Active milk fermented by old vinegar source and compound probiotics and preparation method of active milk fermented by old vinegar source and compound probiotics

Abstract

This invention belongs to the field of biotechnology and provides an active milk fermented with a compound probiotic from Shanxi aged vinegar and a method for its preparation. The method involves processing soybeans, chickpeas, quinoa, oats, and buckwheat from Shanxi using a special germination process, then mixing and grinding them into soy milk. After sterilization and cooling, a compound probiotic fermentation agent from Shanxi aged vinegar is added, and the mixture is fermented at a constant temperature of 37℃ and refrigerated to obtain the final product. The resulting product contains ≥91g CFU / mL of live probiotics, ≥350 mg / 100 g of γ-aminobutyric acid, ≥200 mg / 100 g of total flavonoids, ≥80 mg / L of isoflavone aglycones, ≥5 mg / mL of polypeptides, and ≥8000 U / g of nattokinase. It has a complex flavor of mixed grains and soybeans. In vitro experiments and rat studies have demonstrated that it has potential functions in assisting in lowering blood lipids, lowering blood sugar, regulating intestinal flora, and assisting in regulating blood pressure.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to an active milk made from fermented and sprouted mixed beans and grains using aged vinegar as a compound probiotic and its preparation method. The resulting active milk is rich in active peptides and γ-aminobutyric acid. Background Technology

[0002] The germination process of whole grain seeds involves multiple complex material transformations and has unique physiological effects. The content of some nutrients and functional factors gradually increases under the action of various complex enzymes, while the content of anti-nutritional factors gradually decreases. In germinated whole grains such as red beans, mung beans, black beans, and oats, the content of nutrients such as protein, polyphenols, flavonoids, amino acids, and γ-aminobutyric acid is effectively increased, while the content of anti-nutritional factors such as trypsin inhibitors, phytic acid, and tannins is significantly reduced. Germinated whole grain seeds have certain antioxidant activity, which can effectively scavenge various free radicals and has a certain adjuvant therapeutic effect on complications such as diabetes, hypertension, and hyperlipidemia.

[0003] Gamma-aminobutyric acid (GABA) is a non-protein constituent amino acid and an important inhibitory neurotransmitter in the mammalian nervous system. It possesses various biological functions, such as anti-anxiety, blood pressure reduction, and sleep improvement. Currently, it is widely used in food and dietary supplements in developed countries such as the EU, the US, and Canada. Seed germination is often used to increase the content of bioactive substances in grains. This process is simple and economical. During grain germination, plant physiological activity is vigorous, and the activity of various metabolic-related enzymes is activated, thereby promoting the accumulation of bioactive substances. Before germination, soaking seeds in water creates a low-oxygen environment. Under anaerobic conditions, carbohydrates undergo anaerobic fermentation, accumulating acetic acid, which acidifies the cytoplasm, activates GAD, and promotes GABA accumulation. Microbial fermentation is another major method of GABA enrichment fermentation, including bacteria, fungi, and yeasts. Currently, the main GABA producers are lactic acid bacteria such as *Lactococcus lactis*, *Lactobacillus paracasei*, *Lactobacillus bulgaricus*, and *Lactobacillus plantarum*.

[0004] Invention patent 202211287760.6, entitled "A Chickpea Sauerkraut Powder Rich in γ-Aminobutyric Acid and Its Application", discloses that dried chickpeas are crushed into powder, mixed evenly with lactic acid bacteria and water in a certain proportion, fermented to obtain sauerkraut fermentation liquid, and freeze-dried to obtain chickpea sauerkraut powder rich in γ-aminobutyric acid (GABA). It mainly utilizes the rich amino acids in chickpeas and the free amino acids and GABA produced by the enzyme system of lactic acid bacteria fermentation.

[0005] The patent application number is 202111479124.9, and the invention title is: A method for increasing the content of γ-aminobutyric acid in soybean products and high γ-aminobutyric acid soy milk. It discloses that by soaking soybeans, germinating process and concentration process, the content of γ-aminobutyric acid in soy milk is increased to 45 mg / 100 g.

[0006] The patent application number is 202111159612.1, and the invention title is: A method for preparing fermented soybean milk containing γ-aminobutyric acid and the fermented product obtained. By adding an appropriate amount of monosodium glutamate and inoculating Streptococcus thermophilus and Lactobacillus fermentum, the GABA content in soybean milk reaches 1.3 g / L.

[0007] Currently, most research on fermented soymilk rich in γ-aminobutyric acid (GABA) involves adding or not adding monosodium glutamate (MSG) and inoculating with lactic acid bacteria to enrich GABA. A few studies use soybean sprouting for natural GABA enrichment. However, there is a lack of comparison and optimization of GABA sprouting enrichment technology, screening of high-GABA-producing fermentation strains, and systematic optimization of raw material ratios and fermentation process parameters. This results in low GABA enrichment levels in the products, and an overall lack of richness and harmony in nutritional flavor. Furthermore, there is a scarcity of research on functional mixed grain and bean blends containing GABA-active milk. In addition, there is a lack of research on the retention, transformation, and enrichment changes of other active components during the GABA fermentation process, such as active peptides, flavonoids, isoflavone aglycones, and dietary fiber. This leads to low added value in nutritional function and weak market competitiveness of the products. Summary of the Invention

[0008] This invention provides an active milk fermented with a compound probiotic from Shanxi aged vinegar and a method for its preparation. The resulting active milk is rich in active peptides and γ-aminobutyric acid (GABA). The process involves treating superior Shanxi beans (soybeans, chickpeas) and grains (quinoa, oats, buckwheat) with a special germination process, mixing them in a specific ratio, grinding them into soy milk, sterilizing and cooling the mixture, then inoculating it with a superior compound probiotic fermentation agent from Shanxi aged vinegar. Fermentation is carried out at a constant temperature of 37°C, followed by refrigeration to obtain the final product.

