Oat milk enzymolysis method for promoting proliferation of lactobacillus plantarum and bifidobacterium

Abstract

The invention provides an enzymolysis method of oat milk for promoting proliferation of lactobacillus plantarum and bifidobacterium. According to the invention, through the synergistic effect of in-situ enzymolysis of oat endogenous protease and glucanase and enzymolysis of free enzyme, on the basis, through enzymolysis of proper protease, the obtained oat milk contains oat polypeptide and oat beta-glucan with proper molecular weights, and has remarkable activity of promoting proliferation of lactobacillus plantarum and bifidobacterium.

Description

Technical Field

[0001] The present invention belongs to the technical field of food processing, and specifically relates to a method for preparing enzymatically hydrolyzed oat milk for promoting the proliferation of Lactobacillus plantarum and Bifidobacterium, and more specifically relates to a method for regulating the activity of endogenous enzymes in oats, and utilizing exogenous amylase, saccharifying enzyme and papain to enzymatically hydrolyze the starch in the oats to release glucan and protein, and further enzymatically hydrolyze the protein into active polypeptides, thereby promoting the proliferation of Lactobacillus plantarum and Bifidobacterium. Background Art

[0002] Oats belong to the grass family and contain a variety of nutrients such as protein, fat, minerals, phenolic compounds and β-glucan. They have a variety of biological activities such as lowering blood lipids, lowering blood sugar, regulating lipid metabolism disorders, and preventing and treating atherosclerosis. Oats are rich in dietary fiber, of which the main component of soluble dietary fiber is β-glucan. In recent years, the results of systematic reviews and human clinical trials have shown that continuous consumption of 1.4-6g of β-glucan from oats for 4-6 weeks can significantly increase the content of beneficial bacteria such as bifidobacteria and lactobacilli in the human intestine. At the same time, oat β-glucan improves the intestinal flora and increases the content of beneficial bacteria in the intestine, bringing health effects such as lowering cholesterol and regulating blood sugar. These studies show that oat β-glucan is a kind of prebiotic. However, due to the different ability of different people to decompose oat protein, the protein structure entering the intestine is different. At the same time, the endogenous intestinal microorganisms have different effects on β-glucan and residual insoluble protein hydrolysis, producing proteins and β-glucan hydrolysates of different molecular weights, which have different effects on the proliferation of intestinal microorganisms. However, there has been no systematic study on the differences in the intestinal probiotic effects of dextran with different molecular weights; and it is still unclear whether the probiotic effect of oats is related to the synergistic effect of unabsorbed peptides of oat protein after being acted upon by digestive tract proteases.

[0003] Brown rice germination is a process in which, under sufficient water conditions, it first absorbs water rapidly and expands in volume, activating the activity of endogenous enzymes in the grains, and a series of physiological and biochemical reactions occur under the action of enzymes, and the morphology changes accordingly. It is generally divided into three stages: grain swelling, germination and germination: In the first stage, brown rice grains absorb water rapidly, intracellular substances and matrix undergo hydration, endogenous enzymes such as amylase, protease and phytase begin to be activated, and a series of biochemical reactions occur; in the second stage, after the grains absorb water to a certain extent, the water absorption rate begins to slow down, and the macromolecular nutrients in the grains begin to be decomposed; in the third stage, the embryo begins to grow and germinate slowly, the grain water absorption rate begins to accelerate, and some available nutrients stored in the grains (such as protein, starch and triglycerides, etc.) are decomposed and metabolized under the action of different hydrolases. Studies have found that when oats reach the germination stage, the activities of amylase, cellulase and β-glucanase increase, the cortical fibers are enzymatically softened, cellulose and hemicellulose are degraded, the insoluble dietary fiber content is significantly reduced, β-glucan is degraded, and reducing sugar and soluble sugar increase significantly, thereby increasing the nutrient absorption rate and the in vitro digestibility of starch. Therefore, by regulating the endogenous enzyme activity and water absorption time of oats, it will be possible to control the degree of enzymatic hydrolysis of major substances such as protein and β-glucan. However, the relationship between the biological activity of oat β-glucan and the molecular weight of the enzymatic hydrolyzate is still unclear; the different hydrolysis bond types of the endogenous enzyme system and the exogenous added enzyme during the soaking process of oats make the enzymatic hydrolysis products have significant differences. Summary of the invention

[0004] The purpose of the present invention is to provide an enzymatic hydrolysis method of oat milk that promotes the proliferation of Lactobacillus plantarum and Bifidobacterium by utilizing a combination of "in situ enzymatic hydrolysis of oat endogenous protease and glucanase, free enzyme enzymatic hydrolysis and exogenous protease enzymatic hydrolysis", thereby improving the activity of oat milk in promoting the proliferation of probiotics.

