Method for preparing soybean meal peptide through solid enzymolysis of soybean meal with compound enzyme

Abstract

The invention relates to the technical field of enzyme engineering and food processing, and particularly discloses a method for preparing soybean meal peptide through solid enzymolysis of soybean meal with a compound enzyme. According to the method, alkaline protease, neutral protease and trypsin are compounded into a compound enzyme according to the mass ratio of 1: 1: 1, and the yield and quality of the soybean meal peptide are remarkably improved by optimizing solid enzymolysis process conditions, the temperature is 50 DEG C, the pH is 6.5, the time is 7 hours and the material-liquid ratio is 1: 0.5. The hydrolysis degree reaches 24.61%, the peptide yield reaches 14.77%, the material-liquid ratio is reduced to 1: 0.5 compared with a traditional liquid method, near-zero wastewater discharge is achieved, and the environmental protection cost is reduced. Fourier infrared spectroscopy verifies that the peptide bond structure of the product is complete (the amide I band is 1650 cm <-1 >), and the secondary conformation is mainly beta-folding. The invention provides a low-cost, low-energy-consumption and sustainable soybean meal peptide industrial production scheme for environment-friendly strictly-controlled areas and small and medium-sized enterprises, and has the advantages of high resource value and green process.

Description

Technical Field

[0001] The present invention belongs to the technical fields of enzyme engineering and food processing, and particularly relates to a method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme. Background Art

[0002] Soybean meal is the main by-product after extracting oil from soybeans. It has a protein content as high as 40 - 50% and a balanced amino acid composition, possessing natural advantages for developing bioactive peptides. However, soybean meal is rich in anti-nutritional factors such as trypsin inhibitors and phytic acid. Direct utilization will reduce the protein digestibility (only 60 - 70%) and pose food safety risks. Through the enzymatic hydrolysis process, more than 90% of the anti-nutritional factors can be degraded synchronously, significantly improving the product safety. At the same time, high-active peptide segments are released, achieving a double improvement in resource value and safety.

[0003] Existing soybean meal peptide preparation technologies generally adopt liquid enzymatic hydrolysis methods, with a material-liquid ratio as high as 1:8, having bottleneck problems such as large water consumption and high wastewater treatment costs. For enterprises located in areas with strict environmental control or water-scarce areas, the high energy consumption and wastewater treatment burden (accounting for about 30 - 40% of the total cost) of the liquid enzymatic hydrolysis method are often unbearable; although soybean meal is a low-cost (3000 - 4000 yuan / ton) and stably supplied raw material and can accept phased production debugging, the liquid enzymatic hydrolysis method will still significantly weaken its cost advantage. In contrast, the solid-state enzymatic hydrolysis method can achieve nearly zero wastewater discharge by reducing the water addition amount, directly reducing environmental protection costs, while retaining the cost performance advantage of soybean meal as a raw material.

[0004] When enterprises apply the composite enzyme solid-state enzymatic hydrolysis technology to produce soybean meal peptides, they are mainly restricted by the performance and energy consumption of factory equipment. At the same time, parameters such as the type and ratio of enzymes, enzymatic hydrolysis temperature, enzymatic hydrolysis pH, and enzymatic hydrolysis time have a significant impact on the preparation process of soybean meal peptides. Therefore, developing a solid-state composite enzymatic hydrolysis preparation method and optimizing the process conditions and enzymatic hydrolysis system can effectively improve the high-value utilization efficiency of soybean meal resources. Summary of the Invention

[0005] In order to solve the above problems, the present invention provides a method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme, to solve the problems of high water consumption, high pollution, and equipment energy consumption limitation of the traditional liquid enzymatic hydrolysis method, and provide a low-cost and sustainable industrial production plan for soybean meal peptides for enterprises in areas with strict environmental control and small and medium-sized enterprises.

[0006] To achieve the above object, the present invention adopts the following technical scheme: A method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme, comprising the following steps:

[0007] 1) Dry the soybean meal, screen out impurities, grind it with a pulverizer, and sieve to obtain soybean meal powder;

[0008] 2) Take the composite enzyme;

[0009] 3) Add the complex enzyme in step 2) into the buffer solution and mix evenly.

[0010] 4) Take the sieved soybean meal powder with a material-liquid ratio of 1:0.5 to the buffer solution, and add the complex enzyme buffer solution obtained in step 3).

[0011] 5) Start enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate it, dry it in a constant-temperature drying oven. After the drying is completed, grind it for standby.

[0012] 6) Weigh the soybean meal enzymolysis powder, add deionized water, mix well and place it in a constant-temperature shaking incubator to shake. Then transfer the mixed solution to a 4°C refrigerator for refrigeration. After taking it out, centrifuge for 15 min, and collect the supernatant for standby.

[0013] 7) Purify the supernatant obtained in step 6) using Sephadex G-25.

