阅读说明：本技术 一种具有降血脂活性的无苦味大豆多肽的制备方法 (Preparation method of bitter-free soybean polypeptide with blood fat reducing activity ) 是由 李理 肖聪丽 陈敏 于 2021-09-09 设计创作，主要内容包括：本发明公开了一种具有降血脂活性的无苦味大豆多肽及其制备方法,将大豆分离蛋白与水搅拌后,于80～95℃搅拌均匀,冷却至室温并调节pH值为6～8,加入复合蛋白酶制剂和内切型的蛋白酶制剂,在45～60℃水浴中搅拌5～8h后灭酶；离心后取上清液,经真空冷冻干燥,得大豆多肽；本发明所制备的大豆多肽无苦味、风味良好、溶解性极佳、降血脂活性显著,其分子量＜1000Da范围内的组分可达65％～72％,主要为小分子肽,在一般食品、功能性食品、医药等领域极具开发和应用价值。(The invention discloses a bitter-free soybean polypeptide with blood fat reducing activity and a preparation method thereof, wherein isolated soybean protein and water are stirred, and then are uniformly stirred at 80-95 ℃, cooled to room temperature and adjusted to pH value of 6-8, a compound protease preparation and an internal cutting type protease preparation are added, and the mixture is stirred in a water bath at 45-60 ℃ for 5-8 hours to inactivate enzyme; centrifuging, collecting supernatant, and vacuum freeze drying to obtain soybean polypeptide; the soybean polypeptide prepared by the invention has no bitter taste, good flavor, excellent solubility and remarkable blood fat reducing activity, the components with the molecular weight less than 1000Da can reach 65-72 percent, and the soybean polypeptide is mainly micromolecular peptide and has great development and application values in the fields of general foods, functional foods, medicines and the like.)

1. A preparation method of bitter-free soybean polypeptide with hypolipidemic activity is characterized by comprising the following steps: stirring the isolated soy protein with water, uniformly stirring at 80-95 ℃, cooling to room temperature, adjusting the pH value to 6-8, adding a compound protease preparation and an internal cutting type protease preparation, stirring in a water bath at 45-60 ℃ for 5-8 h, and inactivating enzymes; centrifuging, collecting supernatant, and vacuum freeze drying to obtain soybean polypeptide;

the compound protease preparation is obtained by the following method: taking kilograms and liters as mass and volume units respectively, uniformly mixing bran and water according to the mass-volume ratio of 1: 0.5-1: 1.5, sterilizing, cooling to room temperature, inoculating a mucor seed solution and a lactobacillus seed solution, and culturing in a constant-temperature incubator at 25-35 ℃ for 24-72 hours; after fermentation, adding sterile water into the fermented culture, standing overnight, filtering, collecting filtrate, and centrifuging to obtain supernatant as compound protease preparation;

the Lactobacillus is Lactobacillus plantarum F8(Lactobacillus plantarum F8), Lactobacillus plantarum MF3(Lactobacillus plantarum MF3) or Lactobacillus rhamnosus MF9(Lactobacillus rhamnous MF 9); lactobacillus plantarum MF3, accession number GDMCC 61400; lactobacillus plantarum MF3, accession number GDMCC 61398; lactobacillus rhamnosus MF9 has a accession number GDMCC 61399.

2. The method for preparing soybean polypeptide with blood lipid reducing effect without bitter taste according to claim 1, wherein the Mucor is Actinomucor elegans As3.2778(Actinomucor elegans As3.2778); the preparation method of the mucor seed liquid comprises the following steps: inoculating actinomucor elegans As3.2778 into a PDA inclined test tube, culturing for 24-72 h in a constant-temperature incubator at 25-30 ℃, adding sterile water into the test tube, and oscillating to elute the actinomucor elegans spores on the surface of a culture medium to obtain a spore suspension; adjusting the concentration of the spore suspension to 10⁶～10⁷And (4) obtaining mucor seed liquid.

3. The method for preparing soybean peptide without bitter taste and with hypolipidemic activity according to claim 1, wherein the method for preparing lactobacillus seed solution comprises: inoculating lactobacillus plantarum F8, lactobacillus plantarum MF3 or lactobacillus rhamnosus MF9 into an MRS liquid culture medium, culturing for 12-24 h in a constant-temperature incubator at 28-42 ℃, washing thalli with sterile water, and adjusting the concentration to 10⁸～10⁹And (5) CFU/mL to obtain the lactobacillus seed solution.

4. The method for preparing soybean peptide without bitter taste and with hypolipidemic activity according to claim 1, wherein the inoculation method of the mucor seed solution and the lactobacillus seed solution is as follows: and (2) inoculating the mucor seed liquid into a culture medium independently, or respectively inoculating the mucor seed liquid into the culture medium together with lactobacillus plantarum F8, lactobacillus plantarum MF3 and lactobacillus rhamnosus MF9, wherein the inoculation amount is 4-7% by mass percentage.

5. The method for producing a bitter-free soybean polypeptide having a hypolipidemic activity according to claim 1, wherein the mass-to-volume ratio of the culture to sterile water is 1:4 to 1:8 in kg and l as mass and volume units, respectively.

6. The method for preparing soybean polypeptide having hypolipidemic activity without bitter taste according to claim 1, wherein the centrifugation conditions in step 1) are: the temperature is 4-8 ℃, the rotation speed is 6000-10000 rpm, and the time is 5-20 min.

7. The method of claim 1, wherein the endo-type protease is bromelain or alcalase synthase; the addition amount of the endo-protease is 1000-4000 u/g protein.

8. The method for preparing soybean peptide with blood lipid lowering activity without bitter taste according to claim 7, wherein the bromelain is food grade bromelain produced by Nanning Pombo Biotech limited; the alkaline protease is food grade Alcalase 2.4L alkaline protease produced by Novixin Biotechnology Limited, Denmark.