[0009] This invention is achieved by the following technical solution: a method for preparing active milk from fermented and sprouted mixed beans and grains using Shanxi aged vinegar-based compound probiotics. The mixed beans (soybeans, chickpeas, quinoa, oats, and buckwheat) are treated with a special sprouting process, mixed and ground into soy milk in proportion, sterilized and cooled, and then inoculated with Shanxi aged vinegar-based compound probiotic fermentation agent. The mixture is fermented at a constant temperature of 37°C and then refrigerated to obtain the finished product.

[0010] Furthermore, the following steps are included: (1) Sprouting treatment of miscellaneous beans and grains: Select soybeans, chickpeas, quinoa, oats and buckwheat, remove impurities and broken grains, soak in 0.1% to 0.2% sodium hypochlorite solution for 30 to 60 minutes, and rinse thoroughly with water; add 4 to 6 times the volume of soaking solution, soak at 25℃, soak soybeans and chickpeas for 4 to 6 hours, and quinoa, oats and buckwheat for 8 to 10 hours to allow them to fully absorb water; then sprout soybeans, chickpeas, quinoa, oats and buckwheat grains in the dark at 25℃ and 95% relative humidity. The sprouting time for soybeans and chickpeas is 48 to 60 hours, and the sprouting time for quinoa, oats and buckwheat is 72 to 96 hours. During this period, wash the grains every 12 hours and sprinkle water to prevent the grains from getting moldy and having an odor; The soaking solution is: 1L deionized water, 0.8mmol CaCl2, 2.5mmol VB6 and 0.7g sodium glutamate; (2) Preparation of fermented active milk from sprouted mixed beans and grains: Soybeans, chickpeas, quinoa, oats and buckwheat that have finished sprouting are mixed in a mass ratio of 2:2:1:1:1. Distilled water is added at a ratio of 1:10 to 12 (w / v). The soybeans are ground in a vacuum blender at a speed of 4,000 r / min. The grinding and boiling are intermittent for 30 min. Then, 6% to 8% (w / v) white sugar is added. The mixture is sterilized at 121℃ for 20 min. After cooling to room temperature, 3% to 5% (w / v) compound probiotic starter is added. The mixture is fermented at 37℃ for 5 to 6 h and then stored at 4℃ for 12 h for refrigeration and maturation. This is the finished product. The compound probiotic starter is a mixture of *Lactobacillus plantarum* CGMCC 34901 freeze-dried powder and *Bacillus amyloliquefaciens* CGMCC 15732 freeze-dried powder at a mass ratio of 2:1.

[0011] The *Lactobacillus plantarum* CGMCC 34901 lyophilized powder and *Bacillus amyloliquefaciens* CGMCC 15732 lyophilized powder are active strains for aged vinegar mash. The preparation method for *Lactobacillus plantarum* CGMCC 34901 lyophilized powder and *Bacillus amyloliquefaciens* CGMCC 15732 lyophilized powder is as follows: Activated *Lactobacillus plantarum* CGMCC 34901 or *Bacillus amyloliquefaciens* CGMCC 15732 is inoculated into MRS liquid medium at an inoculation rate of 3%, and incubated statically at 37°C for 24 hours until the bacterial concentration reaches 10⁻⁶. 8 The bacterial sludge was obtained by centrifugation at 8000 r / min for 10 min at CFU / mL. A cryoprotectant was added at a ratio of 3:1 (v / w) to the bacterial sludge, and then freeze-dried to obtain *Lactobacillus plantarum* freeze-dried powder and *Bacillus amyloliquefaciens* freeze-dried powder, both containing active aged vinegar mash. The viable cell concentration reached 10⁻⁶. 10 CFU / g.

[0012] The *Lactobacillus plantarum* CGMCC 34901 strain was deposited at the China General Microbiological Culture Collection Center on June 16, 2025. The colony and cell morphology of *Lactobacillus plantarum* CGMCC 34901 on MRS medium are shown in [Figure 1]. Figure 1 Colony morphology: Round, 1.0-2.0 mm in diameter, milky white, flat or slightly raised, with neat edges and a smooth, matte surface; Cell morphology: Gram-positive bacteria, staining blue, short rod-shaped, cells mostly solitary, paired, or arranged in short chains; 16S rDNA phylogenetic tree of *Lactobacillus plantarum* CGMCC 34901 is shown below. Figure 2 This strain possesses excellent characteristics such as high acid production, high GABA production, high β-glucosidase production, high conversion of soybean isoflavones, and strong cholesterol-lowering ability; The Bacillus amyloliquefaciens CGMCC 15732 is the strain described in the application number 201910354887.7, and the invention title is: A strain of bacteria recorded in a fresh-cut fruit and vegetable preservative composed of antibacterial substances produced by Bacillus licheniformis, Bacillus atrophicus and Bacillus amyloliquefaciens. The protective agents are as follows: Protective agent 1: 5g skim milk powder, 8g whey powder, 50mL water, sterilized at 115℃ for 15min; Protective agent 2: 5g trehalose, 3g sucrose, 5g glucose, 0.5g monosodium glutamate, 50mL water, sterilized at 115℃ for 15min; after the high-temperature sterilized protective agents 1 and 2 are cooled to room temperature, they are mixed in a 1:1 volume ratio to obtain the bacterial cell vacuum freeze-drying protective agent.

