrochloric acid, add 100g of immobilized pepsin gel beads, stir at 100rpm at 40℃ for 3.5h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0056] S5. Adjust the pH of the filtrate to 8.5 with 1M NaOH, add 100g of immobilized trypsin gel beads, and hydrolyze at 45℃ with constant stirring at 100rpm for 1.5h. Filter and remove the gel beads. Adjust the pH of the hydrolysate to neutral, centrifuge to remove waste residue, and use a 5kDa ultrafiltration membrane to remove large molecular weight proteins from the supernatant. Desalt the supernatant using a desalting column and freeze-dry to obtain soybean protein peptides.

[0057] S6. - The soybean protein peptides are stored at low temperature.

[0058] Comparative Example 2

[0059] The preparation of soybean protein peptides by altering the order of enzyme addition includes the following steps:

[0060] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 1L of n-hexane, stir at 30℃ to defatt it twice, 2 hours each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0061] S2. Weigh 16g of sodium alginate, dissolve it in 500mL of distilled water, and after dissolving at 50℃, add 5×10 5 U alkaline protease solution and 2×10 5 U bromelain solution was mixed, and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M Tris-HCl (pH=8.5) to obtain immobilized composite enzyme gel beads. 16g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 2×10⁻⁶ ppm was added. 4 U of pepsin solution was mixed, and then 0.1M CaCl2 solution was added dropwise. The mixture was allowed to stand for 1 hour, and then washed three times with 0.05M citrate buffer (pH=3.0) to obtain immobilized pepsin gel beads. 16g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 6×10⁻⁶ ppm was added. 4 U of trypsin solution was mixed and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M PBS buffer (pH=8.5) to obtain immobilized trypsin gel beads.

[0062] S3. Take 100g of defatted soybean meal, add 800mL of distilled water, adjust the pH to 8.5 with 1M NaOH, add 100g of immobilized trypsin gel beads, stir at 100rpm at 45℃ for 1.5h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0063] S4. Adjust the pH of the filtrate to 3.0 with 1M hydrochloric acid, add 100g of immobilized pepsin gel beads, stir at 100rpm at 40℃ for 3.5h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0064] S5. Adjust the pH of the filtrate to 9.0 with 1M NaOH, add 100g of immobilized complex enzyme gel beads, and hydrolyze at 45℃ and 100rpm for 2 hours. Filter and remove the gel beads. Adjust the pH of the hydrolysate to 7.0, centrifuge to remove waste residue, and use a 5kDa ultrafiltration membrane to remove large protein molecules from the supernatant. Desalt the supernatant using a desalting column and freeze-dry to obtain soybean protein peptides.

[0065] S6. - The soybean protein peptides are stored at low temperature.

[0066] Comparative Example 3

[0067] The preparation of soybean protein peptides using a low-temperature refrigeration method with non-immobilized enzymes includes the following steps:

[0068] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 1L of n-hexane, stir at 30℃ to defatt it twice, 2 hours each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0069] S2. Take 100g of defatted soybean meal, add 800mL of distilled water, adjust the pH to 9.0 with 1M NaOH, and add 5×10 5 U alkaline protease and 2×10 5 U-Bromelain was hydrolyzed at 45°C with constant stirring at 100 rpm for 2 hours, and then inactivated by heating at 95°C for 15 minutes.

[0070] S3. Adjust the pH to 3.0 with 1M hydrochloric acid, then add 2×10 4 U pepsin was hydrolyzed at 40°C with constant stirring at 100 rpm for 3.5 h, and then inactivated by heating at 95°C for 15 min.

[0071] S4. Adjust the pH to 8.5 with 1M NaOH, and add 6×10 4 U trypsin was hydrolyzed at 45℃ with constant stirring at 100 rpm for 1.5 h, and then inactivated by heating at 95℃ for 15 min. The pH of the hydrolysate was adjusted to 7.0, and the residue was removed by centrifugation. The supernatant was then subjected to ultrafiltration with a 5 kDa membrane to remove large protein molecules, followed by desalting using a desalting column and freeze-drying to obtain soybean protein peptides.

[0072] S5. - The soybean protein peptides are stored at low temperature.