[0014] 8) Concentrate the liquid obtained in step 7) using a rotary evaporator.

[0015] 9) After freeze-drying the concentrated solution obtained in step 8), obtain slightly yellow powdery soybean meal peptides.

[0016] In the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a complex enzyme, in step 2), the complex enzyme is one or a mixture of more than one of alkaline protease, neutral protease, trypsin, papain, keratinase, SFQ enzyme, and pectinase.

[0017] In the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a complex enzyme, in step 2), the complex enzyme is an equimolar mixture of alkaline protease, neutral protease, and trypsin.

[0018] In the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a complex enzyme, in step 2), by mass ratio, complex enzyme:soybean meal powder = 1:100.

[0019] In the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a complex enzyme, in step 3), the pH of the buffer solution is 6.5.

[0020] In the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a complex enzyme, in step 3), in the buffer solution, the concentration of the complex enzyme is 0.02 g / mL.

[0021] In the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a complex enzyme, in step 5), both the enzymatic hydrolysis and drying are carried out at 50°C, where the enzymatic hydrolysis time is 7 h and the drying time is overnight.

[0022] In the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme, in step 6), the oscillation time and refrigeration time are 8 h, and the centrifugation conditions are 4 °C and 4000 r / min.

[0023] The slightly yellowish powdery soybean meal peptides prepared by the above method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme.

[0024] Advantages of the present invention: The present invention optimizes the method for preparing soybean peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme through single-factor experiments and orthogonal experiments. With the synergistic action of alkaline, neutral, and trypsin, the enzymatic hydrolysis conditions are optimized (50 °C, pH = 6.5, 7 h), significantly improving the degree of hydrolysis (24.61%) and peptide yield (14.77%) of soybean meal peptides. Compared with the liquid method, the solid-liquid ratio is reduced to 1:0.5, achieving nearly zero wastewater discharge, solving the problems of high water consumption and high pollution, being applicable to the green transformation of small and medium-sized enterprises in environmentally protected and strictly controlled areas, and promoting the high-value utilization of soybean meal resources. Description of the Drawings

[0025] Figure 1 It is a graph showing the relationship between the degree of hydrolysis (DH) and the types of enzymes and their proportion groups.

[0026] Figure 2 It is a graph showing the relationship between the degree of hydrolysis (DH) and the enzymatic hydrolysis temperature.

[0027] Figure 3 It is a graph showing the relationship between the degree of hydrolysis (DH) and the enzymatic hydrolysis time.

[0028] Figure 4 It is a graph showing the relationship between the degree of hydrolysis (DH) and the enzymatic hydrolysis pH.

[0029] Figure 5 It is a graph showing the relationship between different groups of orthogonal experiments and the degree of hydrolysis (DH).

[0030] Figure 6 It is a graph showing the relationship between different groups of orthogonal experiments and the peptide yield.

[0031] Figure 7 It is the finished product of soybean meal peptides.

[0032] Figure 8 It is the Fourier transform infrared spectrum.

[0033] Figure 9 It is the flow chart of the method for preparing soybean meal peptides by solid-state enzymatic hydrolysis with a composite enzyme. Detailed Embodiments

[0034] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0035] The present invention provides a method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of compound enzymes on soybean meal, comprising the following steps:

[0036] S1. Single-factor experiment on preparing soybean meal peptides by solid-state enzymatic hydrolysis of compound enzymes on soybean meal

[0037] Single-factor experiment on the influence of enzyme types and their ratios on enzymatic hydrolysis: Using soybean meal powder as the raw material, single-factor experiments are carried out by changing the enzyme types and their ratios to screen the optimal ratio; seven groups of experiments are designed respectively with the enzyme types and their ratios as alkaline protease: neutral protease: keratinase: pectinase: SFQ enzyme = 4:3:1:1:1, alkaline protease: neutral protease: papain = 1:1:1, alkaline protease: neutral protease: trypsin = 1:1:1, alkaline protease: neutral protease: trypsin = 4:3:3, alkaline protease: neutral protease: papain = 4:3:3, alkaline protease: neutral protease: papain: trypsin = 4:2:1:1, alkaline protease: neutral protease: papain: trypsin = 4:3:1:1;

[0038] Single-factor experiment on the influence of enzymatic hydrolysis temperature on enzymatic hydrolysis: Using soybean meal powder as the raw material, single-factor experiments are carried out by changing the enzymatic hydrolysis temperature to screen the optimal enzymatic hydrolysis temperature; five groups of experiments are designed respectively with the enzymatic hydrolysis temperatures as 40°C, 45°C, 50°C, 55°C, 60°C;

[0039] Single-factor experiment on the influence of enzymatic hydrolysis time on enzymatic hydrolysis: Using soybean meal powder as the raw material, single-factor experiments are carried out by changing the enzymatic hydrolysis time to screen the optimal enzymatic hydrolysis time; five groups of experiments are designed respectively with the enzymatic hydrolysis times as 4h, 5h, 6h, 7h, 8h;