[0005] The enzymatic hydrolysis method of oat milk for promoting the proliferation of Lactobacillus plantarum and Bifidobacterium provided by the present invention comprises the following steps:

[0006] (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats

[0007] ① Heat-shocked oat kernels: Place oat kernels with seed coat in 80-85℃ hot water for 2-3 minutes and quickly put them into tap water to cool to room temperature to obtain heat-shocked oats, wherein oats: hot water = 1g: 20-30mL;

[0008] ② Gibberellic acid soaking: soak the heat-shocked oats at 50℃ for 4-6 hours, remove and drain the water, the water absorption rate is 45%-50%, and the swelling stage of seed germination is completed to obtain soaked oats;

[0009] The soaking liquid is 5-10 mg / L gibberellin, the soaking material-liquid ratio is dry oats: soaking liquid = 1:3-5, and the protease activity in the oats after soaking is ≥70 U / g dry oats, and the glucanase activity is ≥10 U / g dry oats;

[0010] ③ Keep warm and moisturize the seeds and continue in-situ enzymolysis: soak the oats in a heat preservation box with a humidity of 90%-95% and keep warm at 25-28℃ for 5-8 hours to enter the middle stage of seed germination. The protease activity in the oats is ≥120U / g dry oats, and the glucanase activity is ≥20U / g dry oats, so as to obtain oats with high protease and glucanase activities;

[0011] (2) beating and enzymatic hydrolysis of oats with free endogenous enzymes: beating oats with high protease and glucanase activity with water to obtain oat milk with residue, which is then kept at 40-45° C. for 1 hour and filtered to obtain oat milk 1 with free endogenous enzymes;

[0012] (3) Inactivating endogenous enzymes and liquefying starch: heating the oat milk 1 to 80-85° C., adding 80-100 U / mL high-temperature α-amylase, and obtaining oat milk 2 in which endogenous enzymes are inactivated and starch liquefaction is completed;

[0013] (4) Starch full saccharification and papain enzymatic hydrolysis: the oat milk 2 was cooled to 50-55° C., 50-100 U / mL of saccharifying enzyme (50-100 U of saccharifying enzyme was added to each mL of oat milk 2), 0.05-0.10% of papain (0.05-0.10 g of papain was added to each 100 g of oat milk 2), and the mixture was kept warm for enzymatic hydrolysis for 1-1.5 h, and the temperature was raised to 80-85° C. and maintained for 15-20 min to inactivate the enzyme, thereby obtaining oat milk 3;

[0014] The enzymes used in steps (3) and (4) are:

[0015] High temperature α-amylase: Novozymes Biotech LLC, enzyme activity 40000U / g;

[0016] Glucoamylase: Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., enzyme activity 100000U / g;

[0017] Papain: Nanning Pangbo Bioengineering Co., Ltd., enzyme activity 100000U / g;

[0018] (5) Homogenization and sterilization: add or not add sweetener to the oat milk 3, adjust the temperature to 70°C, homogenize at 30 MPa, and sterilize at 121°C for 15 min to obtain enzymatically hydrolyzed oat milk.

[0019] In step (2) of the above method, the weight ratio of oats to water in the pulping is 1:7-8, calculated based on the dry weight of oats; the water temperature of the pulping water is lower than 50°C; and the filtering is performed through a 40-60 mesh sieve;

[0020] In step (3), high temperature α-amylase is added and kept warm for 1 hour.

[0021] The oat milk prepared by the above method also belongs to the protection scope of the present invention.

[0022] The application of the oat milk in promoting the proliferation of probiotics also falls within the protection scope of the present invention.

[0023] In the application, the probiotics include Lactobacillus plantarum and Bifidobacterium.

[0024] The present invention uses the synergistic effect of in-situ enzymolysis of oat endogenous protease and glucanase and free enzyme enzymolysis, and on this basis uses suitable protease enzymolysis to obtain oat milk containing oat polypeptides and oat β-glucan with suitable molecular weights, has significant activity in promoting the proliferation of Lactobacillus plantarum and Bifidobacterium, and has a prebiotic effect. The process is simple and suitable for industrial production, providing a technical reference for large-scale production of oat milk with probiotic proliferation promoting activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 It is the standard curve of glucose content in the present invention.

[0026] Figure 2 It is the protein content standard curve in the present invention.

[0027] Figure 3 It is the sodium periodate standard curve in the present invention.

[0028] Figure 4 This is the standard curve of the relative molecular weight of the polysaccharide in the present invention.

[0029] Figure 5 HPLC spectrum of the polysaccharide of oat milk prepared in Example 1

[0030] Figure 6 This is the HPLC spectrum of the polysaccharide of oat milk prepared in Example 2.

[0031] Figure 7 This is the HPLC spectrum of the polysaccharide of oat milk prepared in Comparative Example 1.

[0032] Figure 8 This is the HPLC spectrum of the polysaccharides from oat milk prepared in Comparative Example 2.

[0033] Fig. 9 This is the HPLC spectrum of the polysaccharides from oat milk prepared in Comparative Example 3.

[0034] Fig.10 This is the HPLC spectrum of the polysaccharides from oat milk prepared in Comparative Example 4.