[0073] Comparative Example 4

[0074] The preparation of soybean protein peptides using only immobilized complex protease includes the following steps:

[0075] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 1L of n-hexane, stir at 30℃ to defatt it twice, 2 hours each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0076] S2. Weigh 16g of sodium alginate, dissolve it in 500mL of distilled water, and after dissolving at 50℃, add 5×10 5 U alkaline protease solution and 2×10 5 U bromelain solution was mixed and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M Tris-HCl (pH=8.5) to obtain immobilized composite enzyme gel beads.

[0077] S3. Take 100g of defatted soybean meal, add 800mL of distilled water, adjust the pH to 9.0 with 1M NaOH, add 100g of immobilized complex enzyme gel beads, and hydrolyze by stirring at 100rpm for 2 hours at 45℃. Filter to obtain the filtrate. Adjust the pH of the filtrate to 7.0, centrifuge to remove waste residue, and use a 5kDa ultrafiltration membrane to remove large molecular weight proteins from the supernatant. Desalt the supernatant using a desalting column and freeze-dry to obtain soybean protein peptides. S5. Store the soybean protein peptides at low temperature.

[0078] Comparative Example 5

[0079] The preparation of soybean protein peptides using only immobilized pepsin and trypsin includes the following steps:

[0080] S1. Take 200g of raw soybean meal, grind it to 80 mesh, add 1L of n-hexane, stir at 30℃ to defatt it twice, 2 hours each time, and recover the solvent by vacuum distillation to obtain defatted soybean meal.

[0081] S2. Weigh 16g of sodium alginate, dissolve it in 500mL of distilled water, and after dissolving at 50℃, add 2×10 4 U of pepsin solution was mixed, and then 0.1M CaCl2 solution was added dropwise. The mixture was allowed to stand for 1 hour, and then washed three times with 0.05M citrate buffer (pH=3.0) to obtain immobilized pepsin gel beads. 16g of sodium alginate was weighed, dissolved in 500mL of distilled water, and after dissolving at 50℃, 6×10⁻⁶ ppm was added. 4 U of trypsin solution was mixed and then 0.1M CaCl2 solution was added dropwise. After standing for 1 hour, the mixture was washed three times with 0.05M PBS buffer (pH=8.5) to obtain immobilized trypsin gel beads.

[0082] S3. Take 100g of defatted soybean meal, add 800mL of distilled water, adjust the pH to 3.0 with 1M hydrochloric acid, add 100g of immobilized pepsin gel beads, stir at 100rpm at 40℃ for 3.5h for enzymatic hydrolysis, filter and remove the gel beads to obtain the filtrate.

[0083] S4. Adjust the pH of the filtrate to 8.5 with 1M NaOH, add 100g of immobilized trypsin gel beads, and hydrolyze at 45℃ and 100rpm for 1.5h with constant temperature stirring. Filter and remove the gel beads. Adjust the pH of the hydrolysate to 7.0, centrifuge to remove waste residue, and use a 5kDa ultrafiltration membrane to remove large protein molecules from the supernatant. Desalt the supernatant using a desalting column and freeze-dry to obtain soybean protein peptides. S5. Store the soybean protein peptides at low temperature.

[0084] Experimental Example 1

[0085] Detection of the enzymatic hydrolysis effect of soybean protein peptides:

[0086] Take 10g of soybean meal, add 100mL of water and mix well. Adjust the pH to 8.5 with NaOH, stir at 100rpm for 25min at 40℃, centrifuge at 5000rpm for 20min, and remove the precipitate. Take the supernatant, adjust the pH to 4.5 with 1M hydrochloric acid, stir at 50rpm for 30min at 40℃, centrifuge at 5000rpm for 5min, retain the precipitate, wash with water and adjust the pH to 7.0. Take a sample for electrophoresis detection. At the same time, take soybean protein peptides prepared in Example 1 for electrophoresis detection. The results are as follows. Figure 1 As shown.

[0087] Soybean meal contains large protein molecules of varying sizes. After enzymatic hydrolysis using the low-temperature refrigeration method described in Example 1, the resulting soybean protein peptides were electrophoresed. The large protein molecules were completely hydrolyzed into small polypeptide molecules, and the bands were located at the bottom of the electrophoresis.