[0040] Single-factor experiment on the influence of enzymatic hydrolysis pH on enzymatic hydrolysis: Using soybean meal powder as the raw material, single-factor experiments are carried out by changing the enzymatic hydrolysis pH to screen the optimal enzymatic hydrolysis time; four groups of experiments are designed respectively with the enzymatic hydrolysis pH values as 6, 6.5, 7, 7.5;

[0041] Preparation of polypeptide enzymatic hydrolysate: After the solid-state enzymatic hydrolysis of soybean meal, it is inactivated and then quickly cooled to room temperature, dried, and ground. According to the solid-liquid ratio of 1:100, the enzymatically hydrolyzed soybean meal powder and water are taken into a conical flask, shaken in a constant-temperature shaker for 8 hours, refrigerated in the refrigerator for 8 hours, centrifuged, and the supernatant is collected. The degree of hydrolysis (DH) is determined by the OPA method, and the protein content is determined by the Coomassie Brilliant Blue method; the optimal enzyme type and its ratio, enzymatic hydrolysis temperature, enzymatic hydrolysis time, and enzymatic hydrolysis pH of the single-factor experiment are determined with the degree of hydrolysis as the index;

[0042] S2. Orthogonal experiment design

[0043] Taking the enzyme type and its ratio, enzymatic hydrolysis temperature, enzymatic hydrolysis time, and enzymatic hydrolysis pH as four key factors, an orthogonal experiment design with four factors and three levels is adopted; the preparation of the polypeptide enzymatic hydrolysate is the same as in step S1;

[0044] S3. Obtaining the optimal scheme

[0045] (1) When the enzyme type and its ratio are alkaline protease: neutral protease: trypsin = 1:1:1, the degree of hydrolysis (DH) is the highest;

[0046] (2) When the enzymatic hydrolysis temperature is 50 °C, the degree of hydrolysis (DH) is the highest;

[0047] (3) When the enzymatic hydrolysis time is 7 hours, the degree of hydrolysis (DH) is the highest;

[0048] (4) When the enzymatic hydrolysis pH is 6.5, the degree of hydrolysis (DH) is the highest;

[0049] (5) When the enzyme type and its ratio are alkaline protease: neutral protease: trypsin = 1:1:1, the enzymatic hydrolysis temperature is 50 °C, the enzymatic hydrolysis time is 7 hours, and the enzymatic hydrolysis pH is 6.5, the degree of hydrolysis (DH) of soybean meal under this enzymatic hydrolysis condition is the highest;

[0050] S4. Determination of peptide yield

[0051] Select the polypeptide enzymatic hydrolysates of all groups in the orthogonal experimental group in step S2, and determine the peptide yield by the biuret reagent method;

[0052] S5. Preparation of soybean meal peptide finished product

[0053] (1) Purification: Combine the polypeptide enzymatic hydrolysates of the top three samples with the highest peptide yield in step S4, perform gel filtration chromatography on Sephadex G-25, elute with deionized water, and collect the purified polypeptide enzymatic hydrolysate;

[0054] (2) Concentration: Concentrate the purified polypeptide enzymatic hydrolysate in step (1) by rotary evaporation (water bath at 30 ± 1 °C, rotation speed of 50 rpm, and the liquid loading volume should be less than 2 / 3 of the bottle volume) to a certain volume;

[0055] (3) Freeze-drying: The concentrated solution is freeze-dried to obtain a slightly yellowish freeze-dried powder of soybean meal peptides;

[0056] S6. Fourier transform infrared spectroscopy analysis of structural characteristics

[0057] Weigh an appropriate amount of the sample and potassium bromide, grind and mix them evenly in an agate mortar, then place them in a tablet press and keep at 15 MPa for 20 s to press into tablets for on-machine monitoring;

[0058] Furthermore, after the soybean meal is enzymolyzed in steps S1 and S2, it is inactivated at 90 °C for 10 min, dried overnight in an oven at 50 °C, ground and bagged for standby. According to a solid-liquid ratio of 1:100, the enzymolyzed soybean meal powder and deionized water are added to a centrifuge tube, shaken in a constant temperature shaker for 8 h, then placed in the refrigerator for refrigeration for 8 h, and then centrifuged at 4000 r / min at 4 °C for 15 min to collect the supernatant;

[0059] Furthermore, in steps S1 and S2: Calculate the protein content of the sample through a standard curve, and calculate the degree of hydrolysis (DH) of the sample using formula (1) and formula (2).

[0060] Furthermore, in steps S1 and S2: Add 5 mL of Coomassie Brilliant Blue, mix for 1 min using a vortex mixer, let stand for 2 min, measure the absorbance of the standard product and the sample at 595 nm, and calculate the protein content using the standard curve.