9. The method for preparing soybean polypeptide having a hypolipidemic activity and no bitterness, according to claim 1, wherein the mass-to-volume ratio of the soybean protein isolate to water after stirring the soybean protein isolate and water is 1:10 to 1:20 in terms of kg and l as mass and volume units; the stirring at 80-95 ℃ is carried out by magnetic stirring in a water bath at 80-95 ℃ for 15-30 min.

10. The method for preparing soybean peptide without bitter taste and with hypolipidemic activity according to claim 1, wherein the enzyme deactivation is carried out at 85-105 ℃ for 10-30 min in terms of kilogram and liter as mass and volume units; the standing overnight is standing overnight in a refrigerator at the temperature of 3-4 ℃; the filtering is carried out by using 4-8 layers of sterile gauze; after centrifugation, the temperature of centrifugation in the supernatant is 4-8 ℃, the rotating speed is 6000-10000 rpm, and the time is 5-20 min.

Technical Field

The invention relates to soybean polypeptide, in particular to a preparation method of soybean polypeptide which has no bitter taste, good flavor and obvious blood fat reducing activity, belonging to the technical field of preparation processes of soybean polypeptide of functional foods.

Background

Hyperlipidemia is a disorder of blood lipid metabolism, and is mainly manifested by high level of Total Cholesterol (TC), Triglyceride (TG), low density lipoprotein cholesterol (LDL-C), and low level of high density lipoprotein cholesterol (HDL-C). Hyperlipidemia usually causes functional disorders and structural damages of endothelial cells, vascular walls and the like, and directly causes a series of diseases seriously harming human health, such as atherosclerosis, coronary heart disease and the like. At present, hyperlipidemia is mainly treated by lipid-lowering chemical drugs, common drugs comprise statins, resins, nicotinic acids, cholic acid chelating agents and the like, and the treatment cost is high, and the toxic and side effects are large. A large number of researches show that the food-derived natural active products including polysaccharides, flavonoids, polyphenols and the like have a certain regulating effect on blood fat, and can be used as functional natural active factors to be applied to the adjuvant treatment of hyperlipidemia and the like.

China is a major producing and consuming country of soybeans, and the soybeans are rich in protein and reasonable in amino acid composition, so that the soybean protein supplement is a high-quality plant protein supplement. The soybean meal protein content after the oil is extracted from the soybeans is further enriched, and the soybean meal protein is an ideal soybean protein resource. The soybean polypeptide is a compound generated by hydrolyzing soybean protein, the average peptide chain length of the soybean polypeptide is 2-10 amino acid residues, and the molecular weight is usually less than 3000 Da. Compared with soybean protein, the soybean polypeptide not only has excellent processing characteristics of high solubility, good stability and the like, but also is easier to digest and absorb by human bodies, has the functional characteristics of oxidation resistance, fatigue resistance, blood pressure reduction, organism immunity enhancement and the like, and is a functional raw material with wide application. At present, the preparation of soybean polypeptide is mainly based on an enzymolysis method, the method has mild reaction conditions, easy control, strong specificity and high product safety, but because enzymatic degradation exposes a large amount of hydrophobic groups in the molecular structure of protein, the polypeptide often has certain bitter taste and other bad flavors, the yield of soluble polypeptide is not high, the application of the soluble polypeptide in the field of food is severely limited, and the production efficiency and the economic benefit are influenced. Therefore, developing a soybean polypeptide product with good flavor, outstanding functionality and high yield is one of the important issues of focusing attention and urgently solving for many years in the industries of food, health care products and the like. At present, polypeptide products are mainly prepared into products with good flavor by methods such as an embedding method, a fermentation method, a multiple enzyme compounding method and the like, and functional characteristics of polypeptides are improved by a refining method, but the methods usually increase production cost or reduce the content of functional components.

Disclosure of Invention

The invention aims to provide a preparation method of soybean polypeptide, which has no bitter taste, good flavor, obvious blood fat reducing activity, simple and convenient process, easy control and suitability for industrial production, aiming at the problems in the prior art.

The functional soybean polypeptide without bitter taste and with remarkable blood fat reducing activity is prepared by automatically preparing the compound protease by a combined fermentation method of mucor and lactobacillus and then carrying out combined enzymolysis on the soybean protein isolate by adopting the compound protease and a commercial enzyme preparation. The invention is characterized in that the preparation of the compound protease. The actinomucor elegans is one of the dominant bacteria commonly used in the traditional fermented foods (such as fermented bean curd and mould bean dregs) in China, has the capability of secreting protease, alpha-amylase, alpha-galactosidase, lipase and other enzyme systems, takes the protease as the main component, and has relatively low activity of other enzymes. The abundant enzyme system produced by actinomucor elegans is one of the important factors for improving and forming the final flavor of the fermentation product. The lactobacillus is the most common strain in the prior dairy products and the traditional fermented plant foods in China, the proteolysis of the lactobacillus is a vital biochemical process in the fermentation process of the dairy products, the bean products and the like, and the lactobacillus has great influence on the final sensory quality of the fermented products. The molecular weight difference of the proteases secreted by different species of lactic acid bacteria is large and is between 30 and 300kDa, the optimal temperature range is generally 40 to 50 ℃, and the optimal pH value is generally 5 to 8. The invention respectively combines and inoculates actinomucor elegans As3.2778 with lactobacillus plantarum F8, lactobacillus plantarum MF3 and lactobacillus rhamnosus MF9 separated from fermented bean products into a bran solid culture medium, cultures for 24-72 h in a constant-temperature incubator at 25-35 ℃, extracts, filters and centrifuges to screen out a compound protease preparation with high protease activity.

