Low-bitterness high-functionality soybean meal protein hydrolysate and application thereof

Abstract

The invention belongs to the technical field of soybean meal products, and particularly relates to a low-bitterness high-functionality soybean meal protein hydrolysate and application thereof. The soybean meal protein hydrolysate is prepared by adopting a specific enzyme and a sequential enzymolysis procedure, and the obtained product has comprehensive performance advantages in the aspects of low bitterness, high oxidation resistance, foamability and emulsibility and keeps necessary nutrient substances, so that good application conditions are provided for application of the soybean meal protein hydrolysate in the fields of foods and food auxiliary materials.

Description

Technical Field

[0001] The present invention belongs to the technical field of soybean meal products, and particularly relates to a low-bitter and highly functional soybean meal protein hydrolysate and its uses. Background Art

[0002] Soybean meal is a by-product after soybean oil extraction, containing rich proteins (40% - 50%) and amino acids (14.3% - 16.5%). In 2024, the output of soybean meal in China reached 75.98 million tons, of which 89.4% was directly used in animal feed production without deep processing. The potential value of soybean meal has not been fully utilized, resulting in great resource waste. Soybean meal protein hydrolysate is a deep-processed product obtained by decomposing the proteins in soybean meal into small peptides and amino acids through chemical hydrolysis, enzymatic hydrolysis, or microbial fermentation processes. Soybean meal protein hydrolysate has been proven to have higher functional activities and physicochemical properties, including solubility, emulsifying property, easy absorbability, as well as antioxidant and angiotensin I-converting enzyme (ACE) inhibitory activities. These properties enable it to have a wider application range in foods, feeds, and biological products, and can be used as food additives, raw materials for health products, or nitrogen sources for fermentation media. Compared with soybean meal, its value and functionality are significantly improved. Although both soybean meal protein hydrolysate and soybean protein hydrolysate are derived from soybeans, due to differences in the composition and characteristics of the hydrolysis raw materials, there are obvious differences in their hydrolysates in terms of composition, processing technology, and application. The solubility and emulsifying property of soybean meal hydrolysate are relatively low, while the antioxidant property and foaming property of soybean protein hydrolysate are stronger.

[0003] In existing production methods of soybean meal protein hydrolysate, the chemical hydrolysis method has low production cost and relatively simple process, but it is difficult to control the degree of hydrolysis, and high temperature and strong acids and bases are likely to cause changes in amino acid composition and loss of nutritional components. The microbial fermentation method uses the metabolic activities of microorganisms to decompose soybean meal protein, which can eliminate the bad flavor and anti-nutritional factors of soybean meal and produce "microbial-derived nutrients", but the fermentation cycle is relatively long, the process control is more complex, and the quality of the hydrolysate is greatly affected by the strain and fermentation conditions. Compared with the above two methods, the enzymatic hydrolysis method uses single or combined protein hydrolases to hydrolyze soybean meal protein under mild conditions, which is not likely to cause amino acid damage, can obtain small peptides with high biological activity, and the degree of hydrolysis is controllable, making it suitable for producing high-value-added functional products to meet the market demand for high-quality and high-biological-activity soybean meal protein hydrolysate.

[0004] However, during the preparation of soybean meal protein hydrolysate by enzymatic hydrolysis, the protein peptide chains are cleaved, hydrophobic amino acid residues are exposed, and small peptides (bitter peptides) that can activate bitter receptors are gradually formed and released, resulting in a certain degree of bitterness in the finally produced soybean meal protein hydrolysate, significantly affecting the taste and flavor of the product and leading to a decrease in consumer acceptance. At the same time, there is a complex correlation between the functional properties (such as emulsifying property, foaming property, antioxidant property, etc.) of the protein hydrolysate and the bitterness intensity. The antioxidant property usually increases significantly with the increase in the degree of enzymatic hydrolysis. However, with the increase in the degree of hydrolysis, the generation of bitter peptides may also increase, resulting in an increase in bitterness intensity. To solve the bitterness limitation in the preparation of soybean meal protein hydrolysate by enzymatic hydrolysis, the existing technology mainly conducts additional debittering treatment on the soybean meal protein hydrolysate, such as removing bitter peptide components by adsorption or separation, or further hydrolyzing the bitter peptides into amino acids to reduce the bitterness intensity. However, due to the structural similarity between bitter peptides and functional peptides, both have similar hydrophobic amino acid residues, which can endow the peptide segments with functional properties and may also cause bitterness. After completely degrading the bitter peptides into amino acids, the bitterness can disappear, but the functional properties such as foaming property may also disappear or weaken due to the too-small size of the protein peptide segments. To sum up, there is a restrictive relationship between the functional properties such as emulsifying property, foaming property, and antioxidant property of the protein hydrolysate and the bitterness intensity. Achieving the balance between its functional properties and bitterness intensity is a complex problem of simultaneous optimization of multiple indicators. It is difficult to optimize other indicators one by one after optimizing one indicator. The reason is that the change of enzymatic hydrolysis parameters will lead to unexpected simultaneous changes in each indicator, and the optimization of bitterness and functionality leads to opposite requirements for the direction of the degree of enzymatic hydrolysis. Therefore, the final effect of multi-index optimization is difficult to predict.