[0061] Furthermore, in steps S1 and S2: Add 3 mL of OPA solution, react in the dark for 2 min, measure the absorbance at 340 nm, use deionized water as a blank control, and calculate the degree of hydrolysis (DH) of the sample using formula (1) and formula (2):

[0062]

[0063] Where: SerineNH2 is mmol serineNH2 / g protein; X is the sample weight; P is the protein content in the sample; N is the dilution factor; V is the volume of the supernatant;

[0064]

[0065] Where: β and α are represented by constants 0.4 and 1 respectively; h tot = 7.72 mmol / g is h tot The millimoles of peptide bonds per gram of raw material protein (mmol / g);

[0066] Further, in step S4: Add 5 mL of 0.4 mol / L trichloroacetic acid, mix and react for 30 min, then centrifuge at 10,000 r / min for 10 min. Take 1 mL of the supernatant, add 4 mL of biuret reagent, let it stand at room temperature for 30 min, measure the absorbance at 540 nm, calculate the peptide content according to the standard curve, and calculate the peptide yield using formula (III):

[0067]

[0068] Further, in step S5: Weigh 12.5 g of Sephadex G-25, swell it in a constant temperature water bath (60 - 80 °C) for 2 h. After it is fully swollen to form uniform colloidal particles, start the peristaltic pump to regulate the elution flow rate for sample loading, ensuring that the liquid level in the column always covers the colloidal surface to prevent cracking. Synchronously start the outlet valve at the lower end of the column and collect the eluate.

[0069] Further, in step S6: The mass ratio of the sample to potassium bromide is in the range of 1:100 to 1:150. Under the conditions of 4000 - 400 cm -1 and a resolution of 4 cm -1 , scan 32 times to obtain a Fourier transform infrared spectrum.

[0070] The present invention will be further described below in conjunction with embodiments:

[0071] Example 1 Single-factor experiment on preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with composite enzymes

[0072] 1 Experimental materials

[0073] Soybean meal (The raw material is dried, impurities are sieved out, ground with a pulverizer, and passed through a 40-mesh sieve to obtain soybean meal powder)

[0074] Enzymes (alkaline protease, neutral protease, trypsin, papain, keratinase, SFQ enzyme, pectinase)

[0075] 2 Single-factor experiment

[0076] 2.1 Single-factor experiment on the effect of enzyme type and its ratio on enzymatic hydrolysis

[0077] 2.1.1 Enzymatic hydrolysis experiment

[0078] Weigh 25 g of soybean meal powder and place it in a beaker for standby. According to the solid-liquid ratio of 1:0.5, take 12.5 mL of buffer solution with pH = 7, add 0.25 g of composite enzyme according to the enzyme-solid ratio of 1:100. After stirring evenly with a magnetic stirrer, add it to the beaker containing 25 g of soybean meal powder. Stir evenly with a glass rod, then pour it into a 250 mL conical flask, wrap the mouth of the conical flask with plastic wrap, make holes, and carry out enzymatic hydrolysis at 50 °C for 7 h. After the enzymatic hydrolysis is completed, inactivate it at 90 °C for 10 min, dry it overnight, and grind it for standby.

[0079] 2.1.2 Preparation of Polypeptide Enzymolysis Solution

[0080] Weigh 0.5 g of enzymolyzed soybean meal powder, add 50 mL of deionized water, oscillate in a constant temperature shaker for 8 h, refrigerate for 8 h, then centrifuge at 4000 r / min for 15 min at 4 °C, and collect the supernatant. Subsequently, measure the degree of hydrolysis by the OPA method to screen out the best enzyme type and its ratio for solid enzymolyzed soybean meal.

[0081] The types and ratios of the above complex enzymes are formulated according to Table 1, and the addition amount should meet the enzyme-to-substrate ratio of 1:100.

[0082] Table 1 Types and Ratios of Enzymes

[0083]

[0084]

[0085] Note: Alkali represents alkaline protease, Zhong represents neutral protease, Papaya represents papain, Yi represents trypsin, Jiao represents keratinase, SFQ represents SFQ enzyme, Guo represents pectinase, and the same applies hereinafter.

[0086] 2.1.3 Determination of Degree of Hydrolysis and Protein Content

[0087] Take 1 mL of polypeptide enzymolysis solution in a test tube, add 5 mL of Coomassie Brilliant Blue solution, mix with a vortex mixer for 1 min, let stand for two minutes, then measure the absorbance at 595 nm, and calculate the protein content of the sample using the standard curve drawn with bovine serum albumin. Take 3 mL of OPA solution and add 400 μL of the supernatant of the polypeptide enzymolysis solution diluted 1000 times, accurately react for 2 min, and then measure the absorbance at 340 nm. At the same time, use the arginine standard solution as a reference and blank test, and finally calculate the degree of hydrolysis with Formula (1) and Formula (2).