[0013] The active milk made from aged vinegar-based compound probiotic fermented sprouted mixed beans and grains prepared by this invention has a probiotic live bacteria count ≥ 91g CFU / mL, γ-aminobutyric acid ≥ 350 mg / 100 g, total flavonoids ≥ 200 mg / 100 g, isoflavone aglycones ≥ 80 mg / L, polypeptides ≥ 5 mg / mL, and nattokinase ≥ 8000 U / g. It has a compound flavor of mixed grains and beans and has the effects of assisting in lowering blood lipids, lowering blood sugar, and regulating intestinal flora.

[0014] Compared with the prior art, the present invention has the following advantages: (1) The present invention uses quinoa as Jinli No. 1, oats as Weiduyou No. 5, and buckwheat as Jinqiaomai (Ku) No. 2, all of which are excellent varieties bred by Shanxi Agricultural University (Shanxi Academy of Agricultural Sciences). By controlling the product quality from the "original grain characteristics", it provides a good foundation for the development of high value-added and high-quality products, precise marketing, and value mining.

[0015] The characteristics of the raw grains are as follows: "Jinli No. 1," a provincially recognized and promoted variety, is high in protein, rich in various amino acids in appropriate proportions and easily absorbed, and rich in unsaturated fatty acids, flavonoids, B and E vitamins, choline, folic acid, β-glucan, etc., and possesses excellent resistance to downy mildew and leaf spot. "Weidu You No. 5," a provincially recognized and promoted variety, contains 22.04% protein and 5.2% β-glucan in its grains, and exhibits high yield, wide adaptability, strong drought and lodging resistance, and relatively strong disease resistance. "Jin Buckwheat (Bitter) No. 2," a provincially recognized and promoted variety, contains 13.45% protein, 3.09% fat, 63.07% starch, and 2.485% total flavonoids in its grains, and possesses excellent agronomic traits and comprehensive resistance.

[0016] This invention mixes legume raw materials—soybeans and chickpeas—with grain raw materials—quinoa, oats, and buckwheat in a certain proportion for co-fermentation. The nutritional factors in the various fermentation substrates complement each other, which is beneficial to the reproduction and metabolism of Bacillus bacteria that mainly produce protease and amylase and lactic acid bacteria that mainly produce acid through sugar fermentation. This results in richer functional nutrients and a more harmonious product flavor.

[0017] (2) This invention innovatively uses cold shock combined with nutrient soaking solution to promote grain germination and GABA enrichment technology. Calcium chloride, vitamins and monosodium glutamate are added to the soaking solution. Compared with natural germination, not only is the germination speed faster and contamination by miscellaneous bacteria prevented, but the GABA enrichment is also significantly increased.

[0018] (3) The plant lactobacillus CGMCC 34901 used in this invention was isolated from the vinegar mash of Shanxi aged vinegar. It not only has excellent fermentation characteristics such as high acid production, high γ-aminobutyric acid production, high β-glucosidase production, high conversion of soybean isoflavones, and good fermentation flavor, but also has excellent probiotic characteristics such as acid resistance, bile salt resistance, strong adhesion, and cholesterol reduction. The amyloliquefaciens CGMCC 15732 used in this invention was isolated from the Daqu of Shanxi aged vinegar. It has excellent characteristics such as strong production of amylase, protease, and nattokinase.

[0019] (4) The fermented sprouted mixed beans and grains active milk prepared by the present invention using aged vinegar as a compound probiotic has been shown in in vitro and rat tests to have good potential functions of anti-oxidation, blood sugar reduction and blood pressure regulation.

[0020] The plant lactobacillus (Lactobacillus plantarum) described in this invention Lactiplantibacillus plantarum It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC 34901 and deposit date of June 16, 2025. Attached Figure Description

[0021] Figure 1Images show the colony and cell morphology of *Lactobacillus plantarum* CGMCC 34901 on MRS agar plates (left image shows colony morphology; right image shows cell morphology). Figure 2 Phylogenetic tree of 16S rDNA of Lactobacillus plantarum CGMCC 34901; Figure 3 The results show the comparison of GABA content before and after germination of miscellaneous beans and grains. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0024] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0026] Example 1: Screening of probiotic lactic acid bacteria from aged vinegar: The fermentation characteristics of 20 lactic acid bacteria strains derived from Shanxi aged vinegar mash, including acid production, GABA production, β-glucosidase production, acid resistance, bile salt resistance, cholesterol reduction, and adhesion, were determined to screen for superior probiotic lactic acid bacteria strains.

[0027] (1) Determination of the acid production, acid tolerance, and bile salt tolerance of the strain Method for determining acid production performance: After the strain was activated by inoculation in MRS liquid medium, it was inoculated into 200 mL of MRS liquid medium with pH 2.0 at a volume fraction of 4%. After static culture at 37℃ for 24 h, the total acid content of the fermentation broth was determined by NaOH titration.

[0028] Methods for determining acid and bile salt tolerance: Bacterial cells were resuspended in MRS liquid medium (pH 2.0 and 0.3% bile salts, respectively) and incubated at 37°C for 4 h. Bacterial suspensions under normal growth conditions were used as controls. OD values ​​at 490 nm were measured using a multi-functional microplate reader. The survival rate was calculated as follows: Survival rate (%) = 100 × OD value of experimental group / OD value of control group.

