Double-bacterium fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, gamma-aminobutyric acid and active probiotics and preparation method of double-bacterium fermented vinegar-soaked black bean freeze-dried powder

Abstract

The invention belongs to the technical field of table vinegar biology, and provides double-bacterium fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, gamma-aminobutyric acid and active probiotics and a preparation method thereof.After black beans are soaked overnight and subjected to high-temperature and high-pressure sterilization, the black beans are inoculated with lactobacillus plantarum and bacillus amyloliquefaciens to be mixed and fermented, then the black beans are soaked with mulberry-mature vinegar aged pulp, the mulberry-mature vinegar aged pulp is subjected to freeze-drying, and the freeze-dried powder is obtained. After soaking is finished, the beans are fished out and mixed with the filtered vinegar-soaked mulberries, the mixture is smashed and subjected to vacuum freeze drying, vinegar-soaked black bean freeze-dried powder is obtained, lactobacillus plantarum sourced from active mature vinegar is added, and the finished product is obtained. The product contains more than or equal to 16000 U / g of nattokinase, more than or equal to 90 mg / L of isoflavone aglycone, more than or equal to 200 mg / 100 g of r-aminobutyric acid, more than or equal to 2000 mg / 100 g of procyanidine and more than or equal to 107 cfu / g of probiotics, is black purple in color and luster, has the composite flavor of mature vinegar, mulberries and beans, and has the effects of reducing blood fat, enhancing immunity and the like.

Description

Technical Field

[0001] The invention belongs to the field of vinegar biotechnology, and specifically relates to a double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, gamma-aminobutyric acid and active probiotics and a preparation method thereof. Background Art

[0002] The unique brewing process of Shanxi aged vinegar makes the product not only have a multi-layered and rich flavor quality of "sour, sweet, mellow, fragrant and fresh", but also is rich in organic acids, amino acids, flavonoids, ligustrazine, melanoidin, γ-aminobutyric acid, lovastatin and other physiologically active ingredients. In many ancient Chinese medical texts, vinegar is widely used in Chinese medical treatment and health care practices, and is often used as an auxiliary material for Chinese medicine soaking. Its main functions are: (1) It can enhance the solubility of the active ingredients in Chinese medicinal materials. For example, after the free alkaloids in Corydalis are soaked in vinegar, they are converted into acetates, which increases the extraction rate of alkaloids by 1 times; (2) It can guide the medicine into the liver meridian, enhance the effects of promoting blood circulation and removing blood stasis, regulating qi and relieving pain. For example, blood circulation and blood stasis-removing medicines such as Curcuma zedoaria, frankincense, and myrrh, and qi-regulating medicines such as Cyperus rotundus and Citrus aurantium are often soaked in vinegar; (3) It can moderate the medicinal properties and reduce toxic side effects. For example, after Euphorbia dahurica and Gelsemium elegans are soaked in vinegar, their laxative effects are moderated; (4) It can remove the fishy smell of the medicine, facilitate the crushing and decoction of the active ingredients, and improve the efficacy. For example, animal bone medicines such as tortoise shell and turtle shell are used as medicine after being soaked in vinegar.

[0003] Black beans are widely produced, mainly in Northeast China, Hebei, Shanxi and Shaanxi. Black beans are not only rich in protein, vitamins, minerals and other nutrients, but also rich in polyphenolic compounds such as anthocyanins. Their nutritional value is far superior to soybeans, and they have a long history of being both medicine and food. The Compendium of Materia Medica records that "black beans have the effects of nourishing the kidney and blood, clearing away heat and detoxifying, promoting blood circulation and removing blood stasis, darkening hair and improving eyesight, and prolonging life". Modern research shows that black beans have antioxidant, blood pressure lowering, anti-tumor and anti-inflammatory effects. As active ingredients such as active peptides, linoleic acid, polyphenols, flavonoids, oligosaccharides in black beans are continuously separated, extracted, analyzed and their active functions verified, the nutritional and health value of black beans has attracted widespread attention from researchers at home and abroad.

[0004] There are many folk recipes about pickled foods in traditional Chinese medicine books, the most typical of which are soybeans, black beans, peanuts, eggs, ginger, etc., of which black beans have the highest nutritional and health value. It is reported that black beans have the functions of beauty, weight loss, kidney tonification, eyesight improvement, and hair blackening, and can effectively improve constipation, high blood pressure, high blood fat, back pain, leg pain, diabetes, chronic fatigue, cold syndrome, high cholesterol, coronary heart disease, etc.