[0035] Fig.11 This is the HPLC spectrum of the polysaccharide of oat milk prepared in Comparative Example 5. DETAILED DESCRIPTION

[0036] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0037] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0038] The enzymes used in the following examples are: high temperature α-amylase: Novozymes Biotechnology Co., Ltd., enzyme activity 40000 U / g; saccharifying enzyme: Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., enzyme activity 100000 U / g; papain: Nanning Pangbo Bioengineering Co., Ltd., enzyme activity 100000 U / g;

[0039] Example 1: Enzymatic hydrolysis of oat milk to promote proliferation of Lactobacillus plantarum and Bifidobacterium

[0040] (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats

[0041] ① Heat-shocked oat kernels: Place oat kernels with seed coat in 85℃ hot water for 2 minutes and then quickly put them into tap water to cool to room temperature to obtain heat-shocked oats, where oats: hot water = 1g: 30mL;

[0042] ② Gibberellic acid soaking: Soak the heat-shocked oats at 50℃ for 6 hours, remove and control the water to complete the imbibition stage of seed germination, and obtain soaked oats with a water absorption rate of 50%. The soaking liquid is 8mg / L gibberellin, and the soaking material-liquid ratio is dry oats: soaking liquid = 1g: 4mL. The protease activity in the soaked oats is 80U / g dry oats, and the glucanase activity is 12U / g dry oats;

[0043] ③ Insulation and moisture retention to regulate the continued in-situ enzymolysis in the seeds: soak the oats in an incubator with a humidity of 95% and a temperature of 28°C for 6 hours to carry out seed germination metabolism, and obtain oats in the middle stage of seed germination. The protease activity in the oats is 140U / g dry oats, and the glucanase activity is 25U / g dry oats;

[0044] (2) Beating and enzymatic hydrolysis of oats with endogenous free enzymes: The oats in the middle stage of seed germination were beaten with water (water temperature 25°C), with the weight ratio of oats to water being 1:8. The oat milk with residue was obtained based on the dry weight of oats. The oat milk was kept at 42°C for 1 h and then passed through a 50-mesh sieve to obtain oat milk 1 with in situ enzymatic hydrolysis of endogenous enzymes;

[0045] (3) Inactivating endogenous enzymes and liquefying starch: heating oat milk 1 to 85° C., adding 100 U / mL high-temperature α-amylase until the oat milk does not turn blue when exposed to iodine, and obtaining oat milk 2 in which endogenous enzymes are inactivated and starch liquefaction is completed;

[0046] (4) Starch full saccharification and papain enzymatic hydrolysis: oat milk 2 was cooled to 50°C, 100 U / mL saccharifying enzyme and 0.05% papain were added, and the mixture was kept at 50°C for 1 h for enzymatic hydrolysis, and then the mixture was heated to 85°C for 15 min to inactivate the enzyme, thereby obtaining oat milk 3;

[0047] (5) Homogenization and sterilization: No sweetener was added to the oat milk 3, the temperature was adjusted to 70°C, homogenized at 30 MPa, and sterilized at 121°C for 15 min to obtain enzymatically hydrolyzed oat milk.

[0048] Example 2: Enzymatic hydrolysis of oat milk to promote proliferation of Lactobacillus plantarum and Bifidobacterium

[0049] (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats

[0050] ① Heat-shocked oat kernels: Place oat kernels with seed coat in 80℃ hot water for 3 minutes and then quickly put them into tap water to cool to room temperature to obtain heat-shocked oats, where oat: hot water = 1g: 20mL;

[0051] ② Gibberellic acid soaking: Soak the heat-shocked oats at 50℃ for 5h, remove and control the water to complete the imbibition stage of seed germination, and obtain soaked oats with a water absorption rate of 48%. The soaking liquid is 10mg / L gibberellin, and the soaking material-liquid ratio is dry oats: soaking liquid = 1:3. The protease activity in the soaked oats is 75U / g dry oats, and the glucanase activity is 13U / g dry oats;

[0052] ③ Insulation and moisture retention to regulate in-situ enzymolysis in seeds: soak oats in an incubator at 26°C and 92% humidity for 7 hours to carry out seed germination metabolism, and obtain oats in the middle stage of seed germination. The protease activity in the oats is 135U / g dry oats, and the glucanase activity is 22U / g dry oats;

[0053] (2) Beating and enzymatic hydrolysis of oats with free endogenous enzymes: The oats in the middle stage of seed germination were beaten with water (water temperature 25°C), with the weight ratio of oats to water being 1:8. The oat milk with residue was obtained based on the dry weight of oats. The oat milk was kept at 40°C for 1 h and then passed through a 50-mesh sieve to obtain oat milk 1 with in situ enzymatic hydrolysis of endogenous enzymes;

[0054] (3) Inactivating endogenous enzymes and liquefying starch: heating oat milk 1 to 80° C., adding 90 U / mL high-temperature α-amylase until the oat milk does not turn blue when exposed to iodine, and obtaining oat milk 2 in which endogenous enzymes are inactivated and starch is liquefied;

[0055] (4) Starch full saccharification and papain enzymatic hydrolysis: oat milk 2 was cooled to 50°C, 100 U / mL saccharifying enzyme and 0.08% papain were added, and the mixture was kept at 50°C for enzymatic hydrolysis for 1 h, and then the mixture was heated to 85°C and kept for 15 min to inactivate the enzyme, thereby obtaining oat milk 3;

[0056] (5) Homogenization and sterilization: No sweetener was added to the oat milk 3, the temperature was adjusted to 70°C, homogenized at 30 MPa, and sterilized at 121°C for 15 min to obtain enzymatically hydrolyzed oat milk.