[0029] (2) Determination of the adhesion and cholesterol-lowering properties of the strain Adhesion performance determination method: After activation by inoculating the strain in MRS liquid medium, it was washed with PBS buffer, resuspended in PBS, and the bacterial sludge concentration was adjusted to OD. 600nm The value was 1, and the mixture was incubated at 37℃. After 4 hours, a sample was taken, and the absorbance (A) of the upper layer of the bacterial suspension at 600 nm was recorded. t The calculation formula is: Self-aggregation rate (%) = (A0 - A t ) / A0×100%, where A0 is the initial absorbance value.

[0030] Cholesterol-lowering ability assay: After activation in MRS liquid medium, the strain was inoculated at a rate of 4% (v / v) into MRS-CHO liquid medium containing a final cholesterol concentration of 30 mg / 100 mL. The culture was anaerobic at 37°C, and samples were taken every 4 hours to measure the OD of the fermentation broth. 600nm Value and cholesterol degradation rate, with culture time as the x-axis, OD 600nm Plot the bacterial growth curve and cholesterol removal rate curve using the vertical axis as the ordinate and the cholesterol degradation rate as the vertical axis.

[0031] Cholesterol in the supernatant was determined using the o-phthalaldehyde colorimetric method, with uninoculated MRS-CHO liquid medium as a control. The cholesterol degradation rate was calculated as follows: Cholesterol degradation rate = (Cholesterol content in the supernatant of uninoculated MRS-CHO medium - Cholesterol content in the supernatant of inoculated MRS-CHO medium) / Cholesterol content in the supernatant of uninoculated MRS-CHO medium × 100%.

[0032] (3) Screening of high-GABA-producing lactic acid bacteria strains Plotting the γ-aminobutyric acid (GABA) standard curve: Prepare γ-aminobutyric acid standard solutions of 0, 0.2, 0.4, 0.6, 0.8, and 1 mg / mL. Take 1 mL of each concentration standard solution and add 1.0 mL of 0.01 mol / L sodium tetraborate buffer (pH 9.0), 1.0 mL of 6% redistilled phenol solution, and 2 mL of 7.5% sodium hypochlorite solution to each solution. Mix well and heat in a boiling water bath for 10 min. Immediately after heating, place the solution in ice water for 5 min. Once the solution turns blue-green, add 1.5 mL of 60% ethanol solution and mix thoroughly by continuous shaking. Measure the absorbance at 645 nm. Plot the standard curve with absorbance on the ordinate and the concentration of each standard solution (mg / mL) on the abscissa.

[0033] Screening for high-yielding γ-aminobutyric acid (GABA)-producing lactic acid bacteria strains: Lactic acid bacteria seed culture was inoculated into MRS medium at a rate of 3% (v / v), incubated statically at 37°C for 24 h, and centrifuged at 12,000 r / min for 10 min to obtain the supernatant. 1 mL of the supernatant was used to determine the GABA-producing capacity of the lactic acid bacteria according to the standard curve assay.

[0034] (4) Determination of the ability of lactic acid bacteria to convert soybean isoflavones Screening of lactic acid bacteria strains with high β-glucosidase production: Soy isoflavones are widely found in legumes. β-glucosidase can break down soy isoflavones in glycoside form into aglycone form that is more active and more easily absorbed by the human body.

[0035] Screening for lactic acid bacteria strains producing high levels of β-glucosidase: Aescin can be hydrolyzed by β-glucosidase into apigenin and glucose. Aescin and Fe... 3+ The reaction produces a brownish-black color. A special agar medium containing esculin and ferric citrate was prepared by adding 0.3% (w / w) of esculin and 0.05% (w / w) of ferric citrate to MRS liquid medium, followed by 1.8% agar. After sterilization, 250 µL was added to each well of a 96-well plate while still hot at approximately 60°C. After the plate solidified, 10 µL of bacterial culture was added to each well in the numbered order. After incubation upside down for 48 hours, wells that turned a deep brownish-black color indicated β-glucosidase-producing strains; the intensity of the color could be used to preliminarily determine the enzyme activity.

[0036] Determination of soybean isoflavone aglycones (reflecting the ability of strains to convert soybean isoflavones): The ability of lactic acid bacteria to convert soybean isoflavones, i.e. the content of soybean isoflavone aglycones produced, is expressed as the total content of daidzein and genistein in fermented soybeans.

[0037] Standard curve plotting: Daidzein and genistein standards were prepared into standard solutions with concentrations of 1, 2, 4, 8, and 16 mg / mL, respectively, and measured by HPLC. The peak area was plotted on the ordinate and the concentration of the standard solution was plotted on the abscissa to obtain the standard curve.

[0038] Determination of soy isoflavone aglycone content in fermented soy milk with different lactic acid bacteria: 5 mL of fermented soy milk was placed in a 50 mL volumetric flask, and 80% methanol solution was added to near the mark. Ultrasonic extraction was performed for 20 min, followed by dilution with 80% methanol. The sample solution was then transferred to a centrifuge tube and centrifuged at 8000 r / min for 15 min. The supernatant was filtered through a 0.45 nm filter, and the filtrate was collected for high-performance liquid chromatography (HPLC). Chromatographic conditions: C 18 Chromatographic column; mobile phase: acetonitrile; aqueous phosphoric acid solution (pH 3.0); flow rate: 1.0 mL / min; UV detection wavelength: 260 nm; injection volume: 10 µL; column temperature: 30 ℃; gradient elution.