[0005] Zhang Xiuhong and others compared and analyzed the effects of five processing conditions, namely, cooking, frying, roasting, high pressure and raw processing, on the flavor and antioxidant activity of black beans soaked in vinegar. Jiang Fan and others optimized the process conditions such as the degree of ripeness and soaking temperature of black beans, and ultimately significantly increased the content of total phenols and total flavonoids in the product. The invention patent with application number 202311159236.5, "A kind of vinegar-soaked black bean food containing active peptides and processing method", obtains a vinegar-soaked black bean product rich in peptides and tyrosinase activity by using airflow to puff black beans and adding acidic proteases.

[0006] In summary, the current processing techniques for vinegar-soaked black bean products mostly follow the traditional method of simple soaking after steaming and frying; or the addition of enzymes is used to increase the content of black bean protein decomposition polypeptides; there is a lack of conversion and enrichment of nutrients and functional ingredients during processing, as well as monitoring of the content and stability of active ingredients in the final product and during storage; moreover, vinegar-soaked beans and vinegar-soaked peanuts on the market are mostly packaged in vacuum bags or glass jars, which have problems such as uneven soaking of contents during the shelf life, drying of the texture, and deterioration of taste and flavor; in addition, there are few studies on the use of probiotics enrichment and fermentation to increase the content of functional factors in vinegar-soaked products, and the optimization of the pickling process system of combined functional vinegar liquid, so as to develop high-value vinegar health products.

[0007] Vinegar, as a carrier of healthy food, is widely recognized by the public. The development of vinegar products that are both medicinal and edible is a major direction for the high-quality development of the vinegar industry. High-quality vinegar-soaked black beans and other vinegar-soaked health products with clear types and contents of effective ingredients have broad and good development prospects. Summary of the invention

[0008] The invention provides a double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, gamma-aminobutyric acid and active probiotics and a preparation method thereof. The black beans are soaked overnight, sterilized at high temperature and high pressure, and then inoculated with excellent probiotic strains of plant lactobacillus and bacillus amyloliquefaciens from Shanxi aged vinegar fermented mash for mixed fermentation, then soaked in mulberry-aged vinegar slurry, after soaking, the beans are fished out and mixed with the previously filtered vinegar-soaked mulberries, crushed, and vacuum freeze-dried to obtain the vinegar-soaked black natto freeze-dried powder, and then the active aged vinegar fermented mash source Lactobacillus plantarum is added to obtain the finished product.

[0009] The present invention innovatively introduces the fermentation of aged vinegar-derived probiotics, soaking of mulberry-aged vinegar pulp, and addition of active probiotic powder into the traditional processing technology of black beans soaked in vinegar. The product is rich in active ingredients such as nattokinase, r-aminobutyric acid, isoflavone aglycones, proanthocyanidins, probiotics, etc., and breaks through the product form of traditional vinegar-soaked black beans. The vinegar-soaked black bean probiotic freeze-dried powder product can be well integrated with breakfast meal replacement powder, beverages and other foods, thereby expanding and enhancing the market space, consumer groups and development prospects of aged vinegar health food.

[0010] The present invention is achieved by the following technical scheme: a method for preparing a double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, gamma-aminobutyric acid and active probiotics, comprising the following steps: (1) Preparing a mulberry-aged vinegar slurry soaking liquid: soaking dried mulberries in 8-year-old aged vinegar slurry at a mass volume ratio of 1:5 for 1 month, filtering out the vinegar liquid, adding 20% ​​honey, 0.03% white sugar, and 0.03% edible salt at a mass volume ratio of the vinegar liquid based on the filtered vinegar liquid, boiling for 40 minutes, and cooling to 60° C. to obtain a mulberry-aged vinegar slurry soaking liquid, and setting aside the filtered vinegar-soaked mulberries; (2) Preparation of freeze-dried powder: The washed black beans were soaked in deionized water overnight, drained with gauze, sterilized at 121°C for 20 min, and after the temperature dropped to ≤40°C, inoculated with 6% Bacillus amyloliquefaciens and 3% Lactobacillus plantarum seed liquid, and placed in a constant temperature incubator at 37°C for 48 h, and then post-ripened at 4°C for 24 h to obtain fermented black beans; The fermented black beans are mixed with the mulberry-aged vinegar soaking liquid obtained in step (1) at a mass volume ratio of 1:1.5. After soaking for 14 days, the beans are taken out and mixed with the vinegar-soaked mulberries obtained in step (1), crushed to 60 mesh, and then vacuum-freeze-dried to obtain vinegar-soaked black bean freeze-dried powder. The vacuum freeze-drying conditions are as follows: freeze-dried thickness 1.5 cm, first placed in a -80°C refrigerator for pre-freezing for 2 hours, then set the cold trap temperature to -51°C, the vacuum degree to 0.09 mbar, and vacuum freeze-dried for 30 to 35 hours to obtain freeze-dried powder; Add active plant lactobacillus CGMCC 15731 freeze-dried powder to the freeze-dried powder, and the amount added should be based on the concentration of active plant lactobacillus in the final product ≥10 7 cfu / g calculation; that is, double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, γ-aminobutyric acid and active probiotics.