[0088] Experiment Example 2

[0089] Sensory evaluation of bitter peptides:

[0090] To conduct a scientific sensory evaluation of the bitterness of the samples, a sensory group of 10 participants (5 men and 5 women, aged 20-30) was selected. The group received a total of 6 hours of training (three 2-hour training sessions held over two weeks). The training consisted of the following steps: ① Training the group using a reference substance (quinine sulfate aqueous solution) to familiarize members with the bitterness properties and establish consensus; ② The group conducted sensory evaluations of the reference substance at different concentrations to reach a consensus on the intensity, using concentrations of 0 and 2.9 × 10⁻⁶. -3 5.8×10 -3 1.2×10 -2 2.4×10 -2 A mmol / L quinine sulfate solution was used as a reference solution, with concentrations ranging from 1 to 5, from lowest to highest. A concentration of 0 corresponded to level "1", and a concentration of 2.4 × 10⁻⁶ corresponded to level "2". -2Bitterness at mmol / L corresponds to a level of "5"; bitterness intensity is assessed using a 5-point intensity scale from 1 to 5: "1" represents none, and "5" represents the strongest. After training, the sensory group conducted sensory evaluations of the samples in the sensory laboratory to determine the bitterness intensity. Before evaluation, sensory evaluators rinsed their mouths with distilled water, held 2-3 mL of sample solution in their mouths for 10 seconds to allow the sample solution to be fully dispersed throughout the oral cavity, primarily allowing the back of the tongue to perceive the taste, and then rinsed their mouths with distilled water after spitting it out. There was a 5-10 minute rest interval between each two samples, and each group member conducted three parallel analyses. To reduce experimental error, group members conducted three sensory evaluations within the same time period over 3 days. The average score of 10 members was used as the taste intensity value to determine the bitterness intensity of different samples, and the results are shown in Table 1 below.

[0091] Table 1. Sensory Evaluation Table of Bitterness

[0092]

[0093] As shown in Table 1, the soybean protein peptides in Examples 1-3 and Comparative Example 3 had no bitter taste, indicating that the combined enzymatic hydrolysis of these four enzymes can effectively remove bitter peptides from soybean protein peptides. Comparative Example 1, which did not use bromelain, had a moderate bitter taste, indicating that bromelain can help reduce some bitter peptides. Comparative Example 2, which changed the order of enzymatic hydrolysis, had a moderate bitter taste, possibly because the trypsin inhibitor in soybeans inhibited the activity of trypsin, resulting in incomplete trypsin hydrolysis and the production of bitter peptides. Comparative Examples 4-5, which used only two enzymes for hydrolysis, had the worst effect and the highest level of bitterness. This experiment proves that using the low-temperature refrigeration method of this patent, combined with the combined enzymatic hydrolysis of soybean protein with four enzymes, can reduce bitter peptides.

[0094] Experimental Example 3

[0095] Detection of soybean saponins:

[0096] Accurately weigh 10 mg of total soybean saponins reference standard and place it in a 200 mL Erlenmeyer flask with a ground glass stopper. Add 100 mL of 50% methanol 2 mol / L hydrochloric acid solution, sonicate to dissolve, and reflux in a water bath for 2 h. After cooling to room temperature, quantitatively transfer to a 100 mL volumetric flask, dilute to the mark with 50% methanol 2 mol / L hydrochloric acid solution, and shake well.

[0097] Accurately transfer 4 mL of the above reference standard hydrolysate into a 10 mL volumetric flask, dilute to the mark with 50% methanol 2 mol / L hydrochloric acid solution, and mix well. Use 50% methanol 2 mol / L hydrochloric acid solution as the reference solution and perform scanning operations at a wavelength of 200–680 nm.

[0098] Accurately transfer 1.0, 2.0, 3.0, 4.0, and 5.0 mL of the soybean saponin reference standard hydrolysis solution into 10 mL volumetric flasks, respectively. Dilute to the mark with 50% methanol 2 mol / L hydrochloric acid solution and mix well. Using 50% methanol 2 mol / L hydrochloric acid solution as a reference solution, measure the absorbance at a wavelength of 276 nm and plot a standard curve as shown below. Figure 2 As shown.