[0039] Fermented soy milk preparation method: Selected soybeans are treated with 0.1% sodium hypochlorite solution and soaked for 30 minutes, then rinsed thoroughly with water; 5 times the volume of deionized water is added, and the mixture is soaked at 25℃ for 4 hours to allow it to fully absorb water. Distilled water is added at a ratio of 1:12 (w / v), and the soybeans are ground into soy milk (using a vacuum blender to make mixed grain and soybean milk at a speed of 4000 r / min, with a 30-minute interval between grinding and boiling). 8% white sugar is added, and the mixture is sterilized at 121℃ for 20 minutes. After cooling to room temperature, 5% (v / v) lactic acid bacteria seed liquid is added, and the mixture is fermented at a constant temperature of 37℃ for 6 hours. After fermentation, the mixture is placed in a refrigerator at 4℃ for 12 hours for post-fermentation to obtain the finished product.

[0040] Preparation method of lactic acid bacteria seed culture: Activated lactic acid bacteria strains were inoculated into MRS liquid medium at an inoculum rate of 3%, and cultured statically at 37℃ for 24 h until the bacterial concentration reached 10⁻⁶. 8 CFU / mL, centrifuged at 8000 r / min for 10 min to obtain bacterial sludge. The bacterial cells were washed twice with 0.85% physiological saline and then resuspended to achieve a concentration of 10. 8 CFU / mL, which is lactic acid bacteria seed culture.

[0041] Table 1 shows the test results. The results indicate that Lactobacillus plantarum CGMCC 34901 has significantly higher abilities than other strains in acid production, GABA production, β-glucosidase production, conversion of soybean isoflavones, acid resistance, bile salt resistance, cholesterol reduction, and adhesion.

[0042] Table 1 Screening of superior probiotic lactic acid bacteria strains from aged vinegar. Example 2: Screening of superior spore-forming bacteria from aged vinegar mash The activities of amylase, protease and nattokinase produced by 20 Bacillus strains derived from aged vinegar mash were determined to screen for superior Bacillus strains.

[0043] 1. Methods for determining the amylase and protease production capacity of the strain: After activation, single colonies of the Bacillus strain were picked and inoculated into nutrient broth medium. After incubation at 37℃ and 160 r / min for 24 h with shaking, a Bacillus seed culture was obtained. The Bacillus seed culture was inoculated into starch medium and casein medium at a ratio of 5% (v / v), respectively. After incubation at 37℃ and 160 r / min with shaking for 48 h, the crude enzyme solution was obtained by centrifugation at 5000 r / min for 15 min. Amylase activity was determined by the DNS colorimetric method, and protease activity was determined by the Folin-phenol colorimetric method.

[0044] 2. Method for determining the nattokinase production capacity of the strain: A certain amount of soybeans was weighed, washed, and soaked overnight. After draining with gauze, the soybeans were autoclaved at 121℃ for 20 min, allowed to cool naturally, and then inoculated with 8% (v / w) Bacillus seed culture. The mixture was incubated at 37℃ for 24 h and then ripened at 4℃ for 24 h. 10 g of fermented natto was dissolved in 90 mL of deionized water, homogenized for 10 s, and extracted at 4℃ for 24 h. The supernatant was then collected by centrifugation at 12,000 r / min for 10 min, and the enzyme activity of nattokinase was determined using the agarose-fibrin method.

[0045] The test results are shown in Table 2. The amylase, protease and nattokinase activities of Bacillus amyloliquefaciens CGMCC 15732 were significantly higher than those of other strains, reaching 61.45 U / mL, 26.14 U / mL and 2886 U / g, respectively.

[0046] Table 2: Screening of Bacillus strains producing high levels of amylase, protease, and nattokinase from aged vinegar. Example 3: Preparation of germinated soybeans, chickpeas, quinoa, oats, and buckwheat: Soybeans, chickpeas, quinoa, oats, and buckwheat were selected, and impurities and broken grains were removed. They were treated with a 0.1%–0.2% sodium hypochlorite solution and soaked for 30–60 minutes, then rinsed thoroughly with water. Five times the volume of the soaking solution was added, and the mixture was soaked at 25°C. Soybeans and chickpeas were soaked for 4 hours, and quinoa, oats, and buckwheat for 10 hours to ensure full water absorption. The soybean, chickpea, quinoa, oat, and buckwheat seeds were then germinated in the dark at 25°C and 95% relative humidity. Germination of soybeans and chickpeas ended after 48 hours, and quinoa, oats, and buckwheat after 72 hours. During this period, the seeds were washed and sprinkled with water every 12 hours to prevent mold and off-odors. The soaking solution consisted of 1 L of deionized water, 0.8 mmol CaCl2, 2.5 mmol VB6, and 0.7 g sodium glutamate. The soaked grains were dried in an oven to constant weight, then pulverized through a 60-mesh pulverizer, and samples were taken to determine the GABA content.

[0047] Determination of GABA content in sprouted grains: 1g of powder was added to a 5mL volumetric flask and diluted to volume. The mixture was extracted at room temperature for 1 hour, allowed to stand for 10 minutes, filtered first with ordinary filter paper, and then filtered through a 0.22μg microporous membrane. The GABA content in the filtrate was determined according to the method described in Example 1. The test results are as follows: Figure 3 As shown, the GABA content of the five grains increased significantly after germination treatment, by approximately 2.8 to 4.5 times.

[0048] Example 4: Preparation of fermented active milk from sprouted mixed beans and grains with compound probiotics derived from aged vinegar: Sprouting soybeans, chickpeas, quinoa, oats, and buckwheat were mixed in a mass ratio of 2:2:1:1:1. Distilled water was added at a ratio of 1:12 (w / v). The mixture was then ground into soy milk (using a vacuum blender to make mixed grain and soybean milk at 4000 r / min, with a 30-minute interval between grinding and boiling). 8% white sugar was added, and the mixture was sterilized at 121℃ for 20 minutes. After cooling to room temperature, 5% (w / v) compound probiotic starter was added. The mixture was fermented at a constant temperature of 37℃ for 6 hours and then stored in a refrigerator at 4℃ for 12 hours for post-fermentation to obtain the finished product.