[0011] The plant lactobacillus is CGMCC 15731, and the amyloliquefaciens is CGMCC 15732. The preparation method of the seed solution of the plant lactobacillus CGMCC 15731 or the amyloliquefaciens CGMCC 15732 is as follows: inoculating the activated plant lactobacillus CGMCC 15731 or the amyloliquefaciens CGMCC 15732 into the MRS liquid culture medium at a rate of 3%, culturing at 37° C. for 24 hours, and the bacterial cell concentration reaches 10 8 cfu / mL, 8000r / min, centrifuge for 10min to obtain bacterial sludge, wash the bacteria twice with 0.85% saline and resuspend the bacteria to a concentration of 10 8 cfu / mL, which is the seed solution.

[0012] The preparation method of the active aged vinegar fermented grains source Lactobacillus plantarum CGMCC 15731 freeze-dried powder is as follows: according to the above-mentioned Lactobacillus plantarum CGMCC 15731 seed solution preparation process, after obtaining bacterial mud, 10% of protective agent is added at a ratio of protective agent / bacterial mud (volume / mass) of 3:1, and vacuum freeze-drying is performed to obtain the active aged vinegar fermented grains source Lactobacillus plantarum CGMCC15731 freeze-dried powder (the concentration of viable bacteria reaches 10 10 cfu / g), the vacuum freeze-drying conditions are the same as the freeze-drying conditions of the above-mentioned vinegar-soaked black bean freeze-dried powder. The preparation method of the protective agent is as follows: protective agent 1: 10g skim milk powder, 100mL water, 115℃, 15min sterilization; protective agent 2: 1.5g trehalose, 0.5g glycerol, 2g sorbitol, 1g maltodextrin, 100mL water, 115℃, 15min sterilization; cool the high-temperature sterilized protective agent 1 and protective agent 2 to room temperature, and mix them in a volume ratio of 1:1 to obtain the protective agent.

[0013] The black beans are from Shenchi black beans in Xinzhou, Shanxi. The 8-year-old aged vinegar syrup has the following contents: total acid ≥6.5 g / 100 mL, non-volatile acid ≥2.5 g / 100 mL, amino nitrogen ≥0.30 g / 100 mL, total ester ≥3.0 g / 100 mL, reducing sugar ≥2.5 g / 100 mL, soluble solids ≥15 g / 100 mL, flavonoids ≥200 mg / 100 g, and ligustrazine ≥90 mg / L.

[0014] The Bacillus amyloliquefaciens described in the present invention is: Application No. 201910354887.7, and the invention name is the Bacillus amyloliquefaciens recorded in a fresh-cut fruit and vegetable preservative composed of antibacterial substances produced by Bacillus licheniformis, Bacillus atrophaeus and Bacillus amyloliquefaciens; the Lactobacillus plantarum is Application No. 201910136386.1, and the invention name is a strain of Shanxi aged vinegar with strong tolerance and high acid production, and the Lactobacillus plantarum recorded in the application.

[0015] The plant lactobacillus CGMCC 15731 described in the present invention is an excellent strain separated from the fermented grains of Shanxi aged vinegar by this laboratory, and has strong tolerance, high acid production, high production of γ-aminobutyric acid, high production of β-glucosidase, high conversion of soybean isoflavones, and good fermentation flavor. The amyloliquefaciens CGMCC 15732 described in the present invention is an excellent strain separated from the fermented grains of Shanxi aged vinegar by this laboratory, and has the advantages of producing nattokinase, strong protease ability, and strong ability to inhibit foreign bacteria.

[0016] The present invention also provides double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, gamma-aminobutyric acid and active probiotics, which is prepared by the method.

[0017] The double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, γ-aminobutyric acid and active probiotics is purple, and the nattokinase is ≥16000 U / g, the γ-aminobutyric acid is ≥200 mg / 100 g, the isoflavone aglycone is ≥90 mg / L, the proanthocyanidins are ≥2000 mg / 100 g, and the probiotics are ≥10 7 cfu / g, black-purple in color, with the complex flavor of aged vinegar, mulberry and bean fragrance, and the effects of lowering blood lipids, enhancing immunity and anti-oxidation.