[0057] Comparative Example 1: In-situ enzymatic hydrolysis of oat milk without heat shock or soaking

[0058] (1) The protease activity in hulled oats is 5 U / g dry oats, and the glucanase activity is 0.33 U / g dry oats;

[0059] (2) beating: directly beating the oats with husks with water (water temperature 25°C), the weight ratio of oats to water being 1:8, and calculating based on the dry weight of oats to obtain oat milk with residues, and the slurry is passed through a 50-mesh sieve to obtain oat milk 1;

[0060] (3) Inactivating endogenous enzymes and liquefying starch: same as in Example 1;

[0061] (4) Starch full saccharification and papain hydrolysis: same as in Example 1;

[0062] (5) Homogenization and sterilization: Same as Example 1.

[0063] Comparative Example 2: Enzymatic oat milk without soaking

[0064] (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats

[0065] ① Heat-shocked oat grains: same as in Example 1;

[0066] ② Gibberellic acid soaking: the same as Example 1; after soaking, the protease activity in the oats was 80 U / g dry oats, and the glucanase activity was 12 U / g dry oats;

[0067] (2) Beating and enzymatic hydrolysis of oats with free endogenous enzymes: Soaked oats were beaten with water (water temperature 25°C), with the weight ratio of oats to water being 1:8, and oat milk with residue was obtained, calculated based on the dry weight of oats. After being kept at 42°C for 1 hour, oat milk 1 with endogenous enzyme in situ enzymatic hydrolysis was obtained.

[0068] (3) Inactivating endogenous enzymes and liquefying starch: same as in Example 1;

[0069] (4) Starch full saccharification and papain hydrolysis: same as in Example 1;

[0070] (5) Homogenization and sterilization: Same as Example 1.

[0071] Comparative Example 3: Oat milk without papain enzymolysis

[0072] (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats

[0073] ① Heat-shocked oat grains: same as in Example 1;

[0074] ② Gibberellic acid soaking: same as Example 1;

[0075] ③ Keep warm and moist to regulate the continued in-situ enzymolysis in the seeds: same as in Example 1;

[0076] (2) Beating and enzymatic hydrolysis of oats with endogenous free enzymes: same as in Example 1;

[0077] (3) Inactivating endogenous enzymes and liquefying starch: same as in Example 1;

[0078] (4) Starch full saccharification: oat milk 2 was cooled to 50°C, 100 U / mL saccharifying enzyme was added and enzymatically hydrolyzed at 50°C for 1 h, and then the temperature was raised to 85°C and maintained for 15 min to inactivate the enzyme, thereby obtaining oat milk 3;

[0079] (5) Homogenization and sterilization: Same as Example 1.

[0080] Comparative Example 4: Oat milk without free enzymatic hydrolysis

[0081] (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats

[0082] ① Heat-shocked oat grains: same as in Example 1;

[0083] ② Gibberellic acid soaking: same as Example 1;

[0084] ③ Keep warm and moist to regulate the continued in-situ enzymolysis in the seeds: same as in Example 1;

[0085] (2) beating: beating oats in the middle stage of seed germination with water (water temperature 25°C), the weight ratio of oats to water is 1:8, and the oat milk with residue is obtained based on the dry weight of oats. The slurry is passed through a 50-mesh sieve to obtain oat milk 1;

[0086] (3) Inactivating endogenous enzymes and liquefying starch: same as in Example 1;

[0087] (4) Starch full saccharification and papain hydrolysis: same as in Example 1;

[0088] (5) Homogenization and sterilization: Same as Example 1.

[0089] Comparative Example 5: Overheating of oat milk by kinase

[0090] (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats

[0091] ① Heat-shocked oat kernels: Place oat kernels with seed coat in 90℃ hot water for 5 minutes and then quickly put them into tap water to cool to room temperature to obtain heat-shocked oats, where oat: hot water = 1g: 30mL;

[0092] ② Gibberellic acid soaking: Soak the heat-shocked oats at 50℃ for 6 hours, remove and control the water to complete the imbibition stage of seed germination, and obtain soaked oats with a water absorption rate of 50%. The soaking liquid is 8mg / L gibberellin, and the soaking material-liquid ratio is dry oats: soaking liquid = 1:4. The protease activity in the soaked oats is 50U / g dry oats, and the glucanase activity is 8U / g dry oats;

[0093] ③ Heat preservation and moisture retention to regulate the continued in-situ enzymolysis in the seeds: soak the oats in an incubator with a humidity of 95% and a temperature of 28°C for 6 hours to carry out seed germination metabolism, and obtain oats in the middle stage of seed germination, in which the protease activity of the oats is 75U / g dry oats and the glucanase activity is 10U / g dry oats;

[0094] (2) Beating and enzymatic hydrolysis of oats with endogenous free enzymes: same as in Example 1;

[0095] (3) Inactivating endogenous enzymes and liquefying starch: same as in Example 1;

[0096] (4) Starch full saccharification and papain hydrolysis: same as in Example 1;

[0097] (5) Homogenization and sterilization: Same as Example 1.

[0098] Related test results of the present invention:

[0099] The oat milk enzymatic hydrolysate was filtered to remove the solids, and the supernatant was taken for index determination.