[0088]

[0089] Where: SerineNH2 is mmol serineNH2 / g protein; X is the sample weight; P is the protein content in the sample; N is the dilution factor; V is the volume of the supernatant.

[0090]

[0091] Where: β and α are represented by constants 0.4 and 1 respectively; h tot = 7.72 mmol / g for h tot mmol of peptide bonds per gram of raw material protein (mmol / g);

[0092] 2.2 Results of Single Factor Experiment on the Influence of Enzyme Types and Their Ratios on Enzymolysis

[0093] Table 2 Degree of hydrolysis of soybean meal enzymatically hydrolyzed by different enzyme types and their proportions

[0094]

[0095]

[0096] As shown in Table 2 and Figure 1 as shown, the degrees of hydrolysis of several polypeptide enzymolysis solutions in the single-factor experiment of the above enzyme types and their proportions are in the following order: alkali: medium: trypsin = 1:1:1 > alkali: medium: papain: trypsin = 4:2:1:1 > alkali: medium: trypsin = 4:3:3 > alkali: medium: papain: trypsin = 4:3:1:1 > alkali: medium: keratin: pectin: SFQ = 4:3:1:1:1 > alkali: medium: papain = 1:1:1 > alkali: medium: papain = 4:3:3.

[0097] When alkaline protease, neutral protease and trypsin are compounded in equal proportions, broad-spectrum site coverage and continuous degradation pathways are achieved through the multi-enzyme synergistic mechanism. Combining equal-proportion mixing to avoid substrate competition and product feedback inhibition to optimize the allocation of enzyme activity resources, compared with the composite system containing papain or keratinase (disperse enzyme activity, insufficient substrate targeting), the hydrolysis efficiency of soybean meal enzymolysis is significantly improved.

[0098] 2.3 Single-factor experiment on the effect of enzymolysis temperature on enzymolysis

[0099] Weigh 25 g of soybean meal powder and place it in a beaker for later use. Take 12.5 mL of buffer solution with pH = 7 according to the solid-liquid ratio of 1:0.5, add 0.25 g of composite enzyme (alkali: medium: trypsin = 1:1:1) according to the enzyme-substrate ratio of 1:100. After stirring evenly with a magnetic stirrer, add it to the beaker containing 25 g of soybean meal powder. After stirring evenly with a glass rod, pour it into a 250 mL conical flask, wrap the mouth of the conical flask with plastic wrap, punch holes, and enzymolyze for 7 h at different enzymolysis temperatures. After the enzymolysis is completed, inactivate at 90 °C for 10 min, dry overnight, and grind for later use.

[0100] Same as the preparation of polypeptide enzymolysis solution in 2.1.

[0101] Same as 2.1.3 for measuring the degree of hydrolysis and protein content.

[0102] The enzymolysis temperature of the above composite enzyme is carried out according to Table 3.

[0103] Table 3 Enzymolysis temperature

[0104]

[0105] 2.4 Results of single-factor experiment on the effect of enzymolysis temperature on enzymolysis

[0106] Table 4 Degree of hydrolysis of soybean meal enzymatically hydrolyzed at different enzymolysis temperatures

[0107]

[0108] As shown in Table 4 and Figure 2 as shown, the degrees of hydrolysis of the several polypeptide hydrolysates in the single-factor experiment of the above enzymatic hydrolysis temperature are in the following order: 50°C > 45°C > 55°C > 40°C > 60°C.

[0109] The degree of hydrolysis of the complex enzyme (alkaline, neutral, trypsin) is the highest at 50°C, attributed to the synergistic effect of the optimal temperature ranges of the three at this temperature: the heat resistance of alkaline protease ensures the stability of the system, and the thermal sensitivity of neutral and trypsin is maintained at 50°C; at the same time, the moderate thermal denaturation of soybean meal protein promotes the exposure of internal cleavage sites, and the temperature does not exceed the critical threshold (≥55°C), avoiding the attenuation of enzyme activity and substrate aggregation (such as the interference caused by the Maillard reaction at 60°C), and finally achieving the optimal balance among the enzymatic reaction rate, substrate accessibility, and system stability.

[0110] 2.5 Single-factor experiment on the effect of enzymatic hydrolysis time on enzymatic hydrolysis

[0111] Weigh 25 g of soybean meal powder and place it in a beaker for later use. Take 12.5 mL of buffer solution with pH = 7 according to the solid-liquid ratio of 1:0.5, add 0.25 g of complex enzyme (alkali:neutral:trypsin = 1:1:1) according to the enzyme-substrate ratio of 1:100, stir evenly with a magnetic stirrer, then add it to the beaker containing 25 g of soybean meal powder, stir evenly with a glass rod, pour it into a 250 mL conical flask, wrap the mouth of the conical flask with plastic wrap, make holes, control different enzymatic hydrolysis times at 50°C. After the enzymatic hydrolysis is completed, inactivate at 90°C for 10 min, dry overnight, and grind for later use.