[0018] Compared with the prior art, the present invention has the following innovative features and beneficial effects: (1) The black beans used in the present invention are black beans from Shenchi, Xinzhou, Shanxi Province, which are famous and high-quality local specialties with a long history and are protected products of national agricultural geographical indications. Shenchi County is located in the loess hilly area, with undulating hills and gullies, an average altitude of 1,500 meters, and a continental monsoon climate with drought, high cold, large temperature difference, and short frost-free period. Shenchi has a cool climate, concentrated rainfall, long-term northwest wind, sufficient sunlight, and a large temperature difference between day and night. Such climatic conditions are just suitable for the production and development of black beans.

[0019] Shenchi black beans have large and full seeds, oval or nearly spherical, shiny black color, and yellow-green cotyledons. The protein content is as high as 38-42g / 100g, the essential linoleic acid content is 4-5g / 100g, and the linolenic acid content is 0.8-1.0g / 100g; it contains 18 kinds of amino acids, especially 8 kinds of amino acids essential to the human body, among which the lysine content can reach 2-3g / 100g; soy isoflavones reach 79.5-83.2mg / 100g, vitamin E reaches 20.1-25.9mg / 100g, calcium reaches 158-187mg / 100g, phosphorus reaches 548-598mg / 100g, and it also contains essential vitamins B1, B2, C, niacin and rich trace elements such as zinc, copper, magnesium, molybdenum, selenium, fluorine, etc.; black bean skin contains anthocyanins, which are a good source of antioxidants and can remove free radicals in the body.

[0020] (2) The present invention uses the excellent plant lactobacillus CGMCC 15731 (strain with strong tolerance, high acid production, high production of r-aminobutyric acid, high production of β-glucosidase, high conversion of soybean isoflavones, and good fermentation flavor) from the fermented mash of Shanxi aged vinegar and the Bacillus amyloliquefaciens CGMCC 15732 (high production of nattokinase and strong protease ability) from the fermented mash to jointly ferment black beans. Compared with single-bacteria fermentation, the contents of nattokinase, isoflavone aglycones, and r-aminobutyric acid in the product are significantly improved, thereby enhancing its nutritional and health functions.

[0021] (3) The vinegar soaking liquid used in the present invention is 8-year-old Shanxi aged vinegar pulp and then added with dried mulberries and soaked for 1 month. The vinegar soaking liquid is not only rich in ligustrazine, flavonoids, and organic acids, which are typical effective ingredients of Shanxi aged vinegar, but also rich in mulberry proanthocyanidins and mulberry fruit flavor.

[0022] (4) The present invention mixes the fermented and soaked black beans with the previously filtered vinegar soaked mulberries, crushes them, freeze-dries them in vacuum, and then adds plant lactobacillus from the vinegar mash of active aged vinegar. This combines the complementary and coordinated advantages of nutrition, function and flavor of the raw materials of vinegar soaked black beans, vinegar soaked mulberries, Shanxi aged vinegar and probiotics, such as miscellaneous beans, fruits and probiotics, and adopts the form of freeze-dried powder, which is not only conducive to maintaining the activity of the functional ingredients in the product, but also expands the diversified product form of vinegar soaked black beans. Finally, the enhanced addition of probiotics in the double-bacteria fermented vinegar soaked black bean freeze-dried powder further improves the nutritional function value of the product such as regulating intestinal flora, lowering blood lipids and anti-oxidation. DETAILED DESCRIPTION

[0023] In order to make the purpose, 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 part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] Unless defined otherwise, 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 belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0025] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified. The instruments and equipment used in the following examples are all conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified.

[0027] Example 1: Screening of high-yield nattokinase spore strains Nattokinase activity determination method: The activity of nattokinase was determined by agarose-fibrin method. 1 mL of 20U / mL thrombin and 2 mL of working solution (0.01 mol / L phosphate buffer and 0.85% saline were mixed at a volume ratio of 1:17) were taken and added to the culture dish; 10 mL of agarose aqueous solution dissolved at 55°C was added to a test tube containing 3 mL of dissolved fibrinogen solution, and then poured into the culture dish containing thrombin, and the culture dish was shaken quickly to mix it evenly. After it solidified, a hole (2 mm in diameter) was made on it, and 10 μL of urokinase standard solution of different concentrations (200, 400, 600, 800, 1000 U / mL, prepared with saline) was loaded into the small hole. Keep the temperature at 37°C for 18 hours, measure the diameter of the dissolved circle, and calculate the area. The activity of nattokinase was calculated using the equation of the standard curve of urokinase fibrinolytic activity, and the fibrinolytic activity unit of urokinase was used as the activity unit of nattokinase.