[0100] (1) Determination of the proliferation rate of Lactobacillus plantarum and Bifidobacterium

[0101] The main bacteria used in this experiment to inhibit bacterial proliferation are Lactobacillus plantarum (CGMCC No. 1.12974) and Bifidobacterium animalis subsp. Lactis (CGMCC No. 1.15623). The above strains are purchased from the China General Microbiological Culture Collection Management Center. The quantitative determination method of the sample enrichment effect is the OD value turbidimetry. Take 0.25mL of sterilized enzymatic oat milk and add it to 4.75mL MRS medium, and replace the glucose in the medium, add 1% of Lactobacillus plantarum seed culture solution by volume, and culture at a constant temperature of 37°C for 18h. After the culture is completed, shake and mix, draw 1mL of culture solution, centrifuge at 8000r / min for 10min, discard the supernatant, add 5mL of sterile saline to dilute, calibrate with sterile saline, and measure the biomass by the absorbance value at 600nm wavelength of UV-visible spectrophotometer. Bifidobacterium was added with 5% seed culture solution by volume, and an anaerobic bag was added into a sealed container at the same time, and cultured at a constant temperature of 37°C for 48 hours. Other conditions were the same as those of Lactobacillus plantarum.

[0102] The OD value of the bacterial solution cultured in MRS medium without oat milk and culture conditions was used as a control, and the formula for calculating the percentage of microbial proliferation was as follows:

[0103]

[0104] A: OD600nm of cultured microorganisms after adding 5% oat milk;

[0105] B: OD600nm of microorganisms cultured in original culture medium.

[0106] (2) Determination of the proliferation rate of intestinal harmful bacteria (Escherichia coli and Staphylococcus aureus)

[0107] ① Bacteria required for the experiment of inhibiting microbial proliferation: The bacteria used in this experiment to inhibit bacterial proliferation mainly include pathogenic Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, which were purchased from the American Type Culture Collection.

[0108] ② Composition of culture medium and culture method for inhibiting microbial proliferation experiment

[0109] Escherichia coli culture medium: 3 g beef extract, 1 g glucose, 10 g peptone, 5 g / L sodium chloride, 1000 mL water, pH 7.4-7.6;

[0110] Staphylococcus aureus culture medium: beef extract 3g, glucose 1g, peptone 5g, water 100mL, pH 7.2-7.4;

[0111] ③ Turbidimetric method was used to determine the inhibitory effect on the proliferation of different microorganisms such as pathogenic Escherichia coli and Staphylococcus aureus: The quantitative determination method for the proliferation effect of oat milk was the OD value turbidimetric method.

[0112] Take 0.25mL of sterilized enzymatic oat milk and add it to 4.75mL of Escherichia coli and Staphylococcus aureus culture medium, add 1% of Escherichia coli and Staphylococcus aureus seed culture medium by volume, and culture at 37°C for 18h. After the culture, shake and mix, take 1mL of culture medium, centrifuge at 8000r / min for 10min, discard the supernatant, dilute with 5mL of sterile saline, calibrate with sterile saline, and determine the biomass by the absorbance value at 600nm wavelength of UV-visible spectrophotometer.

[0113] The OD value of the bacterial solution cultured under the culture medium and culture conditions without oat milk was used as the control, and the formula for calculating the percentage of microbial proliferation was as follows:

[0114]

[0115] A: OD of microorganisms cultured after adding 5% oat milk 600nm ;

[0116] B: OD of the microorganism cultured in the original culture medium 600nm .

[0117] (3) Determination of polysaccharide content:

[0118] Take 4 mL of the supernatant after enzymatic hydrolysis of oat milk (referring to the oat milk after heat shock beating and enzyme hydrolysis, but before homogenization and sterilization), add 4 times the volume of anhydrous ethanol to precipitate, leave overnight, dry, and redissolve in 2 mL of deionized water to obtain a crude oat milk polysaccharide solution. The polysaccharide content was determined by the phenol-sulfuric acid method.

[0119] ① Drawing of standard curve: Accurately pipette 0.2, 0.4, 0.6, 0.8, 1.0 mL of 0.1 mg / mL glucose standard solution into a test tube, and add water to 1.0 mL. After adding 0.5 mL of 6% phenol solution, slowly add 5 mL of concentrated sulfuric acid solution along the wall of the test tube, mix well, boil in water bath for 5 minutes, and cool to room temperature. Use 1.0 mL of distilled water as blank control, and measure the absorbance value at 490 nm wavelength with UV spectrophotometer. After taking the average value of three times, use glucose content as the horizontal axis and OD as the horizontal axis. 490nm As the vertical axis, draw the standard curve as Figure 1 It can be seen that the linear regression equation of the standard curve is y = 8.2243x-0.0087, R 2=0.999, where y is the absorbance value and x is the glucose content (mg / mL).

[0120] ② Determination of polysaccharide in sample solution: Accurately pipette 1 mL of crude polysaccharide solution diluted to a certain multiple, add 0.5 mL of 6% phenol solution, slowly add 5 mL of concentrated sulfuric acid solution along the wall of the test tube, mix well, boil in water bath for 5 minutes, and cool to room temperature. Using the reagent blank as a reference, measure the absorbance value at a wavelength of 490 nm with an ultraviolet spectrophotometer. Calculate the polysaccharide content in the solution based on the standard curve.