[0112] Same as 2.1 Preparation of polypeptide hydrolysate.

[0113] Same as 2.1.3 Determination of degree of hydrolysis and protein content.

[0114] The enzymatic hydrolysis time of the above complex enzyme is carried out according to Table 5.

[0115] Table 5 Enzymatic hydrolysis time

[0116]

[0117]

[0118] 2.6 Results of the single-factor experiment on the effect of enzymatic hydrolysis time on enzymatic hydrolysis

[0119] Table 6 Degrees of hydrolysis of enzymatically hydrolyzed soybean meal at different enzymatic hydrolysis times

[0120]

[0121] As shown in Table 6 and Figure 3As shown, the degrees of hydrolysis (DH) of several polypeptide hydrolysates in the above single-factor experiment on enzymatic hydrolysis time are in the following order: 7h > 6h > 8h > 5h > 4h.

[0122] When the enzymatic hydrolysis time is 7h, the degree of hydrolysis is the highest, mainly because it is at the critical balance point between substrate availability (rapid degradation of high-concentration substrates in the initial stage) and product feedback inhibition effect (competitive inhibition of enzyme activity by the accumulation of small peptides in the later stage). At the same time, the activity of the complex enzyme gradually decays (limited by the thermal stability of neutral and trypsin), resulting in a decrease in the enzymatic reaction rate after 7h. Eventually, 7h becomes the optimal duration for substrate conversion efficiency and system dynamic inhibition.

[0123] 2.7 Results of single-factor experiment on the effect of enzymatic hydrolysis pH on enzymatic hydrolysis

[0124] Weigh 25g of soybean meal powder and set it aside in a beaker. According to the solid-liquid ratio of 1:0.5, take 12.5 mL of buffer solutions with different pH values, add 0.25g of complex enzyme (alkaline: neutral: trypsin = 1:1:1) according to the enzyme-substrate ratio of 1:100. After stirring evenly with a glass rod, add it to the beaker containing 25g of soybean meal powder, stir evenly again, then pour it into a 250 mL conical flask, wrap the mouth of the conical flask with plastic wrap, make holes, and carry out enzymatic hydrolysis at 50°C for 7h. After the enzymatic hydrolysis is completed, inactivate it at 90°C for 10 min, dry it overnight, and grind it for later use.

[0125] Same as 2.1 Preparation of polypeptide hydrolysate.

[0126] Same as 2.1.3 Determination of degree of hydrolysis and protein content.

[0127] The enzymatic hydrolysis pH of the above complex enzyme is carried out according to Table 7.

[0128] Table 7 Enzymatic hydrolysis pH

[0129]

[0130]

[0131] 2.8 Results of single-factor experiment on the effect of enzymatic hydrolysis pH on enzymatic hydrolysis

[0132] Table 8 Degrees of hydrolysis of enzymatically hydrolyzed soybean meal at different enzymatic hydrolysis pH values

[0133]

[0134] As shown in Table 8 and Figure 4 As shown, the degrees of hydrolysis (DH) of several polypeptide hydrolysates in the above single-factor experiment on enzymatic hydrolysis pH are in the following order: 6.5 > 6 > 7.5 > 7.

[0135] The degree of hydrolysis is the highest at pH = 6.5 because it balances the optimal activity of neutral protease (pH = 6 - 7), the weak acid tolerance of trypsin, and the auxiliary effect of alkaline protease in the complex enzyme system (alkali:neutral:trypsin = 1:1:1). At the same time, soybean meal protein moderately aggregates near the isoelectric point, exposing internal enzyme cleavage sites and inhibiting the formation of by-products such as the Maillard reaction, ultimately achieving the optimal matching of enzyme activity synergy, substrate accessibility, and reaction stability.

[0136] Orthogonal experiment of Example 2

[0137] The enzyme types and their ratios, hydrolysis temperature, hydrolysis time, and hydrolysis pH obtained under the optimal enzymatic hydrolysis conditions are the four key factors affecting the degree of hydrolysis (DH) of complex enzyme solid-state enzymatic hydrolysis of soybean meal. After screening through previous experiments, three relatively optimal levels were determined for each factor. An orthogonal experimental design with four factors and three levels was adopted to determine the best combination of these key factors, thereby obtaining the optimal enzymatic hydrolysis conditions.

[0138] The enzyme types and their ratios were set as factor A, the hydrolysis temperature as factor B, the hydrolysis time as factor C, and the hydrolysis pH as factor D.

[0139] An orthogonal experiment table in Table 10 was designed and generated according to Table 9 for the experiment.

[0140] Same as the preparation of polypeptide enzymatic hydrolysate in 2.1.

[0141] Same as the determination of the degree of hydrolysis and protein content in 2.1.3.