[0005] The existing technology often only focuses on the single - aspect consideration and optimization of bitterness or functionality, lacking a preparation method for protein hydrolysates with both taste quality and functional effects. For example, the preparation of soybean meal protein hydrolysate by combining enzymatic method and acid method (CN103478401A) is followed by debittering treatment after hydrolysis, and the high - efficiency preparation process of a low - bitterness and high - quality soybean peptide disclosed in CN 105506045 A also uses activated carbon adsorption for debittering treatment after enzymatic hydrolysis with two endo - and exo - protein composite enzymes, namely neutral protease and compound flavor protease. The above - mentioned schemes do not consider the loss of functional properties of the soybean meal protein hydrolysate after debittering. At the same time, the additional debittering treatment will bring key problems such as increased processes and costs, bitterness recurrence, and loss of nutritional value. Similarly, in the research on strengthening the functional properties of soybean meal protein hydrolysate, such as "Study on the Antioxidant Activity of Degreased Soybean Meal Enzymatic Hydrolysate" (Xu Jing, Jiang Lianzhou, Li Yang, etc. Study on the Antioxidant Activity of Degreased Soybean Meal Enzymatic Hydrolysate [J]. Soybean Science & Technology, 2019, (S1): 147 - 150 + 173.), the enzymatic hydrolysis conditions are optimized with antioxidant activity as the only condition, without considering the bitterness intensity of the hydrolysis product and the acceptability of consumers.

[0006] The above problems lead to the current lack of soybean meal hydrolysate products with both low bitterness and multiple high functionalities, thus limiting their applications in the food field. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a soybean meal protein hydrolysate with low bitterness, high nutritional value, and improved multiple functions, as well as its uses.

[0008] The technical solution of the present invention is as follows:

[0009] First, the present invention provides a low-bitterness and high-functionality soybean meal protein hydrolysate. The preparation method of the soybean meal protein hydrolysate comprises the following steps:

[0010] S1: Crushing soybean meal and preparing it into a soybean meal suspension with distilled water;

[0011] S2: Adjusting the pH value of the soybean meal suspension to 7 - 9 and the temperature to 40°C - 65°C, adding PROTAMEX compound protease for enzymatic hydrolysis for 40 min - 80 min; after inactivating the enzyme, carrying out enzymatic hydrolysis with Flavourzyme for 40 min - 80 min at a pH value of 7 - 9 and a temperature of 40°C - 65°C;

[0012] S3: Inactivating the enzyme after enzymatic hydrolysis is completed and collecting the soybean meal protein hydrolysate.

[0013] Preferably, the Flavourzyme is Flavourzyme.

[0014] Preferably, the PROTAMEX compound protease is added at 0.1% - 1% of the substrate mass.

[0015] Preferably, the Flavourzyme is added at 1% - 3% of the substrate mass.

[0016] Preferably, in step S1, the soybean meal is crushed through a 60-mesh sieve and then prepared into a soybean meal suspension.

[0017] Preferably, in step S2, the pH value of the soybean meal suspension is adjusted with HCl or NaOH solution.

[0018] Secondly, the present invention provides the uses of the above-mentioned low-bitterness and high-functionality soybean meal protein hydrolysate, including:

[0019] (1) Use in the preparation of food antioxidants;

[0020] (2) Use in the preparation of food emulsifiers;

[0021] (3) Use in the preparation of food foaming agents;

[0022] (4) Use in the preparation of food antioxidants, emulsifiers and foaming agents.

[0023] The beneficial effects of the present invention are as follows:

[0024] (1) The low-bitter and highly functional soybean meal protein hydrolysate provided by the present invention has a significantly lower bitterness intensity than the hydrolysates prepared by other methods, high nutritional value, low molecular weight, and is easily absorbed. At the same time, the hydrolysate has stronger emulsifying and foaming properties and better antioxidant properties.

[0025] (2) The method for preparing the hydrolysate provided by the present invention does not require debittering treatment after enzymatic hydrolysis, reduces the operation process, effectively avoids the loss of active ingredients, and has mild and safe production conditions.

[0026] (3) The present invention enzymatically hydrolyzes soybean meal through a specific sequential enzymatic hydrolysis scheme, reducing the proportion of low-molecular peptides related to bitterness within a specific molecular weight range. The bitterness intensity of the obtained soybean meal protein hydrolysate is significantly lower than that of the products obtained by other existing hydrolysis methods. Description of the Drawings

[0027] Figure 1 is the electrophoresis pattern of soybean meal protein hydrolysates treated with different enzymatic hydrolyses;

[0028] Figure 2 is the molecular weight distribution pattern of soybean meal protein hydrolysates treated with different enzymatic hydrolyses;

[0029] Figure 3 is the particle size measurement result diagram of soybean meal protein hydrolysates treated with different enzymatic hydrolyses;

[0030] Figure 4 is the measurement result diagram of the emulsifying property and emulsifying stability of soybean meal protein hydrolysates treated with different enzymatic hydrolyses;

[0031] Figure 5 is the measurement result diagram of the foaming property and foaming stability of soybean meal protein hydrolysates treated with different enzymatic hydrolyses;

[0032] Figure 6 is the measurement result diagram of the 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of soybean meal protein hydrolysates treated with different enzymatic hydrolyses. Detailed Embodiments

[0033] The following further describes the present invention in detail with reference to the embodiments and the drawings, but the embodiments of the present invention are only used for the explanation and illustration of the technical solutions and are not regarded as a limitation of the protection scope.