[0049] Preparation method of compound probiotic starter: Lactobacillus plantarum CGMCC 34901 freeze-dried powder and Bacillus amyloliquefaciens CGMCC 15732 freeze-dried powder are mixed evenly at a mass ratio of 2:1 to obtain the compound probiotic starter.

[0050] The preparation method of the freeze-dried powders of *Lactobacillus plantarum* CGMCC 34901 and *Bacillus amyloliquefaciens* CGMCC 15732, which are the active sources of aged vinegar mash, is as follows: Activated *Lactobacillus plantarum* CGMCC 34901 or *Bacillus amyloliquefaciens* CGMCC 15732 are inoculated into MRS liquid culture medium at an inoculation rate of 3%, and cultured statically at 37°C for 24 h until the bacterial concentration reaches 10⁻⁶. 8 Centrifuge at 8000 r / min for 10 min to obtain bacterial sludge. Add 10% protective agent at a ratio of 3:1 (v / w) and freeze-dry under vacuum to obtain *Lactobacillus plantarum* freeze-dried powder and *Bacillus amyloliquefaciens* (viable count concentration reaching 10) as active aged vinegar mash source. 10 The preparation method of the protectant is as follows: Protectant 1: 5g skim milk powder, 8g whey powder, 50mL water, sterilized at 115℃ for 15min; Protectant 2: 5g trehalose, 3g sucrose, 5g glucose, 0.5g monosodium glutamate, 50mL water, sterilized at 115℃ for 15min; After the high-temperature sterilized protectant 1 and protectant 2 are cooled to room temperature, they are mixed in a 1:1 volume ratio to obtain the cell vacuum freeze-drying protectant.

[0051] Comparative Example 1: Preparation of fermented active milk from unsprouted mixed beans and grains containing aged vinegar-based compound probiotics: Soybeans, chickpeas, quinoa, oats, and buckwheat were selected, and impurities and broken particles were removed. The mixture was treated with a 0.1% sodium hypochlorite solution and soaked for 30 minutes, followed by thorough rinsing with water. Five times the volume of deionized water was added, and the mixture was soaked at 25°C. Soybeans and chickpeas were soaked for 4 hours, and quinoa, oats, and buckwheat were soaked for 10 hours to ensure full water absorption. The mixture was then mixed in a mass ratio of 2:2:1:1:1, and distilled water was added at a ratio of 1:12 (w / v). The remaining preparation methods and operating steps were the same as in Example 4.

[0052] Example 5: Determination of viable count and bioactive components in fermented milk (1) Determination of viable count, GABA, isoflavone aglycone, DPPH scavenging rate, nattokinase and ACE inhibitory activity: The viable count in fermented active milk was determined by MRS dilution plate colony counting method.

[0053] Take 100 mL of fermented active milk sample, centrifuge at 10,000 r / min, 4℃ for 10 min, take the supernatant, and determine the content of GABA, isoflavone aglycone and nattokinase activity in fermented active milk according to the methods in Example 1 and Example 2; use DPPH free radical scavenging ability kit and ACE inhibition activity kit to detect DPPH scavenging rate and ACE inhibition activity in the sample.

[0054] (2) Determination of polypeptide content: Take 100 mL of fermented active milk sample, centrifuge at 10,000 r / min, 4℃ for 10 min, take the supernatant and mix it with 10% trichloroacetic acid (TCA) solution 1:1 (v / v), let it stand for 10 min, and centrifuge at 4,000 r / min for 15 min. Mix the supernatant with biuret reagent 3:2 (v / v), let it stand for 15 min, and measure the absorbance value at 540 nm to calculate the polypeptide content (mg / mL). Use bovine serum albumin as a standard to replace the sample to make a standard curve, and obtain the regression equation: y=0.0158x+0.0977, R 2 =0.9990.

[0055] (3) Determination of the hypoglycemic ability of fermented soy milk Determination of α-amylase inhibition capacity: α-amylase was dissolved in PBS (0.1 mol / L, pH 6.9) buffer. 0.5 mL of the supernatant was mixed with an equal volume of α-amylase (1 U / mL) solution and incubated in a water bath (37℃, 10 min). 0.5 mL of 1% starch solution was added and reacted for 15 min (37℃). Finally, 1 mL of DNS was added and incubated in a boiling water bath for 5 min. The mixture was rapidly cooled and brought to a final volume of 10 mL. The OD value was measured at 540 nm. The α-amylase inhibition rate was calculated using the formula: A c =[1-(A i -A j ) / A0]×100%; Where: A c α-Amylase inhibition rate; A i : Absorbance of sample group; A j : A i Replace the α-amylase in A0 with PBS buffer; A0: Replace A i The sample solution in the sample solution was replaced with distilled water.

[0056] Determination of α-glucosidase inhibitory activity: The lyophilized powder was processed as described above. PNPG and α-glucosidase were dissolved in PBS (0.1 mol / L, pH 6.8) buffer. 0.5 mL of the supernatant was mixed with an equal volume of PBS and PNPG solution (3 mmol / L), and then incubated in a water bath (37℃, 10 min). 0.5 mL of α-glucosidase (0.2 U / mL) was added, and the reaction was allowed to proceed for 40 min. The reaction was terminated with 1.5 mL of Na2CO3 (0.2 mol / L) solution, and the OD value was measured at 405 nm. The α-glucosidase inhibition rate was calculated using the formula: Ac = [1 - (A...] i -A j [Ac / A0]×100%; Where: Ac: α-glucosidase inhibition rate; A i : Absorbance of sample group; A j : A iReplace the α-glucosidase in A0 with PBS buffer; A0: Replace A i The sample solution in the sample solution was replaced with distilled water.