Another feature of the invention resides in the preparation of soy polypeptides. At present, the preparation of soybean polypeptide mainly adopts a commercial protease enzymolysis method, and has the advantages of high enzymolysis efficiency, mild conditions and simple and convenient process. On the one hand, however, due to the fact that the types of commercial protease preparations are few and the acting sites are relatively single, polypeptides produced by enzymolysis tend to be homogeneous and have high bitterness values. On the other hand, the commercial protease is relatively expensive, so that the production cost is sharply increased, and is not suitable for industrial mass production. The invention prepares the soybean polypeptide by using the home-made compound protease and the commercial protease (including bromelain and Alcalase alkaline protease) for combined enzymolysis, thereby not only reducing the production cost, but also having no bitter taste, good flavor and remarkable functionality and being suitable for industrial production.

The purpose of the invention is realized by the following technical scheme:

a preparation method of soybean polypeptide without bitter taste and with hypolipidemic activity comprises: stirring the isolated soy protein with water, uniformly stirring at 80-95 ℃, cooling to room temperature, adjusting the pH value to 6-8, adding a compound protease preparation and an internal cutting type protease preparation, stirring in a water bath at 45-60 ℃ for 5-8 h, and inactivating enzymes; centrifuging, collecting supernatant, and vacuum freeze drying to obtain soybean polypeptide;

the compound protease preparation is obtained by the following method: taking kilograms and liters as mass and volume units respectively, uniformly mixing bran and water according to the mass-volume ratio of 1: 0.5-1: 1.5, sterilizing, cooling to room temperature, inoculating a mucor seed solution and a lactobacillus seed solution, and culturing in a constant-temperature incubator at 25-35 ℃ for 24-72 hours; after fermentation, adding sterile water into the fermented culture, standing overnight, filtering, collecting filtrate, and centrifuging to obtain supernatant as compound protease preparation;

the Lactobacillus is Lactobacillus plantarum F8(Lactobacillus plantarum F8), Lactobacillus plantarum MF3(Lactobacillus plantarum MF3) or Lactobacillus rhamnosus MF9(Lactobacillus rhamnous MF 9); lactobacillus plantarum MF3, accession number GDMCC 61400; lactobacillus plantarum MF3, accession number GDMCC 61398; lactobacillus rhamnosus MF9 has a accession number GDMCC 61399.

The Lactobacillus is Lactobacillus plantarum F8(Lactobacillus plantarum F8) separated from fermented bean curd, the strain number is GDMCC 61400, the Lactobacillus is preserved in Guangdong province microbial strain preservation center, the preservation place is Guangzhou city Michelia Tokyo 100 # 59 building 5 building Guangdong province microbial strain preservation center, and the preservation date is 2020, 12 and 31 days.

Lactobacillus plantarum MF3(Lactobacillus plantarum MF3) is isolated from fermented bean curd, and has a strain preservation number of GDMCC 61398, and is preserved in Guangdong province microorganism strain preservation center at the preservation place of Guangzhou Miehuo No. 100 college No. 59 building, 5 building, Guangdong province microorganism strain preservation center at the preservation date of 2020, 12 months and 31 days.

Lactobacillus rhamnosus MF9(Lactobacillus rhamnous MF9) is isolated from fermented bean curd, and the strain is with the preservation number GDMCC 61399 and is preserved in Guangdong province microbial strain preservation center at the preservation place of Guangzhou city Michelo No. 100 college No. 59 building No. 5 Guangdong province microbial strain preservation center at 12-31 days in 2020.

To further realizeFor the purposes of the invention, preferably, the mucor is Actinomucor elegans As3.2778(Actinomucor elegans As3.2778), and the fermented bean curd producing strain is derived from China center for culture collection and management of industrial microorganisms; the preparation method of the mucor seed liquid comprises the following steps: inoculating actinomucor elegans As3.2778 into a PDA inclined test tube, culturing for 24-72 h in a constant-temperature incubator at 25-30 ℃, adding sterile water into the test tube, and oscillating to elute the actinomucor elegans spores on the surface of a culture medium to obtain a spore suspension; counting under microscope, adjusting the concentration of spore suspension to 10⁶～10⁷And (4) obtaining the Mucor seed liquid in a volume/mL mode.

Preferably, the preparation method of the lactobacillus seed solution comprises the following steps: inoculating lactobacillus plantarum F8, lactobacillus plantarum MF3 or lactobacillus rhamnosus MF9 into an MRS liquid culture medium, culturing for 12-24 h in a constant-temperature incubator at 28-42 ℃, washing thalli with sterile water, and adjusting the concentration to 10⁸～10⁹And (5) CFU/mL to obtain the lactobacillus seed solution.

Preferably, the inoculation mode of the mucor seed solution and the lactobacillus seed solution is as follows: and (2) inoculating the mucor seed liquid into a culture medium independently, or respectively inoculating the mucor seed liquid into the culture medium together with lactobacillus plantarum F8, lactobacillus plantarum MF3 and lactobacillus rhamnosus MF9, wherein the inoculation amount is 4-7% by mass percentage.

Preferably, the mass to volume ratio of the culture to sterile water is 1:4 to 1:8 in kilograms and liters, respectively, as mass and volume units.

Preferably, the centrifugation conditions described in step 1) are: the temperature is 4-8 ℃, the rotation speed is 6000-10000 rpm, and the time is 5-20 min.

Preferably, the endo-type protease is bromelain or alcalase synthase; the addition amount of the endo-protease is 1000-4000 u/g protein.

Preferably, the bromelain is food-grade bromelain produced by Nanning Pombo Biotech limited; the alkaline protease is food grade Alcalase 2.4L alkaline protease produced by Novixin Biotechnology Limited, Denmark.

Preferably, the mass-volume ratio of the soybean protein isolate to water after stirring the soybean protein isolate and water is 1: 10-1: 20 by taking kilograms and liters as mass and volume units; the stirring at 80-95 ℃ is carried out by magnetic stirring in a water bath at 80-95 ℃ for 15-30 min.

Preferably, the enzyme deactivation is carried out for 10-30 min at the temperature of 85-105 ℃ in kilograms and liters as mass and volume units; the standing overnight is standing overnight in a refrigerator at the temperature of 3-4 ℃; the filtering is carried out by using 4-8 layers of sterile gauze; after centrifugation, the temperature of centrifugation in the supernatant is 4-8 ℃, the rotating speed is 6000-10000 rpm, and the time is 5-20 min.