[0099] Accurately weigh 10g of soybean protein peptides from Examples 1-3 and Comparative Examples 1-5 into a Soxhlet extractor, add 200mL of 70% methanol, extract for 5 hours, filter, wash the residue with a small amount of 70% methanol, combine the extract and washings, then evaporate to dryness under reduced pressure, recovering the methanol. Quantitatively transfer the dried product to a 200mL Erlenmeyer flask with a ground glass stopper, add 100mL of 50% methanol / 2mol / L hydrochloric acid solution, sonicate to dissolve, reflux in a water bath for 2 hours, and after cooling to room temperature, quantitatively transfer to a 250mL volumetric flask, dilute to the mark with 50% methanol / 2mol / L hydrochloric acid solution, and shake well. Take 50mL of this solution and place it in a 250mL separatory funnel, extract three times with chloroform, adding 50mL of chloroform each time, combine the chloroform solutions, recover the chloroform and dry, quantitatively transfer this dried product to a 100mL volumetric flask, sonicate to dissolve and dilute to volume with 50% methanol / 2mol / L hydrochloric acid solution, and shake well. Accurately transfer 1.0 mL of this solution into a 25 mL volumetric flask, dilute to the mark with 50% methanol 2 mol / L hydrochloric acid solution, and shake well. Using 50% methanol 2 mol / L hydrochloric acid solution as a reference solution, measure the absorbance at a wavelength of 276 nm. Calculate the total soybean saponin content in soybean protein peptides based on the standard curve, as shown in Table 2.

[0100] Table 2. Total soybean saponin content in soybean protein peptides

[0101]

[0102] As shown in Table 2, Examples 1-3 and Comparative Examples 1, 2, 4 and 5 all had high contents of soybean saponins, with only Comparative Example 3 having the lowest content. This indicates that heating leads to the decomposition and loss of soybean saponins, and also reduces the nutrients in soybean protein peptides. In contrast, the low-temperature refrigeration method of this application can effectively retain more active ingredients in soybeans without heating.

[0103] Experiment Example 4

[0104] Assay for soybean trypsin inhibitor activity:

[0105] Weigh 1g of soybean protein peptides from Examples 1-3 and Comparative Examples 1-5 into 100mL Erlenmeyer flasks, add 50mL of 0.01mol / L sodium hydroxide solution, and shake well. Adjust the pH to 9.5±0.1 with 1mol / L hydrochloric acid solution and 0.1mol / L hydrochloric acid solution, and incubate at 0℃~4℃ for 15h~24h. Remove the sample extract and allow it to reach room temperature, transfer it to a 100mL volumetric flask, and dilute to the mark with water, shaking well. After 15min of precipitation, dilute the sample extract as needed. The dilution depends on the expected TIA value of the sample. Dilute the sample extract to three different concentrations to ensure that at least one of the three inhibition percentages of the TIA value is between 40% and 60%. The trypsin inhibitor activity was determined using a low-temperature refrigeration method, and the results are shown in Table 3.

[0106] Table 3. Trypsin inhibitor activity in soybean protein peptides

[0107]

[0108] As shown in Table 3, Examples 1-3 and Comparative Example 3 all exhibited low trypsin inhibitor activity, indicating that the low-temperature refrigeration method and heating method described in this application can effectively reduce trypsin inhibitor activity. Comparative Examples 4 and 5 showed high trypsin inhibitor activity, indicating that enzymatic hydrolysis with only two enzymes is insufficient to effectively remove trypsin inhibitor activity. Comparative Example 1 lacked bromelain hydrolysis, affecting its inhibitory effect on trypsin inhibitors. Comparative Example 2, by changing the order of enzymatic hydrolysis, also affected its inhibitory effect on trypsin inhibitors, possibly because the inhibitory effect of trypsin inhibitors on trypsin reduces the efficiency of trypsin in cleaving proteins.

[0109] Experimental Example 5

[0110] Antibacterial experiment of soybean protein peptides:

[0111] Streptococcus suis and Clostridium perfringens were cultured in LB medium until OD=0.8. 5 ml of fresh LB medium was added to each test tube, and the tubes were divided into two groups, A and B. Group A was inoculated with 50 μL of Streptococcus suis, and Group B with 50 μL of Clostridium perfringens. The soybean protein peptides from Examples 1-3 and Comparative Examples 1-5 were diluted with PBS to 500 mg / mL. 50 μL was added to each of the A and B test tubes, with three replicates for each substance. A blank control group without soybean protein peptides was also included. After culturing at 37℃ and 180 rpm for 10 h on a shaker, the absorbance was measured at OD600 using a spectrophotometer, and the average value for each group was calculated (results are shown in Table 4 below).