[0121] (4) Determination of polypeptide content:

[0122] Take 5 mL of oat milk enzymatic hydrolysis supernatant (referring to the oat milk before a series of enzyme reactions, homogenization and sterilization), add 5 mL of 10% trichloroacetic acid solution, centrifuge at 8000 rpm for 3 minutes, and then take the supernatant for testing.

[0123] ① Drawing of standard curve: Accurately pipette 0.16, 0.32, 0.48, 0.64, and 0.8 mL of 10 mg / mL bovine serum albumin standard solution into a test tube, add water to 0.8 mL, add 3.2 mL of biuret reagent, mix well, and react at 25°C for 30 minutes. Use 1.0 mL of distilled water as a blank control, measure the absorbance value at a wavelength of 540 nm with an ultraviolet spectrophotometer, and take the average value after three repetitions. Draw a standard curve with protein content as the horizontal axis and OD540 as the vertical axis as shown in the figure. Figure 2 It can be seen that the linear regression equation of the standard curve is y = 8.2243x-0.0087, R 2 =0.999, where y is the absorbance value and x is the protein content (mg / mL).

[0124] ② Determination of polypeptides in sample solution: Accurately pipette 0.8 mL of the sample solution to be tested, add 3.2 mL of biuret reagent, mix well, and react at 25°C for 30 minutes. Using the reagent blank as a reference, measure the absorbance value at a wavelength of 540 nm using an ultraviolet spectrophotometer. Calculate the polypeptide content in the solution based on the standard curve.

[0125] (5) Periodic acid oxidation experiment

[0126] Accurately weigh 321 mg of sodium periodate (NaIO4), dilute to 50 mL with deionized water, which is a 30 mmol / L NaIO4 storage solution, and store in tin foil in the dark. Dilute the storage solution to obtain a 15 mmol / L NaIO4 solution, add 0, 1, 2, 3, 4, and 5 mL of 15 mmol / L NaIO4 solution to the test tubes respectively, make up to 5.0 mL with deionized water, sample 0.1 mL from each tube, dilute 250 times with deionized water to 25 mL, mix well, and measure the absorbance at 223 nm. Use the absorbance value as the ordinate and the NaIO4 concentration (0.012, 0.024, 0.036, 0.048, 0.06 mmol / L) in the mixed solution as the abscissa to draw a standard curve. Figure 3 .

[0127] Weigh 20 mg of oat polysaccharide component, add 12.5 mL of 30 mmol / L NaIO4 solution, dilute to 25 mL with deionized water, make the final concentration of NaIO4 solution reach 15 mmol / L, and react at 4 ° C in the dark. Take a sample of 100uL every 12 hours, dilute 250 times with deionized water, use deionized water as a blank control, and measure the OD value at a wavelength of 223nm. After sampling at intervals until the OD value is constant, add 1.5 mL of ethylene glycol and place it at 4 ° C in the dark for 20 minutes to terminate the oxidation reaction, and calculate the NaIO4 consumption. Take 2 mL of the above NaIO4 oxidation solution, titrate with 0.01 mol / L NaOH solution, use 1 drop of phenolphthalein to indicate the end point, and calculate the amount of formic acid generated.

[0128] (6) Determination of polysaccharide molecular weight

[0129] Take 4 mL of the supernatant after enzymatic hydrolysis of oat milk, add 4 times the volume of anhydrous ethanol to precipitate, leave overnight, dry, and redissolve in 2 mL of deionized water to obtain a crude oat milk polysaccharide solution.

[0130] The molecular weight of polysaccharides was determined using HPLC. The conditions were as follows:

[0131] Chromatograph: Agilent 1260; chromatographic column: AgiLent PL aquageL-OH MIXED-M (300mm×8mm); detector: differential refractometer; mobile phase: 0.1mol / L NaNO3 solution; flow rate: 1.0mL / min; injection volume: 20μL; column temperature: 30℃.

[0132] Use 0.1mol / L NaNO3 solution to dissolve the dextran standards with molecular weights of 3620, 12600, 30200, 63300, and 126000 g / mol, and pass them through a 0.22μm microporous filter and inject them in sequence. Draw a standard curve with LgMw as the ordinate and retention time as the abscissa. The results are as follows: Figure 4The calculation formula of relative molecular weight and retention time of polysaccharide is: y = -0.5281x + 8.9252, R 2 =0.9911, where x is the retention time.

[0133] The oat milk crude polysaccharide solution was diluted with 0.1 mol / L NaNO3 solution to make the polysaccharide content 1.8 mg / mL, passed through a 0.22 μm microporous filter, injected, and the retention time was recorded. The relative molecular mass of the polysaccharide was calculated.