[0034] In the following embodiments, the soybean meal is sourced from Shandong Guohong Biotechnology Co., Ltd.; all enzyme preparations used are commercially available products, among which the compound protease is Compound protease (1.6AU-N / g), the flavor protease is (1000LAPU / g) Flavorzyme and alkaline protease are (2.4AU-A / g) Alkaline protease, papain (800,000U / g), trypsin (250,000U / g), and pepsin (250u / mg) were all purchased from Beijing Solarbio Science & Technology Co., Ltd. Among them, compound protease belongs to endoprotease, and flavorzyme is exoprotease.

[0035] In the quantitative standard of protease addition described in the following examples, the substrate refers to soybean meal powder.

[0036] Example 1 Preparation of soybean meal protein hydrolysate

[0037] 1. Preparation of low-bitter and high-functional soybean meal protein hydrolysate

[0038] Soybean meal was crushed and passed through a 60-mesh sieve to make soybean meal powder;

[0039] Soybean meal powder and distilled water were prepared into a soybean meal suspension at a ratio of 1:10 (g / ml), and the pH was adjusted to 7.5 and the temperature was 50.0°C. Compound protease was added to the soybean meal suspension at 0.5% of the substrate mass, and was subjected to constant-temperature enzymatic hydrolysis at 50.0°C in a water bath. During this period, stirring was maintained and the pH was kept constant. After 70.0 min, it was taken out and inactivated by boiling water bath for 15 min. Flavorzyme was added to the compound protease hydrolysis solution at 1.15% of the substrate mass, the pH was adjusted to 7.2, and it was subjected to constant-temperature enzymatic hydrolysis at 50.0°C in a water bath for 60.0 min. Stirring was also maintained and the pH was kept constant. After inactivating the enzyme by boiling water bath for 15 min, it was centrifuged at 10000.0 rpm at 4.0°C for 20.0 min, and the supernatant was collected and stored at -20.0°C for later use.

[0040] 2. Preparation of other soybean meal protein hydrolysates

[0041] (1) Preparation of soybean meal protein hydrolysate in Comparative Example 1

[0042] The soybean meal protein hydrolysate under the same enzymatic hydrolysis conditions as in Example 1 with a single type of compound protease was prepared. The soybean meal suspension was prepared according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), and the pH was adjusted to 7.5 and the temperature was 50°C. Compound protease was added to the soybean meal suspension at 1.65% of the substrate mass, and was subjected to constant-temperature enzymatic hydrolysis at 50.0°C in a water bath for 130.0 min. During this period, stirring was maintained and the pH was kept constant. After completion, it was inactivated by boiling water bath for 15 min, centrifuged at 10000.0 rpm at 4.0°C for 20.0 min, and the supernatant was collected and stored at -20.0°C for later use.

[0043] (2) Preparation of the soybean meal protein hydrolysate of Comparative Example 2

[0044] The soybean meal protein hydrolysate was prepared by the single alkaline protease hydrolysis method disclosed in the reference document "Study on the Physicochemical Properties of Soybean Protein Isolate Hydrolysates" (Wang Liguo, Liu Rui, Zhang Min, etc. Study on the Physicochemical Properties of Soybean Protein Isolate Hydrolysates [J]. China Food Additives, 2018, (03): 73 - 79.). A soybean meal suspension was prepared at a ratio of soybean meal powder to distilled water of 1:10 (g / ml). Alkaline protease was added to the soybean meal suspension at 1.65% of the substrate mass, the pH was adjusted to 8.0, and after heat inactivation at 55.0 °C for 130.0 min in a water bath, centrifugation was carried out at 10000.0 rpm at 4.0 °C for 20.0 min, the supernatant was collected and stored at -20.0 °C for later use.

[0045] (3) Preparation of the soybean meal protein hydrolysate of Comparative Example 3

[0046] The soybean meal protein hydrolysate was prepared by the single flavor protease hydrolysis method disclosed in the reference document "Study on the Hydrolysis of Soybean Protein Isolate by Flavor Protease and Its Effect on Functional Properties" (Tian Jie, Chai Shasha, Hu Chuanrong, etc. Study on the Hydrolysis of Soybean Protein Isolate by Flavor Protease and Its Effect on Functional Properties [J]. China Oils and Fats, 2015, 40(10): 11 - 14.). A soybean meal suspension was prepared at a ratio of soybean meal powder to distilled water of 1:10 (g / ml). Flavor protease was added to the soybean meal suspension at 0.7% of the substrate mass, the pH was adjusted to 6.5, and after heat inactivation at 50.0 °C for 180.0 min in a water bath, centrifugation was carried out at 10000.0 rpm at 4.0 °C for 20.0 min, the supernatant was collected and stored at -20.0 °C for later use.

[0047] (4) Preparation of the soybean meal protein hydrolysate of Comparative Example 4

[0048] The preparation method of soybean meal protein hydrolysate by alkaline protease and flavor protease combined enzymatic hydrolysis published in the reference "Sensory Characteristics and Antioxidant Activities of Maillard Reaction Products from Soy Protein Hydrolysates with Different Molecular Weight Distribution" (Liu, P., Huang, M., Song, S. et al. Sensory Characteristics and Antioxidant Activities of Maillard Reaction Products from Soy Protein Hydrolysates with Different Molecular Weight Distribution. Food Bioprocess Technol 5, 1775–1789 (2012).): Prepare a soybean meal suspension according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml). Add 0.024 AU / g of alkaline protease to the soybean meal suspension, adjust the pH to 8.0, and react at a constant temperature of 58.0 °C in a water bath for 180 min. Then, add 2.0 LAPU / g of flavor protease to the alkaline protease hydrolysis solution, adjust the pH to 6.5, and react at a constant temperature of 50.0 °C in a water bath for 240 min. After inactivating the enzyme, centrifuge at 10000.0 rpm at 4.0 °C for 20.0 min, collect the supernatant, and store it at -20.0 °C for later use.