[0112] Table 4 Antibacterial Experiment of Soybean Protein Peptides

[0113]

[0114] As shown in Table 4, the soybean protein peptides prepared in Examples 1-3 exhibited better inhibitory effects against Streptococcus suis and Clostridium perfringens. The lack of degradation by some of the enzymes involved affected the antibacterial effect of the obtained soybean protein peptides. The low-temperature refrigeration method using non-immobilized enzymes to degrade soybean protein also affected the antibacterial effect. This may be because non-immobilized enzymes require heating to remove the enzymes, and the heating process causes protein denaturation, affecting the enzyme degradation process and the natural active ingredients present in soybeans, thus weakening the antibacterial effect.

[0115] Experimental Example 6

[0116] Isolation, purification, and antibacterial experiments of soybean protein peptides:

[0117] The soybean protein peptides prepared in Example 1 were dissolved in PBS buffer to a concentration of 500 mg / mL, filtered through a 0.22 μm filter membrane, and purified using a Superdex 30 Increase (10 × 300 mm) chromatography column. The loading volume was 200 μL per column, and the flow rate was 0.8 mL / min. The four peaks A, B, C, and D obtained were collected (e.g., peaks A, B, C, and D). Figure 3 (As shown). The collected solutions of the four peaks were concentrated and diluted to a concentration of 100 mg / mL. 5 mL of fresh LB medium was added to each test tube, and the tubes were divided into two groups: Group I was inoculated with 50 μL of *Streptococcus suis*, and Group II was inoculated with 50 μL of *Clostridium perfringens*. 50 μL of each substance was added to each test tube in Groups I and II, with three replicates for each substance. A blank control group without soybean protein peptides was also included. After incubation at 37℃ and 180 rpm for 10 h on a shaker, the absorbance was measured at OD600 using a spectrophotometer, and the average value for each group was calculated (results are shown in Table 5 below).

[0118] Table 5. Antibacterial experiments of different isolated peptides of soybean protein.

[0119]

[0120] As shown in Table 5, the soybean protein peptides collected from peaks B and C exhibited good inhibitory effects against Streptococcus suis, with peak C showing the best effect. Meanwhile, the soybean protein peptides collected from peaks A and C showed good inhibitory effects against Clostridium perfringens, with peak A showing a better effect than peak C. This experiment demonstrates that enzymatic hydrolysis of soybean protein peptides using these four enzymes yields four main types of soybean protein peptides, each exhibiting inhibitory effects against different bacteria.

[0121] Experimental Example 7

[0122] Treatment of Streptococcus suis infection in farmed pigs with soybean protein peptides:

[0123] In a pig farm in Anhui Province, approximately 30 pigs in two pens contracted Streptococcus suis, exhibiting symptoms such as fever, lethargy, drowsiness, loss of appetite, runny nose, and coughing. Soybean protein peptides prepared using the low-temperature refrigeration method in Example 1 were added at 3% to the pig feed and mixed thoroughly. After 24 hours of feed administration, the pigs' body temperature decreased, their mental state improved, runny nose and coughing symptoms lessened. After 48 hours, their body temperature returned to normal, infection symptoms largely disappeared, feed intake increased, and their overall condition improved. After 72 hours, the pigs fully recovered.

[0124] Experimental Example 8

[0125] Soybean protein peptides for the treatment of Clostridium perfringens infection in farmed pigs:

[0126] In a pig farm in Anhui Province, approximately 20 pigs in one pen were infected with Clostridium perfringens, suddenly developing hemorrhagic diarrhea. Affected pigs exhibited lethargy, decreased or complete loss of appetite, excreting foul-smelling, yellowish-brown or watery feces, fever, and increased thirst. The nasal mucosa was dry, and the skin, extremities, and ear tips were cyanotic. White foam drained from the mouth and nose, and the pigs experienced difficulty breathing. Soybean protein peptides prepared using the low-temperature refrigeration method in Example 1 were added at 3% to the pig feed and mixed thoroughly. This feed was then fed to the pig herd. After 24 hours of feed administration, the body temperature decreased, the diarrhea improved, the feces became yellowish-brown and pasty, and there was no bleeding. After 48 hours, the body temperature returned to normal, the infection symptoms largely disappeared, feed intake increased, and the pigs' condition improved. After 72 hours, the herd fully recovered.