[0134] The results of the determination of the proliferation rate, polypeptide content, and polysaccharide bond type composition ratio of oat milk in the present invention and the comparative example on Lactobacillus plantarum, Bifidobacterium, and intestinal harmful bacteria are as follows:

[0135] As shown in Table 1, the endogenous enzyme activity of oats is properly regulated for in-situ enzymolysis, and the obtained oat milk (embodiment) is prepared by combining exogenous saccharifying enzymes and proteases to cultivate plant lactobacillus proliferation rate exceeding 50%, and the bifidobacterium proliferation rate exceeding 40%, which are significantly higher than the comparative example. Compared with the oat milk without heat shock and soaking in-situ enzymolysis (Comparative Example 1), the plant lactobacillus and bifidobacterium proliferation rates of the embodiment are increased by 126.25%-127.04% and 170.83%-184.82%, respectively, indicating that the oat milk after heat shock soaking and enzymolysis contains more active substances that promote the proliferation of probiotics (plant lactobacillus and bifidobacterium). By comparing the proliferation effect of oat milk in Example 1 with that in Comparative Example 3 on probiotics, the addition of exogenous proteases can decompose oat protein into active peptides, thereby promoting the increase of probiotics. In the process of free enzymolysis (Comparative Example 4), the free enzyme system in oat milk fully plays a role, which is of great significance for improving the effect of oat milk on promoting the proliferation of probiotics. When the heat shock intensity is too large (Comparative Example 5), the endogenous protease and amylase activities of oats are damaged to a certain extent, and the proliferation effect on probiotics is weakened. Under the conditions of the sample dilution used, there is no significant difference in the proliferation rate of intestinal harmful bacteria (Escherichia coli and Staphylococcus aureus) cultured in the oat milk of the embodiment and the comparative example. In summary, the oat milk prepared by the present invention can promote the proliferation of Lactobacillus plantarum and Bifidobacterium.

[0136] Table 1 Oat milk of Examples 1-2 of the present invention and Comparative Examples 1-5 for microbial proliferation rate

[0137] Lactobacillus plantarum (%) Bifidobacterium (%) Escherichia coli (%) Staphylococcus aureus (%) Example 1 52.15±2.33 41.22±2.21 3.21±2.55 3.22±2.23 Example 2 51.97±1.78 43.35±2.67 2.56±2.43 3.25±2.28 Comparative Example 1 22.97±3.22 15.22±1.23 4.43±2.28 4.18±2.34 Comparative Example 2 28.44±2.56 23.75±3.11 3.41±3.19 3.12±3.19 Comparative Example 3 27.31±1.21 21.64±1.69 1.25±2.35 3.55±2.21 Comparative Example 4 32.59±1.59 29.73±1.98 1.28±3.32 4.57±3.31 Comparative Example 5 26.98±2.96 23.78±2.38 2.27±2.27 2.87±2.32

[0138] As can be seen from Table 2, the oat milk obtained after enzymatic hydrolysis by the method of this patent has a polysaccharide content greater than 38 mg / mL and a polypeptide content greater than 9.5 mg / mL. Compared with the oat milk without heat shock and soaking (Comparative Example 1), the polysaccharide content in the oat milk of the embodiment is increased by 50.85%-68.18%, and the polypeptide content is increased by more than 5 times. Compared with the oat milk without enzymatic hydrolysis after germination after soaking (Comparative Example 2), the polypeptide content of the oat milk (Example 1) after germination of seeds after soaking is increased by 91.62%, indicating that seed germination regulates the activity of endogenous proteases in oats. Compared with the oat milk without papain enzymatic hydrolysis (Comparative Example 3), the polypeptide content is increased by 119.17%, indicating that adding an appropriate amount of exogenous protease is beneficial to the decomposition of oat protein into active polypeptides. The polypeptide content in the oat milk without free enzymatic hydrolysis (Comparative Example 4) is lower than that in the oat milk with free enzymatic hydrolysis, indicating that the release of free enzymes after beating plays a key role in the decomposition of macromolecules in oats into small molecular polysaccharides and polypeptides. The polysaccharide and polypeptide contents in the overheated oat milk were lower than those in the examples, indicating that overheating destroyed the endogenous enzyme activity of oats, thereby affecting the decomposition of oat protein and starch. In summary, the method provided by the present invention can increase the polysaccharide and polypeptide contents in oat milk.

[0139] Table 2 Polysaccharide and polypeptide contents in oat milk of Examples 1-2 of the present invention and Comparative Examples 1-5

[0140]

[0141]

[0142] The types and proportions of the bond types of the oat milk polysaccharides of the present invention and the comparative examples are shown in Table 3. As can be seen from the table, the 1→6 connection ratios of the polysaccharides in the oat milk obtained in Examples 1 and 2 are 54.68% and 56.70% respectively, and the 1→3 connection ratios are 20.18% and 24.88% respectively, which are lower than the oat milk obtained by enzymatic hydrolysis without heat shock and soaking (Comparative Example 1), oat milk without free enzymatic hydrolysis (Comparative Example 4) and overheating (Comparative Example 5), indicating that the proportions of 1→6 and 1→3 connections in the polysaccharide structure of the examples are relatively low. Oat β-glucan is connected by 1→3 and 1→4 glycosidic bonds. Although the proportion of 1→3 glycosidic bond connection of the polysaccharides in the oat milk of the examples is relatively low, the proportions of 1→2 and 1→4 connections are significantly higher than those in Comparative Example 1, indicating that appropriate heat shock and biotin soaking can adjust the composition and activity of the endogenous glucanase system in the seed imbibition and germination stages, and increase the polysaccharides with 1→2 and 1→4 connections.