[0049] (5) Preparation of soybean meal protein hydrolysate in Comparative Example 5

[0050] Select trypsin as the endoprotease to prepare soybean meal protein hydrolysate under the same enzymatic hydrolysis conditions as in Example 1. Prepare a soybean meal suspension according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), adjust the pH to 7.5, and the temperature to 50 °C. Add trypsin to the soybean meal suspension at 0.5% of the substrate mass, and carry out enzymatic hydrolysis at a constant temperature of 50.0 °C in a water bath for 70.0 min. After inactivating the enzyme in a boiling water bath for 15 min, add flavor protease to the trypsin hydrolysis solution at 1.15% of the substrate mass, adjust the pH to 7.2, and react at a constant temperature of 50.0 °C in a water bath for 60.0 min. After inactivating the enzyme, centrifuge at 10000.0 rpm at 4.0 °C for 20.0 min, collect the supernatant, and store it at -20.0 °C for later use.

[0051] (6) Preparation of soybean meal protein hydrolysate in Comparative Example 6

[0052] Papain was selected as the endoprotease to prepare soybean meal protein hydrolysate under the same enzymatic hydrolysis conditions as in Example 1. A soybean meal suspension was prepared according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), the pH was adjusted to 7.5, and the temperature was 50 °C. Papain was added to the soybean meal suspension at 0.5% of the substrate mass, and the enzymatic hydrolysis was carried out at a constant temperature of 50.0 °C in a water bath for 70.0 min. After inactivating the enzyme in a boiling water bath for 15 min, flavor protease was added to the papain hydrolysate at 1.15% of the substrate mass, the pH was adjusted to 7.2, and the reaction was carried out at a constant temperature of 50.0 °C in a water bath for 60.0 min. After inactivating the enzyme, centrifugation was carried out at 10000.0 rpm at 4.0 °C for 20.0 min, and the supernatant was collected and stored at -20.0 °C for later use.

[0053] (7) Preparation of soybean meal protein hydrolysate in Comparative Example 7

[0054] Pepsin was selected as the endoprotease to prepare soybean meal protein hydrolysate under the same enzymatic hydrolysis conditions as in Example 1. A soybean meal suspension was prepared according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), the pH was adjusted to 7.5, and the temperature was 50 °C. Pepsin was added to the soybean meal suspension at 0.5% of the substrate mass, and the enzymatic hydrolysis was carried out at a constant temperature of 50.0 °C in a water bath for 70.0 min. After inactivating the enzyme in a boiling water bath for 15 min, flavor protease was added to the pepsin hydrolysate at 1.15% of the substrate mass, the pH was adjusted to 7.2, and the reaction was carried out at a constant temperature of 50.0 °C in a water bath for 60.0 min. After inactivating the enzyme, centrifugation was carried out at 10000.0 rpm at 4.0 °C for 20.0 min, and the supernatant was collected and stored at -20.0 °C for later use.

[0055] (8) Preparation of soybean meal protein hydrolysate in Comparative Example 8

[0056] Soybean meal protein hydrolysate with the same types of enzymatic hydrolysis and sequential enzymatic hydrolysis method as in Example 1 but different enzymatic hydrolysis conditions was prepared. A soybean meal suspension was prepared according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), the pH was adjusted to 6.0, and the temperature was 50 °C. Compound protease was added to the soybean meal suspension at 0.5% of the substrate mass, and the enzymatic hydrolysis was carried out at a constant temperature of 50.0 °C in a water bath for 70.0 min. After inactivating the enzyme in a boiling water bath for 15 min, flavor protease was added to the compound protease hydrolysate at 1.15% of the substrate mass, the pH was adjusted to 6.0, and the reaction was carried out at a constant temperature of 50.0 °C in a water bath for 60.0 min. After inactivating the enzyme, centrifugation was carried out at 10000.0 rpm at 4.0 °C for 20.0 min, and the supernatant was collected and stored at -20.0 °C for later use.

[0057] (9) Preparation of soybean meal protein hydrolysate in Comparative Example 9

[0058] Prepare a soybean meal protein hydrolyzate with the same types of enzymatic hydrolysis and sequential enzymatic hydrolysis method as in Example 1, but with different enzymatic hydrolysis conditions. Prepare a soybean meal suspension according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), adjust the pH to 7.5, and the temperature to 65.0 °C. Add compound protease to the soybean meal suspension at 0.5% of the substrate mass, and carry out constant-temperature enzymatic hydrolysis at 65 °C in a water bath for 70.0 min. After inactivating the enzyme in a boiling water bath for 15 min, add flavor protease to the compound protease hydrolysis solution at 1.15% of the substrate mass, adjust the pH to 7.2, carry out a constant-temperature reaction in a water bath at 65.0 °C for 60.0 min, inactivate the enzyme, and then centrifuge at 10000.0 rpm at 4.0 °C for 20.0 min, collect the supernatant, and store it at -20.0 °C for later use.