[0127] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis, characterized in that, Includes the following steps: S1. Take raw soybean meal, crush it, add n-hexane, stir to defatt it, and recover the solvent by vacuum distillation to obtain defatted soybean meal; S2. Immobilized composite enzyme gel beads were prepared using sodium alginate solution, alkaline protease, and bromelain; immobilized pepsin gel beads were prepared using sodium alginate solution and pepsin; immobilized trypsin gel beads were prepared using sodium alginate solution and trypsin. S3. Take defatted soybean meal, add distilled water and mix well. Adjust the pH to 8.5-9.5 with NaOH. Add immobilized complex enzyme gel beads and perform enzymatic hydrolysis by stirring at a constant temperature for the first time. Filter and remove the gel beads to obtain the first filtrate. S4. Adjust the pH of the first filtrate to 2.0-4.0 with hydrochloric acid, add immobilized pepsin gel beads, stir and hydrolyze at a constant temperature for the second time, filter and remove the gel beads to obtain the second filtrate; S5. Adjust the pH of the second filtrate to 8.0-9.0 with NaOH, add immobilized trypsin gel beads, and perform a third enzymatic hydrolysis with constant temperature stirring. Filter and remove the gel beads. Centrifuge the hydrolysate to remove waste residue, filter the supernatant through an ultrafiltration membrane, and then desalt the filtrate using a desalting column to obtain soybean protein peptides. S6. - The soybean protein peptides are stored at low temperature.

2. The low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis according to claim 1, characterized in that, In step S1, the soybean meal is pulverized to pass through an 80-mesh sieve; the ratio of soybean meal to n-hexane is 1g:(4-6)mL; the temperature for stirring and defatting is 25-35℃, and the time is 1.5-2.5h.

3. The low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis according to claim 1, characterized in that, In step S2, the ratio of sodium alginate to water in the sodium alginate solution is (2.5–4.0) g: 100 mL; the ratio of alkaline protease to sodium alginate solution is (4 × 10⁻⁶ g / mL). 5 -6×10 5 U: 500mL; the ratio of bromelain to sodium alginate solution is (1.5 × 10⁻⁶). 5 -2.5×10 5 U: 500mL; the ratio of pepsin to sodium alginate solution is (1.5 × 10⁻⁶). 4 -2.5×10 4 U: 500mL; the ratio of trypsin to sodium alginate solution is (5 × 10⁻⁶). 4 -7×10 4 U: 500mL.

4. The low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis according to claim 1, characterized in that, In S3, the ratio of defatted soybean meal to distilled water is 1g:(7-9)mL; the mass ratio of immobilized composite enzyme gel beads to defatted soybean meal is (0.8-1.2):1; the first constant temperature stirring enzymatic hydrolysis temperature is 40-50℃, the stirring speed is 80-120rpm, and the enzymatic hydrolysis time is 1.5-2.5h.

5. The low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis according to claim 1, characterized in that, In S4, the mass ratio of the immobilized pepsin gel beads to defatted soybean meal is (0.8-1.2):1; the second isothermal stirring enzymatic hydrolysis temperature is 35-45℃, the stirring speed is 80-120rpm, and the enzymatic hydrolysis time is 3-4h.

6. The low-temperature refrigeration method for preparing soybean protein peptides by enzymatic hydrolysis according to claim 1, characterized in that, In S5; the mass ratio of the immobilized trypsin gel beads to defatted soybean meal is (0.8-1.2):1; the third isothermal stirring enzymatic hydrolysis temperature is 40-55℃, the stirring speed is 80-120rpm, and the enzymatic hydrolysis time is 1-2h; the ultrafiltration membrane has a molecular weight cutoff of 5kDa.

7. The application of soybean protein peptides prepared by the low-temperature refrigeration method for enzymatic hydrolysis of soybean protein peptides according to any one of claims 1-6 in pig feed additives.

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