Compared with the prior art, the invention has the following advantages:

firstly, the invention adopts a combined fermentation method of mucor and lactobacillus to prepare the compound protease preparation, the enzyme system of the enzyme preparation is rich, and the effects of bitter taste modification, flavor improvement and the like of polypeptide products are obvious.

Secondly, the soybean polypeptide is prepared by adopting the combined enzymolysis method of the compound protease and the commercial enzyme preparation, the enzymolysis efficiency is high, the process is simple and convenient, the obtained soybean polypeptide has no bitter taste, good flavor and obvious functionality, and the soybean polypeptide is mainly composed of small molecular peptides with the molecular weight less than 1000Da and is easy to digest and absorb.

Thirdly, the soybean polypeptide obtained by the invention has good in-vitro hypolipidemic activity. A high cholesterol cell model is constructed by HepG2 human liver cancer cells, a lipid-lowering drug simvastatin is used as an additive group, and the soybean polypeptide is verified to be capable of reducing the contents of intracellular Triglyceride (TG), Total Cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) to a certain extent, and simultaneously improving the content of high-density lipoprotein cholesterol (HDL-C) and the activity of superoxide dismutase (SOD), wherein the effect of reducing the contents of TC, TG and LDL-C in cells and promoting the generation of HDL-C and the activity of SOD by 10 mu g/ml of the soybean polypeptide MB8 is similar to that of 5mg/ml of simvastatin, and the soybean polypeptide MB has very high application and development prospects.

Drawings

FIG. 1 is an SDS-PAGE pattern of the soybean polypeptide of the present invention, wherein 1 is large marker, 2 is soybean protein, 3 is M8 (comparative example 4), 6 is MB8 (example 1), 10 is MA3 (example 2), 11 is MA9 (example 3), 12 is A, 13 is B, and 14 is small marker.

FIG. 2 is a graph showing the effect of soybean polypeptides of the present invention on the Total Cholesterol (TC) level in HepG2 cells (example 1, example 2, example 3, comparative example 5);

FIG. 3 is a graph showing the effect of soybean polypeptides of the present invention on the intracellular Triglyceride (TG) content of HepG2 (example 1, example 2, example 3, comparative example 5);

FIG. 4 is a graph showing the effect of soy polypeptides of the present invention on the intracellular low density lipoprotein cholesterol (LDL-C) content of HepG2 (examples 1, 2, 3, and 5);

FIG. 5 is a graph showing the effect of soybean polypeptides of the present invention on the intracellular high density lipoprotein cholesterol (HDL-C) content of HepG2 (example 1, example 2, example 3, comparative example 5);

FIG. 6 is a graph showing the effect of soybean polypeptides of the present invention on the intracellular superoxide dismutase (SOD) activity of HepG2 (example 1, example 2, example 3, comparative example 5).

Detailed Description

In order to clearly and intuitively understand the present invention, the following further describes the present invention with reference to examples, but the embodiments of the present invention are not limited thereto.

In the following examples, the degree of hydrolysis of soybean polypeptide was determined by OPA method, and the specific procedure was: adding 3mL of prepared OPA reagent into a test tube, then adding 400 mu L of serine standard solution into a standard tube, adding 400 mu L of deionized water into a blank tube, adding 400 mu L of test solution into a test tube, respectively carrying out 3 parallel tests, and averaging, wherein ODblank is approximately equal to 0.07, ODstand is approximately equal to 0.8 under the normal condition, otherwise, the OPA reagent needs to be prepared again. When deionized water, serine and the solution to be detected are added into 3mL of OPA reagent, the mixture is uniformly mixed for 5s, and the light absorption value of 340nm is immediately read after the mixture is statically reacted for 2 min. The degree of hydrolysis is calculated according to the following formula:

the experiment takes soy protein as substrate, alpha is taken as coefficient, 0.970 is taken, beta is taken as coefficient, 0.342 h is taken_totThe value is 7.8. Wherein, X represents the weight of the sample, g;

protein content in P-sample,%;

0.1-sample volume converted to L.

In the following examples, the total protein content of the lyophilized powder of the soybean protein enzymatic hydrolysate is determined by using the kjeldahl method with reference to the standard GB/T5009.5-2010, and the method is as follows: a certain amount of sample was weighed into a digestion tube, followed by 10mL of concentrated sulfuric acid in a fume hood, and a spatula of mixed catalyst (copper sulfate and potassium sulfate) was added. Putting the digestion tube into a digestion furnace, then opening condensed water, heating until the solution is digested to be transparent and clear, taking out the digestion tube and cooling. After the digestion tube is connected with an automatic azotometer, the alkali adding knob and the steam knob are respectively pressed, 25mL of 4% boric acid is added into the other conical flask, and ten drops of methylene red-green indicator are dripped into the conical flask. After completion of the alkali addition procedure, the erlenmeyer flask (blue) was removed for acid titration and the results were recorded and the protein content and protein loss rate were calculated as follows.

In the formula, V1-volume of hydrochloric acid standard solution consumed by sample titration, mL;

v2-blank titration consumes hydrochloric acid standard solution volume, mL;

c, hydrochloric acid standard titration solution concentration, mol/L;

m-mass of azotic sample, g;

m1 — protein content of soy protein;

m2 — protein content of soy polypeptide;

in the following examples, the bitterness evaluation method was: quinine is used as a standard substance, the values of the quinine are respectively 0, 1, 2, 3, 4 and 5 corresponding concentrations, namely 0, 20, 40, 200, 400 and 800ppm, before the quinine is used, 2 men and 2 women are selected as tasting objects of the experiment, before bitter taste experiment evaluation, the tasting objects are trained, the experimenting objects need to eat light diet, and the standard substances with different concentrations are tasted and scored every day until the deviation between the scoring and the theoretical value is controlled to be about 5%, then the different samples to be tested are tasted and scored (0-5 points), and the average value is obtained after three parallels are made.