[0028] Screening of high-yield nattokinase spore strains: Weigh a certain amount of black beans, wash and soak overnight; drain with gauze, autoclave and steam at 121℃ for 20 min, cool naturally, inoculate with 8% spore-forming bacteria seed liquid, culture at 37℃ for 24 h, and ripen in a refrigerator at 4℃ for 24 h. Weigh 10g of fermented natto and dissolve it in 90mL of deionized water, homogenize for 10 s, extract at 4℃ for 24 h, and then collect the supernatant after centrifugation at 12,000 r / min for 10 min to determine the enzyme activity value of nattokinase.

[0029] Preparation method of spore-forming bacteria seed solution: inoculate the activated spore-forming bacteria strain into MRS liquid culture medium at a rate of 3%, and culture at 37°C for 24 hours until the bacterial concentration reaches 10 8 cfu / mL, 8000r / min, centrifuge for 10min to obtain bacterial sludge, wash the bacteria twice with 0.85% saline and resuspend the bacteria to a concentration of 10 8 cfu / mL, i.e., spore-forming bacteria seed solution.

[0030] The nattokinase activity of 20 spore-forming fungi isolated from Shanxi aged vinegar or vinegar mash was measured, and finally a high-yield nattokinase-producing Bacillus amyloliquefaciens CP2014 (deposit number: CGMCC 15732) was screened out, and its nattokinase activity could reach 2886±51.14 U / g.

[0031] Table 1: Determination of nattokinase activity of 20 strains of spore-forming bacteria from aged vinegar Daqu or vinegar mash Example 2: Screening of lactic acid bacteria that produce high levels of γ-aminobutyric acid and β-glucosidase According to the following method, the γ-aminobutyric acid, β-glucosidase and soybean isoflavone conversion abilities of 10 lactic acid bacteria strains isolated from Shanxi aged vinegar mash were determined to screen out high-yield strains.

[0032] γ-aminobutyric acid standard curve drawing: prepare 0, 0.2, 0.4, 0.6, 0.8, 1 mg / mL γ-aminobutyric acid standard solutions, take 1 mL of different concentrations of standard solution, add 1.0 mL of 0.01 mol / L sodium tetraborate buffer (pH 9), 1.0 mL of 6% redistilled phenol solution, and 2 mL of 7.5% sodium hypochlorite solution, mix well, heat in a boiling water bath for 10 minutes, take out and immediately put into ice water for 5 minutes, wait for the solution to turn blue-green, then add 1.5 mL of 60% ethanol solution, shake and mix continuously, and measure its absorbance at 645 nm. Draw the standard curve with the absorbance as the ordinate and the concentration of each standard solution (mg / mL) as the abscissa.

[0033] Screening of lactic acid bacteria strains with high γ-aminobutyric acid production: 3% of lactic acid bacteria seed solution was inoculated into MRS medium, cultured at 37°C for 24 h, and centrifuged at 12,000 r / min for 10 min to obtain the supernatant. Take 1 mL of the supernatant and determine the ability of lactic acid bacteria to produce GABA according to the standard curve determination method.

[0034] Screening of lactic acid bacteria strains with high production of β-glucosidase: Esculin can be hydrolyzed into aesculin and glucose by β-glucosidase, and aesculin reacts with Fe 3+ The reaction is brown-black. Add 0.3% aesculin and 0.05% ferric citrate by mass to MRS liquid medium, and then add 1.8% agar to prepare a special agar medium with aesculin and ferric citrate. After sterilization, add 250µL to each well of a 96-well plate while it is still hot at about 60°C. After the plate solidifies, take 10µL of the bacterial solution and add it to the 96-well plate in the order of numbering. After inverted culture for 48 hours, the holes turn dark brown-black, which is a strain producing β-glucosidase. The enzyme activity can be preliminarily judged based on the color depth.

[0035] Rescreening of lactic acid bacteria strains with high conversion of soy isoflavones: The oxygen glycosidic bond of glycoside-type soy isoflavones will be stripped of the glucose group to become soy isoflavone aglycones under the action of β-glucosidase secreted by lactic acid bacteria. The lactic acid bacteria strains to be tested are used to ferment black beans, and the ability of different strains to convert soy isoflavones in black beans is compared.