[0142] Table 9 Orthogonal experiment factor level design

[0143]

[0144] Table 10 Orthogonal experiment table

[0145]

[0146] Table 11 Orthogonal experiment results and range analysis results

[0147]

[0148]

[0149] Note: Samples Ⅰ - Ⅸ correspond to 1 - 9 in the orthogonal experiment table in Table 10 in sequence, and the same applies hereinafter. Figure 5 and Figure 6 The same applies hereinafter.

[0150] By the magnitudes of the R values in Table 11: R4 > R1 > R2 > R3, it can be concluded that the order of the influence of the various factors in the above orthogonal experiment on the effect of complex enzyme solid-state enzymatic hydrolysis of soybean meal is: enzymatic hydrolysis pH > enzyme type and its ratio > enzymatic hydrolysis temperature > enzymatic hydrolysis time. According to the degrees of hydrolysis, R values, and K values, the optimal enzymatic hydrolysis conditions are alkaline protease: neutral protease: trypsin = 1:1:1, enzymatic hydrolysis temperature 50°C, enzymatic hydrolysis time 7 h, and enzymatic hydrolysis pH 6.5. From Table 11 and Figure 5 it can be seen that the top three samples in terms of degree of hydrolysis are: Sample III, Sample I, and Sample IX. The soybean meal peptide finished products are prepared from the samples using the above enzymatic hydrolysis conditions for subsequent testing.

[0151] Example 3 Determination of Peptide Yield

[0152] 1 Experimental Method

[0153] 1.1 Standard Curve Drawing

[0154] Prepare a 0.0 - 2.0 mg / mL Gly-Gly-Tyr-Arg tetrapeptide standard solution with 5% TCA. Take 6.0 mL and add 4.0 mL of biuret reagent. After mixing evenly, let it stand for 10 min, centrifuge at 2000 r / min for 10 min, take the supernatant, measure the OD value at 540 nm, and use 0.0 mg / mL as the blank control to draw the standard curve of peptide content X (mg / mL) against Y (OD value).

[0155] 1.2 Determination of Polypeptide Content in Samples

[0156] Take an equal volume of soybean meal polypeptide enzymatic hydrolysis solution and 5% TCA, mix well with a vortex mixer for 1 min, let it stand and react for 30 min. After the reaction, centrifuge at 1000 r / min for 10 min, take 1 mL of the supernatant, add 4 mL of biuret reagent and mix well, then let it stand at room temperature for 30 min, measure the absorbance value at 540 nm, and obtain the concentration (mg / mL) of soybean meal polypeptide in the sample by referring to the standard curve, and calculate the polypeptide content in the sample. Finally, the peptide yield is obtained.

[0157] 2 Experimental Results

[0158] Table 12 Peptide Yield Results of Different Groups

[0159]

[0160]

[0161] The results are shown in Table 12 and Figure 6As shown, the peptide yields of the above peptide yield determination experiments were as follows: Sample III > Sample I > Sample IX > Sample IV > Sample VIII > Sample V > Sample VI > Sample VII > Sample II. The Pearson correlation coefficient between the degree of hydrolysis and the peptide yield was 0.71, indicating a certain positive correlation between the two, but not a strictly linear relationship. This may be because the high-degree hydrolysis products cause aggregation and precipitation, reducing the content of soluble peptides, so some samples have a high degree of hydrolysis but a low peptide yield.

[0162] Example 4 Purification and Concentration

[0163] 1 Preparation of Polypeptide Enzymolysis Solution

[0164] Weigh 2.5 g of enzymolyzed soybean meal powder into a 250 mL conical flask, add 250 mL of deionized water, stir evenly, place it in a constant temperature shaking incubator and shake for 8 h, take it out and refrigerate for 8 h, then centrifuge at 4 °C and 4000 r / min for 15 min, and collect the supernatant.

[0165] 2 Purification

[0166] Weigh 12.5 g of Sephadex G-25, swell it in a constant temperature water bath (60 - 80 °C) for 2 h to form uniform colloidal particles. Subsequently, use wet packing to fill the chromatography column, continuously wash it with gravity sedimentation combined with distilled water, gradually remove the air bubbles in the column and balance the column bed until the surface is flat and dense, and retain a 2 cm liquid layer at the top of the column to maintain the stability of the column bed. After the column bed is stable, use a wide-mouth pipette to slowly add the sample along the inner wall of the chromatography column, avoiding directly impacting the gel surface and causing bed disturbance. Turn on the peristaltic pump to control the elution flow rate to ensure that the liquid level in the column always covers the gel surface to prevent cracking. Synchronously start the outlet valve at the lower end of the column and collect the eluate.