In the following examples, SDS-PAGE electrophoresis was carried out: weighing appropriate amount of soybean polypeptide to obtain solution with certain concentration, mixing 5 μ l of the solution with 2X electrophoresis sample buffer solution (total protein concentration is 3mg/mL), heating at 95 deg.C for 10min in pcr instrument, and centrifuging. Electrophoresis was performed using 15% separation gel and 5% concentration gel. And (3) adding 6 mu l of sample mixed with the loading solution into the electrophoresis slot, running at a constant voltage of 50mV for 2-3h, then keeping the constant voltage of 150mV for about 60min, and stopping electrophoresis when the sample strip moves to the bottom of the gel. After electrophoresis, the gel is stained with 0.1% (w/v) Coomassie brilliant blue R-250 staining solution for 30min, and then decolorized until the background is colorless, and the gel is stored by imaging through a gel imaging system.

In the following examples, the analysis method of the molecular weight distribution of soybean polypeptide is: according to the method of national standard GB/T22492-2008 and appropriate changes, high performance gel filtration chromatography is adopted for determination, porous fillers are used as stationary phases, separation is carried out according to the difference of relative molecular mass of samples, detection is carried out under the condition that the ultraviolet absorption wavelength is 220nm, and then GPC data processing software is adopted for calculating the relative molecular mass and the distribution range of the soybean polypeptide.

In the following examples, the pancreatic lipase inhibition rate of soybean polypeptides was determined as follows: redissolving soybean polypeptide to a certain concentration, adding 4mL of substrate emulsion and 5mL of phosphate buffer solution with pH 7.5 into a 100mL conical flask, mechanically stirring for 5min at a constant temperature of 37 ℃, adding 1mL of pancreatic lipase, quickly and uniformly mixing, accurately reacting for 30min at 37 ℃, and stopping the reaction with 15mL of 95% ethanol. Titration was performed to neutrality with NaOH solution (0.025mol/L) and the volume of NaOH solution consumed was recorded. Lipase activity was calculated according to the following formula:

pancreatic lipase activity 1000 (A-B) M/TW

In the formula, A represents the average volume of sodium hydroxide titration solution consumed by a sample parallel test group, mL;

b, blank test consumes the volume of sodium hydroxide titration solution, mL;

m is the concentration of sodium hydroxide titration solution, mol/L;

w-sample size of lipase, g;

t-reaction time, min.

In the following examples, the measurement of the contents of triglyceride and cholesterol was performed according to the instructions of Nanjing build-up kit, and the specific procedure was as follows: taking a 96-well plate, adding 2.5 mu L of distilled water into a blank tube, adding 2.5 mu L of standard solution into a calibration tube, adding 2.5 mu L of solution to be detected into a sample tube, then adding 250 mu L of working solution into the sample tube, repeating the three groups to be parallel, and measuring the light absorption value at 490nm by using an enzyme-labeling instrument, wherein the calculation formula is as follows:

triglyceride content (mmol/L) ═ (A1-A2) × 2.26/((A3-A2) × protein concentration of sample to be tested)

Total cholesterol content (mmol/L) ═ (a1-a2) × 5.17/((A3-a2) × protein concentration of test sample)

In the formula, A1 represents the absorbance of a sample to be measured at 490 nm;

a2-absorbance at 490nm for blank tubes;

a3-absorbance at 490nm of standard tube;

2.26 — concentration of triglyceride standard;

5.17-concentration of cholesterol standard.

In the following examples, the determination of the contents of low-density lipoprotein cholesterol and high-density lipoprotein cholesterol was performed according to the instructions of Nanjing's kit, and the details are as follows: taking a 96-well plate, adding 2.5 mu L of distilled water into a blank tube, adding 2.5 mu L of standard solution into a calibration tube, adding 2.5 mu L of solution to be detected into a sample tube, then adding 180 mu L of working solution R1 into the sample tube, incubating for 10min at 37 ℃, measuring the OD value of the sample tube at 490nm, then adding 60 mu L of working solution R2, repeating the three groups to be parallel, and measuring the light absorption value of the sample tube at 490nm by using an enzyme-labeling instrument, wherein the calculation formula is as follows:

low density lipoprotein content (mmol/L) ═ a1-a2) × 4.3/((A3-a2) × protein concentration of sample to be tested)

High density lipoprotein content (mmol/L) ═ A1-A2) × 1.8/((A3-A2) × protein concentration of sample to be tested)

In the formula, A1 represents the absorbance of a sample to be measured at 490 nm;

a2-absorbance at 490nm for blank tubes;

a3-absorbance at 490nm of standard tube;

4.3-concentration of low density lipoprotein standard;

1.8-concentration of high density lipoprotein standard.

In the following examples, the determination of superoxide dismutase activity was performed according to the instructions of Nanjing as a kit, and the details are as follows: taking a 96-well plate, adding 20 mu L of distilled water into a control hole and a control blank hole, adding 20 mu L of a sample to be detected into a measurement hole and a measurement blank hole, adding 20 mu L of enzyme working solution into the control hole and the measurement hole, adding 20 mu L of enzyme diluent into the control blank hole and the measurement blank hole, adding 200 mu L of substrate application solution into each hole, uniformly mixing, incubating for 20min at 37 ℃, repeating three groups to be parallel, and measuring the light absorption value at 450nm by using an enzyme reader, wherein the calculation formula is as follows:

SOD inhibition (%) (a1-a2-A3+ a4) × 100%/(a 1-a2)

SOD activity (U/mgprot) ═ SOD inhibition rate ÷ 50% × n ÷ concentration of sample to be measured

Wherein, A1-absorbance at 450nm in control wells;

a2 — absorbance at 450nm of control blank wells;

a3-determination of the absorbance of the well at 450 nm;

a4-measurement of the absorbance at 450nm of blank wells;

n-dilution of the reaction system, 24.