[0036] Preparation method of lactic acid bacteria seed liquid: inoculate the activated lactic acid bacteria into MRS liquid culture medium at a rate of 3%, culture at 37℃ for 24h, and then the bacterial concentration reaches 10 8cfu / mL, 8000r / min, centrifuge for 10min to obtain bacterial sludge, wash the bacteria twice with 0.85% saline and resuspend the bacteria to a concentration of 10 8 cfu / mL, that is, lactic acid bacteria seed liquid.

[0037] Weigh a certain amount of black beans, wash and soak overnight; drain with gauze, sterilize and cook at 121℃ for 20min, cool naturally, inoculate with 6% lactic acid bacteria seed liquid, culture at 37℃ for 24h, and ripen in a refrigerator at 4℃ for 24h. Weigh 10g of fermented black beans and dissolve in 90mL of deionized water, homogenize for 10s, extract at 4℃ for 24h, and collect the supernatant by centrifugation at 12,000 r / min for 10min.

[0038] Take 5 mL of supernatant in a 50 mL volumetric flask and add 80% methanol solution to the mark. Ultrasonic extraction for 20 minutes, dilute with 80% methanol, shake well, take the sample solution and place it in a centrifuge tube, centrifuge at 8000r / min for 15 minutes, take the supernatant and filter through a 0.45nm filter membrane, collect the filtrate and enter high performance liquid chromatography. Chromatographic conditions: C 18 Chromatographic column; mobile phase: acetonitrile; phosphoric acid aqueous solution (pH 3.0); flow rate: 1.0mL / min; UV detection wavelength: 260nm; injection volume: 10µL; column temperature: 30℃; gradient elution.

[0039] The ability of lactic acid bacteria to transform soy isoflavones, that is, the content of soy isoflavone aglycones produced, is expressed as the total content of daidzein and genistein in the fermented beans. The daidzein and genistein standards were prepared into standard solutions with a concentration of 1, 2, 4, 8, and 16 mg / mL for HPLC determination, and the standard curve was drawn with the peak area as the ordinate and the concentration of the standard solution as the abscissa.

[0040] The ability of 10 lactic acid bacteria strains isolated from Shanxi Laochen vinegar to produce r-aminobutyric acid, β-glucosidase and convert soy isoflavones was tested. Finally, a strain of Lactobacillus plantarum CP7 (deposit number: CGMCC15731) with high production of GABA and soy isoflavone aglycones was selected, with the production of GABA and soy isoflavone aglycones reaching 2.5 mg / mL and 70 mg / L, respectively.

[0041] Table 2: Determination of the ability of 10 Shanxi Laochen vinegar lactic acid bacteria strains to produce r-aminobutyric acid, β-glucosidase and soybean isoflavone aglycones Example 3: Preparation of freeze-dried black bean powder fermented with double bacteria and soaked in vinegar: Preparation method of mulberry-aged vinegar pulp soaking liquid: dried mulberries: 8-year-old aged vinegar pulp is 1:5 (mass volume ratio), soak for 1 month, filter out the vinegar liquid, add 20% (mass volume ratio) of honey, 0.03% (mass volume ratio) of white sugar, and 0.03% (mass volume ratio) of edible salt, boil for 40 minutes, cool to 60℃, and you can get mulberry-aged vinegar pulp soaking liquid. The mulberries soaked in vinegar are set aside.

[0042] The washed black beans were soaked in deionized water overnight, sterilized at high temperature and high pressure (121°C, 105 kPa) for 20 minutes, and after the temperature dropped below 40°C, 6% amyloliquefaciens and 3% Lactobacillus plantarum seed solutions were inoculated respectively, and placed in a constant temperature incubator at 37°C for 48 hours, and then post-ripened at 4°C for 24 hours to obtain fermented black beans. The soaking solution was added at a ratio of 1:1.5 (mass volume ratio) of fermented black beans to mulberry-aged vinegar slurry soaking solution. After soaking for 14 days, the beans were taken out and mixed with the vinegar-soaked mulberries filtered out of the mulberry-aged vinegar slurry soaking solution, crushed, and vacuum freeze-dried to obtain vinegar-soaked black bean freeze-dried powder, and then Lactobacillus plantarum, a vinegar-sour source of active aged vinegar, was added to obtain the finished product.

[0043] Comparative Example 1: Preparation of freeze-dried black bean powder fermented with commercial common natto bacteria: only 6% commercial natto Bacillus subtilis was inoculated for black bean fermentation, and the rest of the preparation method and operation steps were the same as those in Example 3.