[0143] Table 3 Types and proportions of polysaccharides in oat milk of Examples 1-2 of the present invention and Comparative Examples 1-5

[0144]

[0145] Figure 5-Figure 11The polysaccharide HPLC diagram of oat milk obtained from Examples 1 and 2 Comparative Examples 1-5. The HPLC results are summarized in Table 4. It can be seen from the figure and the table that the proportion of small molecular weight glucans with a molecular weight of about 700Da in the oat polysaccharides of the embodiment is significantly higher than that of the comparative example. In the oat milk without heat shock and soaking enzymatic hydrolysis (Comparative Example 1), the proportion of 18-20kDa molecular weight polysaccharides exceeds 55%, while the content of 700Da molecular weight polysaccharides is only 6.2%, which is significantly lower than that of Example 1, indicating that moderate heat shock soaking enzymatic hydrolysis can effectively increase the proportion of small molecular weight glucans in oat polysaccharides. The proportion of 700Da molecular weight polysaccharides in the oat milk prepared without soaking and germination in Comparative Example 2 is lower than that in the embodiment, indicating that germination and activation of glucanase after soaking is conducive to the decomposition and production of small molecular weight active polysaccharides. The molecular weight and proportion of oat polysaccharides in the oat milk prepared without adding exogenous papain (Comparative Example 3) are similar to those in the embodiment. Combining the results of the content of oat milk polysaccharides and the types and proportions of bond types, it can be seen that papain has little effect on the active polysaccharides of oats. In the oat milk obtained without free enzymatic hydrolysis (Comparative Example 4), the proportion of 700Da molecular weight polysaccharides was only 3.4%, indicating that more active 700Da oat glucans can be produced during free enzymatic hydrolysis. Comparative Example 5 used oat seeds with higher intensity heat shock to obtain oat milk. As a result, excessive heat shock slightly reduced the proportion of 700Da molecular weight polysaccharides and increased the proportion of 6-7kDa molecular weight polysaccharides, indicating that excessive heat shock is not conducive to obtaining active small molecular weight glucans.

[0146] Table 4 Determination of molecular weight of polysaccharides in oat milk of Examples 1-2 of the present invention and Comparative Examples 1-5

[0147]

[0148] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for enzymatic hydrolysis of oat milk, comprising the following steps: (1) Regulation of endogenous enzyme activity and in situ enzymatic hydrolysis in oats ① Heat-shocked oat kernels: Place oat kernels with seed coat in 80-85℃ hot water for 2-3 minutes and quickly put them into tap water to cool to room temperature to obtain heat-shocked oats. Oats: hot water = 1g: 20-30mL; ② Gibberellic acid soaking: soak the heat-shocked oats at 50℃ for 4-6 hours, remove and drain the water, the water absorption rate is 45%-50%, and the swelling stage of seed germination is completed to obtain soaked oats; The soaking liquid is 5-10 mg / L gibberellin, the soaking material-liquid ratio is dry oats: soaking liquid = 1:3-5, the protease activity in the soaked oats is ≥70 U / g dry oats, and the glucanase activity is ≥10 U / g dry oats; ③ Keep warm and moisturize the seeds and continue in-situ enzymolysis: soak the oats in a heat preservation box with a humidity of 90%-95% and keep warm at 25-28℃ for 5-8 hours to enter the middle stage of seed germination. The protease activity in the oats is ≥120U / g dry oats, and the glucanase activity is ≥20U / g dry oats, so as to obtain oats with high protease and glucanase activities; (2) beating and enzymatic hydrolysis of oats with free endogenous enzymes: beating oats with high protease and glucanase activity with water to obtain oat milk with residue, which is then kept at 40-45° C. for 1 hour and filtered to obtain oat milk 1 with free endogenous enzymes; (3) Inactivating endogenous enzymes and liquefying starch: heating the oat milk 1 to 80-85° C., adding 80-100 U / mL high-temperature α-amylase, and obtaining oat milk 2 in which endogenous enzymes are inactivated and starch liquefaction is completed; (4) Starch full saccharification and papain enzymolysis: cool the oat milk 2 to 50-55° C., add 50-100 U / mL saccharifying enzyme and 0.05-0.10% papain, keep the temperature for enzymolysis for 1-1.5 h, raise the temperature to 80-85° C. and keep for 15-20 min to inactivate the enzyme, and obtain oat milk 3; The enzymes used in steps (3) and (4) are: High temperature α-amylase: Novozymes Biotech LLC, enzyme activity 40000U / g; Glucoamylase: Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd., enzyme activity 100000U / g; Papain: Nanning Pangbo Bioengineering Co., Ltd., enzyme activity 100000U / g; (5) Homogenization and sterilization: add or not add sweetener to the oat milk 3, adjust the temperature to 70°C, homogenize at 30 MPa, and sterilize at 121°C for 15 min to obtain enzymatically hydrolyzed oat milk.

2. The method according to claim 1, characterized in that In step (2), the weight ratio of oats to water in the beating is 1:7-8, calculated based on the dry weight of oats; the water temperature of the beating water is lower than 50° C.; and the filtering is performed through a 40-60 mesh sieve; In step (3), high temperature α-amylase is added and kept warm for 1 hour.

3. Oat milk prepared by the enzymatic hydrolysis method according to claim 1 or 2.

4. Use of the oat milk according to claim 3 in promoting the proliferation of probiotics.

5. The use according to claim 4, characterized in that: The probiotics include Lactobacillus plantarum and Bifidobacterium.

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