The embodiment 1, 2 and 3 of the invention mainly relate to the polypeptide function test prepared by the screened enzymolysis scheme, wherein the control group is a positive control group, a high-cholesterol culture medium is used, and the medicine group is simvastatin hypolipidemic medicine.

Comparative example 1

(1) Weighing 5.0g of soy protein isolate, dissolving in 100mL of water, magnetically stirring in a water bath at 80 ℃ for 15min to uniformly disperse the solution, cooling to room temperature, and adjusting the pH value to 6.0. 2000u/g protein of Alcalase alkaline protease was added and stirred magnetically for 5h in a water bath at 55 ℃. And after the enzymolysis is finished, putting the soybean peptide into water bath at 95 ℃ to inactivate enzyme for 20min, cooling to room temperature, centrifuging for 10min at 8000rpm, and obtaining supernate, namely the soybean peptide A.

(2) As can be seen from physical analysis and sensory evaluation, the degree of hydrolysis of the soybean polypeptide A is 53.85%, and SDS-PAGE results show that the protein band of the soybean polypeptide A completely disappears (FIG. 1, lane 12), which indicates that the protein is degraded into a polypeptide with a molecular weight of less than 3.3 KD; further analysis showed that the soy polypeptide protein loss rate (protein in the pellet as a proportion of total protein after centrifugation) was 11.70% for the enzyme preparation, and the bitterness score was 3.50 for the sensory evaluation.

FIG. 1 is an SDS-PAGE pattern of the soybean polypeptides in the examples and comparative examples of the present invention, which indicates the degradation ability of the protein, and there is no band at all, i.e., the protein is completely degraded. Where 1 is a large marker, 2 is soy protein, 3 is M8 (comparative example 4), 6 is MB8 (example 1), 10 is MA3 (example 2), 11 is MA9 (example 3), 12 is A, 13 is B, 14 is a small marker. The following examples and comparative examples relate to the same situation as in fig. 1.

(3) The result of biochemical analysis shows that the inhibition rate of the soybean polypeptide A on the pancreatic lipase is 15.42% (the sample concentration is 0.01 mg/mL).

Comparative example 2

(1) Weighing 5.0g of soy protein isolate, dissolving in 100mL of water, magnetically stirring in a water bath at 80 ℃ for 15min to uniformly disperse the solution, cooling to room temperature, and adjusting the pH value to 6.0. Adding 2000u/g protein bromelain, magnetically stirring in 55 deg.C water bath for 6 hr, and inactivating enzyme in 95 deg.C water bath for 20min after enzymolysis. Cooling to room temperature, centrifuging at 8000rpm for 10min, and collecting supernatant as soybean polypeptide B.

(2) As can be seen from physiological analysis and sensory evaluation, the degree of hydrolysis of the soybean polypeptide B is 14.58%, SDS-PAGE results show that the protein band is only slightly degraded (FIG. 1, lane 13, compared with the soybean protein in lane 2, the protein band is all in, but the color is light, which indicates that the protein is degraded, but the degradation degree is not high), and further analysis shows that the loss rate of the soybean polypeptide protein prepared by the enzyme method is 22.56%, and the sensory evaluation result shows that the bitterness score is 2.00.

(3) The result of biochemical analysis shows that the inhibition rate of the soybean polypeptide B on the pancreatic lipase is 12.17% (the sample concentration is 0.01 mg/mL).

Comparative example 3

Weighing 10.0g of bran, placing the bran into a 250mL triangular flask, adding 11mL of distilled water, uniformly mixing, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating 1mL of actinomucor elegans As3.2778 seed solution, and culturing in a constant-temperature incubator at 28 ℃ for 72 h. After the fermentation is finished, adding 100mL of sterile water into the fermented culture, standing overnight in a refrigerator at 4 ℃, then filtering by 8 layers of gauze, collecting the filtrate and centrifuging to obtain supernatant, namely crude enzyme liquid, wherein the activity of the protease is 28.8U/mL.

Comparative example 4

Weighing 10.0g of bran, placing the bran into a 250mL triangular flask, adding 10mL of distilled water, uniformly mixing, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating 1mL of actinomucor elegans As3.2778 seed solution and 1mL of lactobacillus plantarum F8(GDMCC 61400) seed solution, and culturing in a constant temperature incubator at 28 ℃ for 72 h. After the fermentation is finished, adding 100mL of sterile water into the fermented culture, standing overnight in a refrigerator at 4 ℃, then filtering by 8 layers of gauze, collecting the filtrate and centrifuging to obtain supernatant, namely crude enzyme liquid, wherein the activity of the protease is 39.0U/mL. Vacuum freeze drying to obtain compound protease preparation P8.

(2) Weighing 5.0g of isolated soy protein, dissolving in 100mL of water, magnetically stirring in a water bath at 80 ℃ for 15min to uniformly disperse the solution, cooling to room temperature, adjusting the pH value to 6.0, adding 2000u/g of protein compound protease P8, magnetically stirring in a water bath at 55 ℃ for 6h, and after enzymolysis, inactivating the enzyme in a water bath at 95 ℃ for 20 min. Cooling to room temperature, centrifuging at 8000rpm for 10min, and vacuum freeze drying the supernatant to obtain soybean polypeptide M8.

(3) It can be seen from physiological analysis and sensory evaluation that the degree of hydrolysis of soybean polypeptide M8 is 38.03%, and SDS-PAGE results show that most of the protein band disappears, but a clear band still exists at 25KD (lane 3 in FIG. 1), indicating that the degradation of the protein is not complete. Further analysis shows that the loss rate of the soybean polypeptide protein prepared by the enzyme method is 18.01 percent, and the bitterness score is 2.40.

(4) The result of biochemical analysis shows that the inhibition rate of the soybean polypeptide M8 on pancreatic lipase is 12.03% (the sample concentration is 0.01 mg/mL).