[0044] Comparative Example 2: Preparation of freeze-dried black bean powder fermented with Bacillus amyloliquefaciens: Only 6% Bacillus amyloliquefaciens was inoculated for black bean fermentation, and the rest of the preparation method and operation steps were the same as those in Example 3.

[0045] The results showed that compared with single-bacteria fermentation, the content of nattokinase, γ-aminobutyric acid, isoflavone aglycones and proanthocyanidins in black beans soaked in vinegar by double-bacteria fermentation was significantly increased. Compared with the commercially commonly used natto strains, the preferred Bacillus amyloliquefaciens CGMCC 15732 nattokinase of the present invention was significantly increased.

[0046] Table 3: Determination of functional components of probiotic fermented black bean vinegar-soaked freeze-dried powder Example 4: Changes in functional components of freeze-dried black bean powder fermented with double bacteria and soaked in vinegar during storage: The results are shown in Table 4. After storage at 4°C for 9 months, the contents of nattokinase (13600±25.12 U / g), isoflavone aglycones (77.1±2.16 mg / L), γ-aminobutyric acid (191±15.12 mg / 100 g) and proanthocyanidins (1760±42.05 mg / 100 g) were 85%, 85.7%, 90% and 80% of the initial contents of the product, respectively. The number of viable probiotics remained at 10 7After storage at 25°C for 9 months, the contents of nattokinase (9800±34.40 U / g), isoflavone aglycones (54.1±1.14 mg / L), γ-aminobutyric acid (145±16.32 mg / 100g) and proanthocyanidins (1325±10.05 mg / 100 g) were 61.25%, 60.24%, 68.39% and 60.22% of the initial contents of the product, respectively. The number of viable probiotics was 10 5 cfu / g, decreased by two orders of magnitude.

[0047] Table 4 Changes in functional components of double-bacteria fermented black bean soaked in vinegar freeze-dried powder during storage Example 5: Blood lipid lowering test of double bacteria fermented vinegar soaked black bean freeze-dried powder: Healthy SD rats (150 ± 20) g were randomly divided into a model group of 10 rats and an experimental group of 10 rats after adaptive feeding for 7 days. The feeding temperature was 22 ± 1 ° C and the relative humidity was 55 ± 5%. Both the model group and the experimental group were fed with high-fat feed. The experimental group was gavaged with vinegar soaked black bean freeze-dried powder liquid (prepared according to Example 3 for double bacteria fermented vinegar soaked black bean freeze-dried powder, dissolved in normal saline) every day. The gavage dose was 0.3g vinegar soaked black bean freeze-dried powder / 200g body weight, and the gavage liquid volume was 5mL / kg body weight. The model group was gavaged with an equal amount of normal saline. After gavage for 40 days, the rats were fasted for 12 hours, blood was collected from the femoral artery of the rats, centrifuged (3000r / min, 15min), and serum was obtained to determine the serum total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) content of the rats.

[0048] Basic feed: protein 32%, fat 5%, cellulose 2%, Ca 1.8%, P 1.2%, nitrogen-free extract 58%.

[0049] High-fat feed: basic feed 83.5%, cholesterol 1%, lard 10%, ox bile salt 0.3%, propylthiouracil 0.2%, sucrose 5%.

[0050] The measurement results are shown in Table 5. The results show that compared with the model group, the total cholesterol, triglycerides and low-density lipoprotein-cholesterol in the serum of the experimental group rats were significantly reduced, and there was no significant difference in the high-density lipoprotein-cholesterol content between the two groups.

[0051] Table 5 Effects of freeze-dried black bean powder fermented with double bacteria and soaked in vinegar on blood lipid levels in rats Example 6: Experiment on the efficacy of double-bacteria fermented vinegar-soaked black bean freeze-dried powder in enhancing the body's immunity: healthy Kunming mice, male, weighing 20±2g, kept at 22±1°C, and 55±5% relative humidity. A normal control group and an experimental group were set up, with 10 mice in each group, and they were allowed to eat and drink freely. The experimental group was gavaged with vinegar-soaked black bean freeze-dried powder (prepared according to Example 3, double-bacteria fermented vinegar-soaked black bean freeze-dried powder, dissolved in normal saline), with a gavage dose of 0.3g vinegar-soaked black bean freeze-dried powder / 200g body weight, and a gavage liquid volume of 5mL / kg body weight. The normal control group was gavaged with an equal amount of normal saline, and the gavage was continued for 30 days, and the indicators such as mouse NK cell activity and mouse spleen index were measured.