[0057] Table 3 shows that, compared with the unsprouted treatment, the viable bacteria count and the content of various functional active ingredients in the fermented milk of mixed beans and grains after sprouting treatment were significantly increased. The viable bacteria count, GABA, total flavonoids, isoflavone aglycones, polypeptides, and nattokinase content increased to 9.15 lg CFU / mL, 380.24 mg / 100 g, 200.25 mg / 100 g, 95.30 mg / L, 5.35 mg / mL, and 8450.32 U / g, respectively. Meanwhile, it exhibited excellent in vitro bioactivity: DPPH scavenging rate, ACE inhibitory activity, α-amylase inhibitory capacity, and α-glucosidase inhibition rate reached 80.36%, 62.15%, 40.14%, and 46.28%, respectively, indicating that it has good potential functions in antioxidation, assisting in lowering blood sugar, and assisting in regulating blood pressure.

[0058] Table 3: Determination of viable bacteria count and bioactive components in fermented milk from aged vinegar-based compound probiotic sprouted beans and grains Example 5: Functional testing of fermented and sprouted mixed bean and grain active milk derived from aged vinegar and compound probiotics to assist in lowering blood lipids, lowering blood sugar, and regulating intestinal flora. Thirty healthy 8-week-old rats (150±20) g were randomly divided into a normal control group (n=10), a model group (n=10), and an experimental group (n=10). The normal control group was fed a regular diet, while the model and experimental groups were fed a high-fat diet. For the first 6 weeks, the experimental group was administered a compound probiotic fermented sprouted mixed bean and grain active milk (prepared according to the method in Example 4) by gavage daily at a volume of 5 mL / kg body weight, while the normal control group was administered an equal volume of physiological saline by gavage. This gavage was continued for 30 days. At week 7, the model and experimental groups were induced by intraperitoneal injection of streptozotocin (STZ) (40 mg / kg), while the control group was injected with physiological saline. The rats were fed until week 13. After the experiment, the rats were sacrificed, and specimens were collected for measurement.

[0059] Blood glucose, blood lipids, and hormone levels in rats were measured using a kit. Fresh feces were aseptically collected the night before euthanasia of the rats to determine the viable bacterial count. Lactobacillus was cultured aerobically at 37°C for 48 h on Lactobacillus selective medium; Bifidobacterium was cultured anaerobically at 37°C for 48 h on BBL agar medium; and Enterobacteriaceae was cultured aerobically at 37°C for 48 h on eosin methylene blue agar before counting.

[0060] High-fat feed: 45% corn flour, 1% vitamins, 20% lard, 20% casein, 4% bone meal, and 10% sucrose.

[0061] Common feed: 50% cornmeal, 25% wheat bran, 20% soybeans, 2% bone meal, 2% fish meal, and 1% vitamins.

[0062] As shown in Table 4, fermented active milk made from sprouted mixed beans and grains with compound probiotics from aged vinegar can significantly reduce the concentrations of serum TC, LDL-C, and TG in rats fed a high-fat diet.

[0063] Table 4: Effects of fermented and sprouted mixed bean and grain active milk derived from aged vinegar and compound probiotics on serum TC, HDL-C, LDL-C and TG in diabetic rats Note: Mean (SD, standard deviation) (n = 10); compared to the model group, P < 0.01 indicates a significant difference.

[0064] The effects of fermented sprouted mixed bean and grain active milk containing Lao Chen vinegar and probiotics on fasting blood glucose and glucose tolerance in diabetic rats are shown in Table 5. The results showed that compared with the normal control group, the fasting blood glucose level of the model group rats was significantly increased, while the fermented sprouted mixed bean and grain active milk had a hypoglycemic effect. There were no significant changes in fasting blood glucose levels at weeks 7 and 13 in the control and model groups, but the fasting blood glucose level in the rat group administered fermented sprouted mixed bean and grain active milk by gavage was significantly lower at week 13 compared to week 7, and glucose tolerance (AUC: area under the curve) significantly recovered at the end of week 13.

[0065] Table 5: Effects of fermented and sprouted mixed bean and grain active milk derived from aged vinegar and probiotics on fasting blood glucose and glucose tolerance in diabetic rats. Type 2 diabetes mellitus is a non-insulin-dependent form of diabetes mellitus, characterized by hyperglycemia, insulin resistance, and relative insulin deficiency. As shown in Table 6, serum insulin, glucagon, and leptin levels in the model group rats were significantly higher than those in the normal control group (P<0.05). After gavage treatment with fermented active milk from sprouted mixed beans and grains, the levels of these three hormones decreased. Furthermore, the levels of glucagon-like peptide-1 and tyrosinase in the diabetes model group were significantly lower than those in the normal control group (P<0.05), while gavage treatment with fermented active milk from sprouted mixed beans and grains significantly increased the levels of these two hormones (P<0.05).

[0066] Table 6: Effects of fermented and sprouted mixed bean and grain active milk derived from aged vinegar and probiotic compound on hormone levels in diabetic rats As shown in Table 7, compared with the model group, the serum ALT and AST activities and liver SOD and GSH of rats that were given fermented active milk containing aged vinegar compound probiotics were significantly restored, while the production of MDA was reduced.