Comparative example 5

(1) Taking a logarithmic phase high-cholesterol HepG2 model cell for plating, placing the model cell in a carbon dioxide incubator for culturing for 24h, adding serum-free and serum-free MEM (high-sugar type, Gibco) as a blank group, adding 20 mu g/m L high-cholesterol serum-free MEM (high-sugar type, Gibco) as a control group, adding 5mg/mL simvastatin serum-free MEM (high-sugar type, Gibco) as an addition group, and continuing culturing for 24h in the carbon dioxide incubator.

(2) And after the culture is finished, removing the supernatant, adding 0.25% trypsin for digestion, centrifuging, removing the supernatant, carrying out heavy suspension by adopting a PBS buffer solution and centrifuging, repeating the steps for three times, adding a lysate triton-100, and measuring the contents of TC, TG and LDL-C, HDL-C and the SOD activity of the lysate. The results showed that the drug-added group was able to significantly decrease intracellular TG and LDL-C concentrations, significantly increase HDL-C concentrations and SOD viability (P < 0.05) (fig. 2, fig. 3, fig. 4, fig. 5, fig. 6).

Example 1

(1) Weighing 10.0g of bran, placing the bran into a 250mL triangular flask, adding 10mL of distilled water, uniformly mixing, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating 1mL of actinomucor elegans As3.2778 seed solution and 1mL of lactobacillus plantarum F8(GDMCC 61400) seed solution, and culturing in a constant temperature incubator at 28 ℃ for 72 h. After the fermentation is finished, adding 100mL of sterile water into the fermented culture, standing overnight in a refrigerator at 4 ℃, then filtering by 8 layers of gauze, collecting the filtrate and centrifuging to obtain supernatant, namely crude enzyme liquid, wherein the activity of the protease is 39.0U/mL. Vacuum freeze drying to obtain compound protease preparation P8.

(2) Weighing 5.0g of isolated soy protein, dissolving in 100mL of water, magnetically stirring in a water bath at 80 ℃ for 15min to uniformly disperse the solution, cooling to room temperature, adjusting the pH value to 6.0, adding 1000u/g of protein compound protease P8 and 1000u/g of protein bromelain, magnetically stirring in a water bath at 55 ℃ for 6h, and after enzymolysis, putting in a water bath at 95 ℃ to inactivate enzyme for 20 min. Cooling to room temperature, centrifuging at 8000rpm for 10min, and vacuum freeze drying the supernatant to obtain soybean polypeptide MB 8.

(3) Through physical analysis and sensory evaluation, the hydrolysis degree of the soybean polypeptide MB8 is 55.51%, the polypeptide molecules with the molecular weight of less than 1000Da account for 64.9% (see table 1), the SDS-PAGE result shows that the protein band completely disappears (figure 1, lane 6), and further analysis shows that the soybean polypeptide protein prepared by the enzyme method has the loss rate of 9.05% and the bitterness score of less than 1.

(4) The result of biochemical analysis shows that the inhibition rate of the soybean polypeptide MB8 on pancreatic lipase is 28.83% (the sample concentration is 0.01 mg/mL).

(4) Through a high-cholesterol HepG2 model cell experiment, the result shows that the soybean polypeptide MB8 shows a dose-dependent relation on the contents of TC, TG, LDL-C and HDL-C in cells, and in the concentration gradient range of 10, 50, 100, 200 and 500 mug/mL, the contents of TC, TG and LDL-C are reduced along with the increase of the concentration of a sample and are obviously higher than that of a medicine adding group (simvastatin) (P is less than 0.05) (figure 2, figure 3 and figure 4); as the concentration of the sample increased, the HDL-C content also increased (FIG. 5).

5) Through a high-cholesterol HepG2 model cell experiment, the result shows that the SOD activity of the soybean polypeptide MB8 in the concentration range of 10-500 is obviously higher than that of a drug-added group (simvastatin) (P is less than 0.05) (figure 6), and the SOD activity is gradually increased from 4.76 +/-0.61U/mL to 26.70 +/-0.24U/mL along with the continuous increase of the concentration, and the increase is as high as 82.17%.

Example 2

(1) Weighing 10.0g of bran, placing the bran into a 250mL triangular flask, adding 10mL of distilled water, uniformly mixing, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating 1mL of actinomucor elegans As3.2778 seed solution and 1mL of lactobacillus plantarum MF3(GDMCC 61398) seed solution, and culturing in a constant temperature incubator at 28 ℃ for 72 h. After the fermentation is finished, 100mL of sterile water is added into the fermented culture, the mixture is kept still in a refrigerator at 4 ℃ overnight, then 8 layers of gauze are used for filtering, the filtrate is collected and centrifuged, and the obtained supernatant is crude enzyme liquid, wherein the activity of the active protease is 34.2U/mL, and the activity of the alkaline protease is 36.0U/mL. Vacuum freeze drying to obtain compound protease preparation P3.

(2) Weighing 5.0g of isolated soy protein, dissolving in 100mL of water, magnetically stirring in a water bath at 80 ℃ for 15min to uniformly disperse the solution, cooling to room temperature, adjusting the pH value to 6.0, adding 1000u/g of protein compound protease P3 and 1000u/g of protein Alcalase alkaline protease, magnetically stirring in a water bath at 55 ℃ for 6h, and after enzymolysis, inactivating the enzyme in a water bath at 95 ℃ for 20 min. Cooling to room temperature, centrifuging at 8000rpm for 10min to obtain supernatant as soybean polypeptide solution, and vacuum freeze drying to obtain soybean polypeptide MA 3.

(3) Through physical analysis and sensory evaluation, the hydrolysis degree of the soybean polypeptide MA3 is 74.67%, the polypeptide molecules with the molecular weight of less than 1000Da account for 70.1% (see table 1), the SDS-PAGE result shows that the protein band completely disappears (figure 1, lane 10), and further analysis shows that the soybean polypeptide protein prepared by the enzyme method has the loss rate of 8.49%, and the bitterness score is less than 1.