[0167] 3 Concentration

[0168] Transfer the purified liquid in Step 2 to a rotary evaporation flask (the liquid loading amount is strictly controlled below 2 / 3 of the nominal volume of the flask body to avoid the risk of overflow during high-speed rotation). Set the temperature of the constant temperature water bath to 30 ± 1 °C, adjust the rotary motor to a constant speed of 50 rmp. After installing the rotary evaporation flask (ensure good airtightness), turn on the vacuum pump. After the system pressure gradient is stable, synchronously start the rotary evaporator and monitor the liquid state in the bottle in real time. When the solvent in the system evaporates to close to the preset concentrated volume threshold, collect the concentrated sample and freeze-dry it for standby.

[0169] 4 Obtaining the Sample

[0170] Put the freeze-dried powder into a packaging bag, such as Figure 7 , which is light in texture, slightly yellowish and off-white in color, and extremely soluble in water. However, since it is easy to absorb moisture when exposed to air and is prone to deterioration at room temperature, it should be stored under low-temperature and dry conditions.

[0171] Structural Characteristics of Fourier Transform Infrared Spectroscopy Analysis of Samples in Example 5

[0172] 1 Experimental Method

[0173] Weigh 1 mg of the top three samples in terms of peptide content and potassium bromide into an agate mortar, grind and mix them evenly (the mass ratio of the sample to potassium bromide is within the range of 1:100 - 1:150). Then place them in a tablet press and keep at 15 MPa for 20 s to press into tablets for on-machine monitoring. At 4000 - 400 cm -1 , with a resolution of 4 cm -1 , scan 32 times to obtain the Fourier transform infrared spectrogram.

[0174] 2 Experimental Results

[0175] The results are as Figure 8 shown. There are stretching vibrations of N - H and O - H bonds near 3400 cm -1 for all three samples, which may be due to the presence of peptide bonds in the samples. There is also an asymmetric stretching vibration of C - H near 2900 cm -1 for the samples. By analyzing the absorption characteristics of the amide I band (1600 - 1700 cm-1) and the amide III band (1220 - 1330 cm-1), important information about the protein secondary structure can be provided. There is a strong absorption peak near 1650 cm -1 for all three samples, which is the amide I band (C=O stretching vibration), a distinct characteristic of the peptide bond (-CONH-). There is an absorption peak near 1250 cm -1 in the amide III band, with a significant intensity. It can be speculated that the samples are soybean meal peptides and the secondary structure is mainly dominated by β-sheets.

Claims

1. A method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme, characterized in that, It includes the following steps: 1) Dry the soybean meal, sieve out impurities, grind it with a pulverizer, and sieve to obtain soybean meal powder; 2) Take the complex enzyme; 3) Add the complex enzyme in step 2) to the buffer solution and mix evenly; 4) Take the soybean meal powder after sieving with a material-liquid ratio of 1:0.5 to the buffer solution, and add the complex enzyme buffer solution obtained in step 3); 5) Start enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate it, dry it in a constant-temperature drying oven. After the drying is completed, grind it for standby; 6) Weigh the enzymatically hydrolyzed soybean meal powder, add deionized water, mix well and place it in a constant-temperature shaking incubator to shake. Then transfer the mixed solution to a 4°C refrigerator for refrigeration. After taking it out, centrifuge for 15 min, and collect the supernatant for standby; 7) Purify the supernatant obtained in step 6) using Sephadex G-25; 8) Concentrate the liquid obtained in step 7) using a rotary evaporator; 9) After freeze-drying the concentrated solution obtained in step 8), obtain slightly yellow powdery soybean meal peptides.

2. The method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of a composite enzyme on soybean meal according to claim 1, wherein In step 2), the complex enzyme is one or a mixture of more than one of alkaline protease, neutral protease, trypsin, papain, keratinase, SFQ enzyme, and pectinase.

3. A method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme according to claim 2, wherein In step 2), the complex enzyme is an equal proportion mixture of alkaline protease, neutral protease, and trypsin.

4. A method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme according to claim 2, characterized in that, In step 2), by mass ratio, complex enzyme:soybean meal powder = 1:

100.

5. A method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme according to claim 1, characterized in that, In step 3), the pH of the buffer solution is 6.

5.

6. The method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of a composite enzyme on soybean meal according to claim 1, characterized in that, In step 3), in the buffer solution, the concentration of the complex enzyme is 0.02 g / mL.

7. A method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme according to claim 1, characterized in that, In step 5), the enzymatic hydrolysis and drying are both carried out at 50°C, where the enzymatic hydrolysis time is 7 h and the drying time is overnight.

8. A method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a composite enzyme according to claim 1, characterized in that, In step 6), the shaking time and refrigeration time are 8 h, and the centrifugation conditions are 4°C and 4000 r / min.

9. Slightly yellow powdery soybean meal peptides prepared by the method for preparing soybean meal peptides by solid-state enzymatic hydrolysis of soybean meal with a complex enzyme according to any one of claims 1-8.

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