[0059] (10) Preparation of soybean meal protein hydrolyzate of Comparative Example 10

[0060] Prepare a soybean meal protein hydrolyzate with the same types of enzymatic hydrolysis and sequential enzymatic hydrolysis method as in Example 1, but with different enzymatic hydrolysis conditions. Prepare a soybean meal suspension according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), adjust the pH to 7.5, and the temperature to 50 °C. Add compound protease to the soybean meal suspension at 0.1% of the substrate mass, and carry out constant-temperature enzymatic hydrolysis at 55 °C in a water bath for 70.0 min. After inactivating the enzyme in a boiling water bath for 15 min, add flavor protease to the compound protease hydrolysis solution at 1.15% of the substrate mass, adjust the pH to 7.2, carry out a constant-temperature reaction in a water bath at 50.0 °C for 60.0 min, inactivate the enzyme, and then centrifuge at 10000.0 rpm at 4.0 °C for 20.0 min, collect the supernatant, and store it at -20.0 °C for later use.

[0061] (11) Preparation of soybean meal protein hydrolyzate of Comparative Example 11

[0062] Prepare a soybean meal protein hydrolyzate with the same types of enzymatic hydrolysis and sequential enzymatic hydrolysis method as in Example 1, but with different enzymatic hydrolysis conditions. Prepare a soybean meal suspension according to the ratio of soybean meal powder to distilled water of 1:10 (g / ml), adjust the pH to 7.5, and the temperature to 50 °C. Add compound protease to the soybean meal suspension at 0.5% of the substrate mass, and carry out constant-temperature enzymatic hydrolysis at 55 °C in a water bath for 70.0 min. After inactivating the enzyme in a boiling water bath for 15 min, add flavor protease to the compound protease hydrolysis solution at 2.50% of the substrate mass, adjust the pH to 7.2, carry out a constant-temperature reaction in a water bath at 50.0 °C for 60.0 min, inactivate the enzyme, and then centrifuge at 10000.0 rpm at 4.0 °C for 20.0 min, collect the supernatant, and store it at -20.0 °C for later use.

[0063] Example 2, Test Example 1, Bitter taste sensory evaluation

[0064] Using quinine as the reference substance, prepare a quinine solution with a concentration of 0.1×10 -4 g / ml as the standard solution, and set the bitterness value score to 0-10. Score according to the bitterness of the standard solution. The evaluation group consists of 10 people (5 males and 5 females). After rinsing their mouths, the evaluators use a disposable straw to take 2.0 mL of the soybean meal hydrolysate and place it in their mouths. Perform a rinsing action throughout the mouth so that the back of the tongue can feel the bitterness of the bitter substances. After 10.0 s, spit out the sample, rinse the mouth with water 3 times, taste the standard solution with a similar bitterness level, confirm whether the bitterness is similar, and repeatedly compare it with the standard bitterness value solution until the bitterness value is determined. The result is the average value evaluated by 10 people.

[0065] Table 1 Sensory bitterness intensity of soybean meal protein hydrolysates prepared under different methods or conditions

[0066]

[0067] The bitterness intensity of soybean meal protein hydrolysates under different enzymatic hydrolysis treatments was analyzed by sensory evaluation, and the results are shown in Table 1. Under the sequential enzymatic hydrolysis of compound protease and flavor protease, the bitterness score of the soybean meal protein hydrolysate obtained in Example 1 was relatively low, around 0.5. The bitterness scores of the soybean meal protein hydrolysates of Comparative Examples 1-4 obtained by the single-enzyme enzymatic hydrolysis or combined enzymatic hydrolysis methods published in the literature were significantly higher than those of the examples. Among them, the bitterness score of the product of the single alkaline protease hydrolysis in Comparative Example 2 was the highest. Because the alkaline protease itself has a relatively broad selection of hydrolysis sites and a higher degree of hydrolysis, the content of hydrophobic amino groups and bitter peptides in the hydrolysate is higher. In Comparative Example 1 and Comparative Example 3, compound protease and flavor protease were used for single-enzyme enzymatic hydrolysis respectively. The results showed that the bitterness scores of the hydrolysates generated by using the above two enzymes alone were much higher than the bitterness value of the sequential enzymatic hydrolysis product of Example 1, indicating the superiority of the sequential enzymatic hydrolysis method of the present invention. Comparative Example 4 is the combined enzymatic hydrolysis method of alkaline protease and flavor protease published in the literature. Compared with Comparative Example 2 using alkaline protease alone, its bitterness value was significantly reduced, but it was still higher than that of Example 1 of the present invention, which proved the superiority of the selected enzyme combination and sequential enzymatic hydrolysis method of the present invention. Comparative Examples 5-7 were sequential enzymatic hydrolysis with different combinations of endonucleases and flavor protease, and their bitterness scores were all lower than that of Example 1, which proved the superiority of the selected enzyme combination of the present invention. Comparative Examples 8-11 were enzymatic hydrolysis conditions not limited by the present invention. When the enzyme group and enzymatic hydrolysis method were the same, if the enzymatic hydrolysis conditions including pH, temperature, enzyme addition amount, enzyme ratio, etc. exceeded the scope defined by the present invention, the bitterness score of the prepared soybean meal hydrolysate was also higher than that of the example, indicating the superiority of the enzymatic hydrolysis conditions of the present invention.