(4) The result of biochemical analysis shows that the inhibition rate of the soybean polypeptide MA3 on pancreatic lipase is 28.42% (the sample concentration is 0.01 mg/mL).

(4) Through a high-cholesterol HepG2 model cell experiment, the result shows that the soybean polypeptide MA3 has a remarkable reduction effect on the content of TG and LDL-C in cells, and is remarkably higher than that of an additive group (simvastatin) (P is less than 0.05) (figure 3, figure 4).

(5) Through a high-cholesterol HepG2 model cell experiment, the result shows that the cell SOD activity of the soybean polypeptide MA3 in the concentration range of 100-500 is obviously higher than that of a drug-added group (simvastatin) (P < 0.05) (figure 6).

Example 3

(1) Weighing 10.0g of bran, placing the bran into a 250mL triangular flask, adding 10mL of distilled water, uniformly mixing, sterilizing at 121 ℃ for 30min, cooling to room temperature, inoculating 1mL of actinomucor elegans As3.2778 seed solution and 1mL of lactobacillus rhamnosus MF9(GDMCC 61399) seed solution, and culturing in a constant-temperature incubator at 28 ℃ for 72 h. And after the fermentation is finished, adding 100mL of sterile water into the fermented culture, standing overnight in a refrigerator at 4 ℃, filtering by using 8 layers of gauze, collecting filtrate and centrifuging to obtain supernatant, namely crude enzyme solution, and carrying out vacuum freeze drying to obtain the compound protease preparation P9.

(2) Weighing 5.0g of isolated soy protein, dissolving in 100mL of water, magnetically stirring in a water bath at 80 ℃ for 15min to uniformly disperse the solution, cooling to room temperature, adjusting the pH value to 6.0, adding 1000u/g of protein compound protease P9 and 1000u/g of protein Alcalase alkaline protease, magnetically stirring in a water bath at 55 ℃ for 6h, and after enzymolysis, inactivating the enzyme in a water bath at 95 ℃ for 20 min. Cooling to room temperature, centrifuging at 8000rpm for 10min to obtain supernatant as soybean polypeptide solution, and vacuum freeze drying to obtain soybean polypeptide MA 9.

(3) The results of physical analysis and sensory evaluation show that the hydrolysis degree of the soybean polypeptide MA9 is 56.85%, the polypeptide molecules with the molecular weight of less than 1000Da account for 71.9% (see table 1), the SDS-PAGE result shows that the protein band completely disappears (figure 1, lane 11), and further analysis shows that the soybean polypeptide protein prepared by the enzyme method has the loss rate of 6.27%, and the bitterness score is less than 1.

(4) The result of biochemical analysis shows that the inhibition rate of the soybean polypeptide MA9 on pancreatic lipase is 31.08% (the sample concentration is 0.01 mg/mL).

(4) The results of high-cholesterol HepG2 model cell experiments show that the soybean polypeptide MA9 has obvious effect of reducing the content of TG and LDL-C in cells (figure 3 and figure 4).

(5) Through a high-cholesterol HepG2 model cell experiment, the result shows that the soybean polypeptide MA9 has a significant effect on the SOD activity of cells (fig. 6).

As can be seen from the above examples and comparative examples, 1) the degree of hydrolysis of soybean protein isolate using the commercial enzyme preparations Alcalase alkaline protease and bromelain was 53.85% and 14.58%, respectively, and the corresponding bitterness score was 3.50 and 2.00, respectively, with a noticeable bitterness that was not acceptable to consumers (comparative example 1 and comparative example 2). The soybean protein isolate is subjected to enzymolysis by adopting the composite protease of the invention in combination with bromelain and Alcalase alkaline protease respectively, and the obtained soybean polypeptides MB8, MA3 and MA9 have bitterness scores of less than 1 and basically have no bitterness. And compared with the preparation without the commercial enzyme (comparative example 1 and comparative example 2), the hydrolysis degree and the protein utilization rate of the preparation are both obviously improved. In particular, the enzyme production capacity can be remarkably improved by adopting the combined fermentation of the mucor and the lactic acid bacteria, the enzyme production capacity of the mucor cultured alone is 28.3U/ml of crude enzyme liquid (comparative example 3), and the enzyme production capacity of the mucor cultured together with the lactic acid bacteria is improved to nearly 40U/ml of crude enzyme liquid (see comparative example 4, example 1, example 2 and example 3), so that the production efficiency is greatly improved.

2) The soybean polypeptides MB8, MA3 and MA9 obtained by adopting the composite protease of the invention to carry out combined enzymolysis on soybean protein isolate respectively with bromelain and Alcalase alkaline protease have the inhibition rates of 28.83%, 28.42% and 31.08% on pancreatic lipase (example 1, example 2 and example 3), and compared with the polypeptides prepared by adopting commercial enzyme preparations (15.42% and 12.17%), the inhibition rates on the pancreatic lipase are obviously improved (comparative example 1 and comparative example 2), which shows that the polypeptides prepared by the self-made composite enzyme preparation probably have strong lipid-lowering activity.

3) Within a certain concentration range, the soybean polypeptides MB8, MA3 and MA9 can reduce the content of TC, TG and LDL-C in cells of a high-cholesterol HepG2 model, improve the content of HDL-C and SOD activity in the cells and have good in-vitro hypolipidemic activity (example 1, example 2, example 3 and comparative example 5). The blood lipid-lowering activity of the soybean polypeptide MB8 is the most remarkable, and the effect of 10 mu g/mL of the soybean polypeptide MB8 on reducing the content of TC, TG and LDL-C in cells and promoting the generation of HDL-C and SOD activity is similar to that of 5mg/mL of simvastatin, so that the soybean polypeptide MB8 has high application and development values (example 1).

The specific molecular weight distribution of the soybean polypeptide obtained in the examples of the present invention is shown in Table 1.

TABLE 1 molecular weight distribution of Soybean Polypeptides

The embodiments of the present invention are not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents and are included in the scope of the present invention.