[0067] Table 7: Effects of fermented and sprouted mixed bean and grain active milk derived from aged vinegar and compound probiotics on serum ALT, AST activity and liver SOD, GSH and MDA in diabetic rats Note: Mean (SD, standard deviation) (n = 10); compared to the model group, P < 0.01 indicates a significant difference.

[0068] Table 8 shows that, compared with the control group, the number of Lactobacillus and Bifidobacterium in the feces of rats in the model group was significantly reduced. Meanwhile, the fermented and sprouted mixed bean and grain active milk containing compound probiotics increased the number of beneficial bacteria and reduced the number of harmful bacteria, demonstrating a significant effect on improving the intestinal flora of diabetic rats. Studies have confirmed that clinical consumption of yogurt or lactic acid bacteria preparations can increase intestinal probiotics, inhibit the invasion and colonization of pathogenic bacteria to a certain extent, regulate the balance of intestinal flora, improve the intestinal mucosal barrier function, and effectively alleviate and improve diabetic symptoms.

[0069] Table 8: Effects of fermented and sprouted mixed bean and grain active milk derived from aged vinegar and compound probiotics on serum intestinal flora in diabetic rats Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing an active milk fermented and sprouted mixed beans and grains using aged vinegar as a source of compound probiotics, characterized in that: Soybeans, chickpeas, quinoa, oats, and buckwheat are germinated, then mixed and ground into soy milk in a certain proportion. After sterilization and cooling, the soy milk is inoculated with Shanxi aged vinegar-based compound probiotic fermentation agent, fermented at a constant temperature of 37°C, and then refrigerated to obtain the finished product.

2. The preparation method according to claim 1, characterized in that: Specifically, the steps include the following: (1) Sprouting treatment of miscellaneous beans and grains: Select soybeans, chickpeas, quinoa, oats and buckwheat, remove impurities and broken grains, soak in 0.1% to 0.2% sodium hypochlorite solution for 30 to 60 minutes, and rinse thoroughly with water; add 4 to 6 times the volume of soaking solution, soak at 25℃, soak soybeans and chickpeas for 4 to 6 hours, and quinoa, oats and buckwheat for 8 to 10 hours to allow them to fully absorb water; then sprout soybeans, chickpeas, quinoa, oats and buckwheat grains in the dark at 25℃ and 95% relative humidity. The sprouting time for soybeans and chickpeas is 48 to 60 hours, and the sprouting time for quinoa, oats and buckwheat is 72 to 96 hours. During this period, wash the grains every 12 hours and sprinkle water to prevent the grains from getting moldy and having an odor; The soaking solution is: 1L deionized water, 0.8mmol CaCl2, 2.5mmol VB6 and 0.7g sodium glutamate; (2) Preparation of fermented active milk from sprouted mixed beans and grains: Soybeans, chickpeas, quinoa, oats and buckwheat that have finished sprouting are mixed in a mass ratio of 2:2:1:1:

1. Distilled water is added at a ratio of 1:10 to 12 w / v. The soybeans are ground in a vacuum blender at a speed of 4,000 r / min. The grinding and boiling are intermittent for 30 min. Then, 6% to 8% w / v white sugar is added. The mixture is sterilized at 121℃ for 20 min. After cooling to room temperature, 3% to 5% v / v compound probiotic starter is added. The mixture is fermented at 37℃ for 5 to 6 h and then stored at 4℃ for 12 h for refrigeration and maturation. This is the finished product. The compound probiotic starter is a mixture of *Lactobacillus plantarum* CGMCC 34901 freeze-dried powder and *Bacillus amyloliquefaciens* CGMCC 15732 freeze-dried powder at a mass ratio of 2:

1.

3. The preparation method according to claim 2, characterized in that: The *Lactobacillus plantarum* CGMCC 34901 lyophilized powder and *Bacillus amyloliquefaciens* CGMCC 15732 lyophilized powder are active strains for aged vinegar mash. The preparation method for *Lactobacillus plantarum* CGMCC 34901 lyophilized powder and *Bacillus amyloliquefaciens* CGMCC 15732 lyophilized powder is as follows: Activated *Lactobacillus plantarum* CGMCC 34901 or *Bacillus amyloliquefaciens* CGMCC 15732 is inoculated into MRS liquid medium at an inoculation rate of 3%, and cultured statically at 37°C for 24 h until the bacterial concentration reaches 10⁻⁶. 8 CFU / mL, centrifuged at 8000 r / min for 10 min to obtain bacterial sludge. A preservative was added at a v / w ratio of 3:1 (bacterial cell freeze-drying preservative to bacterial sludge), and then freeze-dried under vacuum to obtain *Lactobacillus plantarum* freeze-dried powder and *Bacillus amyloliquefaciens* freeze-dried powder, both containing active aged vinegar mash. The viable cell concentration reached 10-1. 10 CFU / g; The protective agents are as follows: Protective agent 1: 5g skim milk powder, 8g whey powder, 50mL water, sterilized at 115℃ for 15min; Protective agent 2: 5g trehalose, 3g sucrose, 5g glucose, 0.5g monosodium glutamate, 50mL water, sterilized at 115℃ for 15min; after the high-temperature sterilized protective agents 1 and 2 are cooled to room temperature, they are mixed in a 1:1 volume ratio to obtain the bacterial cell vacuum freeze-drying protective agent.

4. Active milk made from fermented and sprouted mixed beans and grains using a compound probiotic fermented with aged vinegar source, prepared by any one of the methods described in claims 1-3.

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