[0068] Example 3 Test Example 2 Electronic tongue bitterness analysis

[0069] Testing was carried out using a taste sensing system SA402B (Insent Inc., Atsugi chi, Japan). The electronic tongue consists of a bitterness sensor (SB2C00), an astringency sensor (SB2AE1), and a saltiness sensor (SB2CT0). A 0.01 g / mL sample was used for electronic tongue measurement. Potassium chloride (2.2365 rpm) and tartaric acid (0.045 rpm) were mixed with 1000.0 mL of deionized water to prepare a control solution. The results were compared with the data measured from the unhydrolyzed soybean meal as the zero point, and the bitterness response value and the bitterness aftertaste response value were selected as the key criteria for comparison.

[0070] Table 2 Bitterness and bitterness aftertaste of soybean meal protein hydrolysates prepared by different methods or conditions measured by electronic tongue

[0071]

[0072] In the electronic tongue test, the bitterness value mainly describes the immediate feeling when entering the mouth, while the bitterness aftertaste value represents the persistence and residual degree of bitterness. As shown in Table 2, the comparison results of the electronic tongue bitterness values of each test sample are consistent with the sensory evaluation results, proving the effectiveness of bitterness detection. In Example 1, the bitterness value is 10.08 and the bitterness aftertaste value is 1.59. In Comparative Examples 1-3, the single-enzyme hydrolysis method published in the literature has a bitterness value in the range of 15.25 - 18.3 and a bitterness aftertaste value in the range of 5.13 - 6.71, which are significantly higher than those in Example 1, indicating the superiority of the sequential enzyme hydrolysis method for producing low-bitterness protein hydrolysates in the present invention. In Comparative Example 4, according to the combined enzyme hydrolysis method published in the literature, the bitterness value and the bitterness aftertaste value are 14.64 and 4.14 respectively, which are also higher than those in Example 1. In Comparative Examples 5-7, sequential enzyme hydrolysis is carried out with different combinations of endonucleases and flavor proteases, and their bitterness values are in the range of 14.24 - 14.96 and the bitterness aftertaste values are in the range of 4.05 - 4.32, both of which are lower than those in Example 1, which proves the superiority of the selected enzyme combination in the present invention. Finally, in Comparative Examples 8-11, the enzyme hydrolysis conditions are not limited by the present invention. When the enzyme group and the enzyme hydrolysis method are the same, if the enzyme hydrolysis conditions, including pH, temperature, enzyme addition amount, enzyme ratio, etc., exceed the range defined by the present invention, the bitterness value and the bitterness aftertaste value of the prepared soybean meal hydrolysate are also higher than those in Example 1, indicating the superiority of the enzyme hydrolysis conditions in the present invention.

[0073] Example 3 Test Example 3 SDS-PAGE analysis

[0074] The enzymatic hydrolysis process of soybean meal hydrolysates prepared by different methods or under different conditions was analyzed by SDS-PAGE. The concentrations of the stacking gel and the separating gel were selected as 5.0% and 12.0%. After mixing 100.0 μL of the soybean meal hydrolysate with 25.0 μL of 5× protein loading buffer and boiling for 6.0 min, 10.0 μL was taken for loading (the total amount of loaded protein was 50.0 μg). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to determine the protein band distribution of the samples under reducing conditions.

[0075] The results are shown in Figure 1 . M is the standard protein Mark band, SM is the sample before the start of hydrolysis, and the numbers represent the samples at different enzymatic hydrolysis times. As shown in the figure, after the start of hydrolysis of each sample, the protein bands with a molecular weight exceeding 35 kDa gradually disappeared, new protein bands were generated in the range of 35.0 - 17.0 kDa, and the protein bands became darker in the range of 17.0 - 11.0 kDa. This indicates that the soybean meal proteins with large molecular weights are hydrolyzed into small molecular weight proteins or peptides under the action of enzymes, which are more easily absorbed by the human body. Since the same enzyme combination and sequential enzymatic hydrolysis method were used, the band change trends of Example 1 and Comparative Example 11 were similar. The differences in enzymatic hydrolysis conditions led to differences in the final bands. The enzyme addition amount in Comparative Example 11 was higher, and the band at 35 kDa disappeared more completely. In comparison, Comparative Example 4 was enzymatically hydrolyzed with a combination of alkaline protease and flavor protease published in the literature, and the molecular weight distribution range of its final bands was larger than that of Example 1, and the retention degree of the large molecular weight protein bands was higher, and the hydrolysis effect was lower than that of Example 1. Similarly, Comparative Examples 5 - 7 were sequentially enzymatically hydrolyzed with different combinations of endonucleases and flavor proteases, and more large molecular weight protein bands remained after the enzymatic hydrolysis. Among them, the sequential enzymatic hydrolysis effect of trypsin and flavor protease in Comparative Example 5 was the worst, and the bands in the range of 35 - 75 kDa did not disappear. The above results illustrate the superiority of the specific enzyme combination sequential enzymatic hydrolysis method of the present invention in producing protein hydrolysates for digestion and absorption under defined enzymatic hydrolysis conditions.

[0076] Determination of the molecular weight distribution of soybean meal protein in Test Example 4 of Example 4

[0077] The molecular weight (MW) distribution of soybean meal protein hydrolysates prepared under different methods or conditions was determined using an HPLC system (Waters 2695 / 2996, Waters, USA). HPLC was performed using a BioCore SEC-300 (5 μm, 7.8 × 300 mm) column, eluting the samples with 0.15 mol / L sodium phosphate buffer (pH 7.0) at a column temperature of 25.0 °C and a flow rate of 0.7 mL / min, and measuring at 280.0 nm. The standards were IgM 750000.0 Da, IgG 150000.0 Da, BSA 66000.0 Da, cytochrome c 12365.0 Da, small peptide 1 5319.0 Da, small peptide 2 3246.0 Da, and tyrosine 181.0 Da.

[0078] Figure 2 The molecular weight distribution of the test samples is shown, which is consistent with the results of SDS-PAGE analysis. Compared with Comparative Examples 4-7, Example 1 had the highest proportion of small molecular weight bands (≤0.5 kDa) and the lowest proportion of large molecular weight bands (>50 kDa), indicating excellent protein hydrolysis effect and the ability to convert macromolecular proteins into small molecular proteins or peptides. At the same time, research reports that polypeptide bitterness is closely related to its molecular weight distribution, and the bitterness of soy peptides is mainly related to medium molecular weight peptides of 0.5 - 4 kDa, with lower molecular weight components having lower bitterness than higher molecular weight components. Example 1 had the largest proportion of peptides within ≤0.5 kDa, which was 55.49%, and the lowest total proportion of peptides in the medium molecular weight range of 0.5 - 4 kDa, which was consistent with the bitterness measurement results.

[0079] Example 5, Test Example 5 Amino Acid Analysis

[0080] The amino acid composition of soybean meal protein hydrolysates was determined using an amino acid analyzer, and the contents of essential amino acids and hydrophobic amino acids were calculated. The samples were hydrolyzed with 6.0 mol / L HCl at 110.0 °C in a sealed vacuum glass tube for 24.0 h, and the hydrolysis products were derivatized with phenyl isothiocyanate. The free amino acid components were obtained by mixing the samples with 10.0% trichloroacetic acid, precipitating for 2.0 h, and then centrifuging at 11000.0 rpm for 15.0 min. The supernatant was adjusted to pH 2.0 with aqueous HCl solution and filtered through a 0.45 μm microfiltration membrane.

[0081] Table 3 Amino Acid Composition of Soybean Meal Protein Hydrolysates Prepared under Different Methods or Conditions

[0082]

[0083]

[0084] As shown in Table 3, the ratios of essential amino acids to total amino acids (E / T) in all samples were between 31.84% and 37.5%, indicating that the proportions of essential amino acids in these samples were relatively high and they had good nutritional value. The AAS of Comparative Example 4 was the highest (50.8), indicating that its amino acid composition was closest to the reference protein pattern and had high nutritional value. The AAS of Example 1 was 48.03, also showing good amino acid balance. Comparative Example 11, which used the same enzyme combination and sequential enzymatic hydrolysis method as Example 1, was still lower than Example 1 in terms of E / T and AAS. The above results prove that the low-bitter soybean meal protein hydrolysate prepared by the present invention has high nutritional value.

[0085] Example 6 Determination of particle size in Test Example 6

[0086] Dilute the soybean meal protein hydrolysate to 0.4 mg / mL, and use a nanoparticle size analyzer to measure the particle size of the sample. Measurement conditions: temperature is 25.0 °C, and the refractive index of the protein is 1.145.

[0087] The results of the particle size determination of the soybean meal protein hydrolysate are as Figure 3 shown. The particle size values of each group of hydrolysates were significantly smaller than those of the unhydrolyzed soybean meal (P < 0.05). Among them, the particle size value of Example 1 was the lowest, and that of Comparative Example 5 was the highest. The smaller particle size helps to improve the adsorption rate at the interface and the number of exposed active sites, and improve the solubility, emulsifying property, foaming property and antioxidant property of the protein hydrolysate. The above results prove the superiority of the low-bitter soybean meal protein hydrolysate prepared by the present invention in terms of particle size.

[0088] Example 7 Determination of emulsifying property in Test Example 7

[0089] Add 10.0 mL of the hydrolyzate at 8.0 mg / mL and 250.0 μL of soybean oil (40:1; v / v) into a clean 50.0 mL beaker. Set the rotation speed of the high-speed homogenizer to 15,000.0 rpm, and continuously stir the oil-water mixture in the above beaker with the high-speed homogenizer for 2.0 min. After the stirring stops, immediately use a 50.0 μL microinjector to absorb the liquid at the bottom of the emulsion, mix it with 8.0 mL of 0.1% (w / v) sodium dodecyl sulfate solution with a vortex oscillator, and measure its absorbance at a wavelength of 500.0 nm. After 10.0 min, absorb the liquid from the bottom again and measure its absorbance. Use the sodium dodecyl sulfate solution with the same concentration as the blank control. Calculate the emulsifying activity (EAI) and emulsifying stability (ESI) according to the following formula:

[0090]

[0091] ESI(%) = A 10 / A0 × 100

[0092] Where: C is the sample concentration (g / mL); — the proportion of the oil phase in the emulsion; D is the dilution factor; A0 is the initial absorbance value of the emulsion; A 10 — the absorbance value of the emulsion after 10 min

[0093] Figure 4 is the emulsifying property of the soybean meal protein hydrolysate prepared by different enzyme combinations. The size of EAI is related to the ability of proteins or polypeptides to adsorb at the oil-water interface, while ESI is related to the stability of proteins or polypeptides. As Figure 4 shown, after enzymatic hydrolysis of soybean meal, the EAI of its hydrolysate was significantly higher than that of the unhydrolyzed soybean meal (P < 0.05). This is because enzymatic hydrolysis produces more low-molecular peptides, increasing the adsorption of the hydrolysate at the oil-water interface. Example 1 showed the best emulsifying performance, with EAI and ESI being 215.05 g / m 2 and 149.27 min, respectively. This may be due to the relatively large number of low-molecular-weight peptide segments in the hydrolysate of Example 1, which is beneficial to the formation and stability of the emulsion. The low-bitter soybean meal protein hydrolysate prepared in Example 1 of the present invention simultaneously has significantly excellent emulsifying properties, which are superior to other published methods.

[0094] Example 8 Determination of the foaming property in Test Example 8

[0095] Take 20.0 mL of a sample solution with a concentration of 0.02 g / ml into a flat-bottom centrifuge tube, and continuously stir with a high-speed homogenizer at a speed of 10000.0 rpm for 2.0 min. Immediately after the stirring is completed, measure the foam height at this time with a graduated cylinder and record it as V1. After 30 min, measure its foam height again and record it as V2.

[0096] The formula for the foaming capacity (FC) is as follows:

[0097]

[0098] The formula for the foaming stability (FS) is as follows:

[0099]

[0100] Figure 5 is the foaming property and foaming stability of the soybean meal protein hydrolysate prepared by different enzyme combinations. Foaming property is an important functional property of proteins, which can endow foods with excellent texture properties and a uniform and delicate taste, and plays an important role in food processing fields such as bakery products, ice cream, beer, and aerated candies. As Figure 5As shown, Example 1 is significantly superior to the unhydrolyzed protein and each comparative example. Example 1 has the highest FC and FS, which are 255.15% and 59.91% respectively. Followed by Comparative Example 11, with FC and FS being 212.89% and 47.32% respectively. The above results prove that the low-bitter soybean meal protein hydrolyzate prepared by the present invention has significantly excellent foaming properties at the same time, which is superior to other published methods.

[0101] Example 9 Test Example 9 Determination of DPPH Free Radical Scavenging Activity

[0102] Prepare 0.2 mmol / L DPPH solution with absolute ethanol for standby. Mix 100.0 μL of the sample with an equal volume of DPPH solution, react in the dark for 30.0 min, and measure the absorbance A1 at a wavelength of 517.0 nm. Use deionized water to replace the sample as a blank control to measure the absorbance A0. The DPPH free radical scavenging activity is calculated using the following equation:

[0103] DPPH scavenging rate (%) = [(A0 - A1)] / A0 × 100

[0104] Where A1 sample is the absorbance of the sample; A0 blank is the absorbance of the blank.

[0105] Figure 6 For the DPPH free radical scavenging activity of soybean meal protein hydrolyzates prepared by different enzyme combinations. DPPH free radical scavenging is a commonly used method for measuring antioxidant activity. DPPH is a stable free radical that is scavenged by pairing with a single electron of an antioxidant. The antioxidant ability of unhydrolyzed soybean meal protein is weak, mainly because its structure is relatively complete and few active groups are exposed. The DPPH free radical scavenging ability of each hydrolyzate obtained after enzymatic hydrolysis has been significantly improved. Among the groups of samples, Example 1 showed the highest DPPH free radical scavenging activity of 73.69%, followed by Comparative Example 11 and Comparative Example 4, which may be related to the large number of low molecular weight peptide segments produced by hydrolysis with specific enzyme combinations. The above results prove that the low-bitter soybean meal protein hydrolyzate prepared by the present invention has excellent antioxidant properties at the same time, which is superior to other published methods.

Claims

1. A low-bitter and highly functional soybean meal protein hydrolyzate, characterized in that, The preparation method of the soybean meal protein hydrolyzate comprises the following steps: S1: Crush the soybean meal and prepare it into a soybean meal suspension with distilled water; S2: Adjust the pH value of the soybean meal suspension to 7-9 and the temperature to 40°C-65°C, add PROTAMEX compound protease and enzymatically hydrolyze for 40 min-80 min; after inactivating the enzyme, use Flavourzyme to enzymatically hydrolyze for 40 min-80 min at pH 7-9 and 40°C-65°C; S3: Inactivate the enzyme after the enzymatic hydrolysis is completed and collect the soybean meal protein hydrolyzate.

2. The low-bitter and high-functional soybean meal protein hydrolyzate according to claim 1, characterized in that, The flavor protease is Flavourzyme flavor protease.

3. A low-bitter and high-functional soybean meal protein hydrolyzate according to claim 1, characterized in that, The PROTAMEX compound protease is added in an amount of 0.1%-1% based on the mass of the substrate.

4. A low-bitter and highly functional soybean meal protein hydrolyzate according to claim 1, wherein the Flavourzyme is added in an amount of 1%-3% based on the mass of the substrate.

5. A low-bitter and highly functional soybean meal protein hydrolyzate according to claim 1, wherein, In the step S1, the soybean meal is crushed through a 60-mesh sieve and then prepared into a soybean meal suspension.

6. The low-bitter and high-functional soybean meal protein hydrolyzate according to claim 1, characterized in that, In the step S2, the pH value of the soybean meal suspension is adjusted with HCl or NaOH solution.

7. Use of a low-bitter and highly functional soybean meal protein hydrolyzate according to claim 1 in the preparation of a food antioxidant.

8. Use of a low-bitter and highly functional soybean meal protein hydrolyzate according to claim 1 in the preparation of a food emulsifier.

9. Use of a low-bitter and highly functional soybean meal protein hydrolyzate according to claim 1 in the preparation of a food foaming agent.

10. Use of a low-bitter and highly functional soybean meal protein hydrolyzate according to claim 1 in the preparation of a food antioxidant, emulsifier and foaming agent.

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