[0052] The results of the effects of double-bacteria fermented vinegar-soaked black bean freeze-dried powder on the immune ability of mice are shown in Table 6. It can be seen from Table 6 that compared with the control group, the NK cell activity and the content of anti-inflammatory factors IFN-α and IL-10 in the experimental group were significantly increased, indicating that the double-bacteria fermented vinegar-soaked black bean freeze-dried powder has an immune-enhancing effect. There was no significant difference in body weight and spleen index between the two groups of mice.

[0053] Table 6 Effects of freeze-dried black bean powder fermented with double bacteria and soaked in vinegar on the immune ability of mice Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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 a double-fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, γ-aminobutyric acid and active probiotics, characterized in that: The steps include: (1) Preparing a mulberry-aged vinegar slurry soaking liquid: soaking dried mulberries in 8-year-old aged vinegar slurry at a mass volume ratio of 1:5 for 1 month, filtering out the vinegar liquid, adding 20% ​​honey, 0.03% white sugar, and 0.03% edible salt at a mass volume ratio of the vinegar liquid based on the filtered vinegar liquid, boiling for 40 minutes, and cooling to 60° C. to obtain a mulberry-aged vinegar slurry soaking liquid, and setting aside the filtered vinegar-soaked mulberries; (2) Preparation of freeze-dried powder: Wash the black beans and soak them in deionized water overnight. Take out the soaked black beans and sterilize them at 121°C and 105 kPa for 20 min. After the temperature drops to ≤40°C, inoculate with 6% Bacillus amyloliquefaciens and 3% Lactobacillus plantarum seed solution and place them in a constant temperature incubator at 37°C for 48 h. Then, post-ripen them at 4°C for 24 h to obtain fermented black beans. The fermented black beans are mixed with the mulberry-aged vinegar soaking liquid obtained in step (1) at a mass volume ratio of 1:1.

5. After soaking for 14 days, the beans are taken out and mixed with the vinegar-soaked mulberries obtained in step (1), crushed to 60 mesh, and then vacuum-freeze-dried to obtain vinegar-soaked black bean freeze-dried powder; the vacuum freeze-drying conditions are as follows: freeze-dried thickness 1.5 cm, first placed in a -80°C refrigerator for pre-freezing for 2 hours, then the cold trap temperature is set to -51°C, the vacuum degree is 0.09 mbar, and vacuum freeze-dried for 30-35 hours to obtain the freeze-dried powder; Add active plant lactobacillus CGMCC 15731 freeze-dried powder to the freeze-dried powder, and the amount added should be based on the concentration of active plant lactobacillus in the final product ≥10 7 Calculated by cfu / g, it is the double-fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, γ-aminobutyric acid and active probiotics.

2. The preparation method according to claim 1, characterized in that: The plant lactobacillus is CGMCC 15731, and the amyloliquefaciens is CGMCC 15732. The preparation method of the seed solution of the plant lactobacillus CGMCC 15731 or the amyloliquefaciens CGMCC15732 is as follows: inoculating the activated plant lactobacillus CGMCC 15731 or the amyloliquefaciens CGMCC15732 into the MRS liquid culture medium at a rate of 3%, culturing at 37° C. for 24 hours, and culturing the bacterial cell concentration to 10 8 cfu / mL, 8000r / min, centrifuge for 10min to obtain bacterial sludge, wash the bacteria twice with 0.85% saline and resuspend the bacteria to a concentration of 10 8 cfu / mL, which is the seed solution.

3. The preparation method according to claim 2, characterized in that: The preparation method of the active aged vinegar fermented grains source Lactobacillus plantarum CGMCC 15731 freeze-dried powder is as follows: the Lactobacillus plantarum CGMCC 15731 bacterial mud is added with 10% of a protective agent at a volume / mass ratio of protective agent / bacterial mud of 3:1, and vacuum freeze-dried to obtain the active aged vinegar fermented grains source Lactobacillus plantarum CGMCC15731 freeze-dried powder, and the viable bacterial count concentration reaches 10 10 cfu / g, vacuum freeze-drying conditions: freeze-dried thickness 1.5 cm, first placed in a -80℃ refrigerator for 2 hours, then set the cold trap temperature to -51℃, vacuum degree 0.09 mbar, and vacuum freeze-dry for 30-35 hours; The preparation method of the protective agent is as follows: protective agent 1: 10g skim milk powder, 100mL water, 115°C, sterilization for 15min; protective agent 2: 1.5g trehalose, 0.5g glycerol, 2g sorbitol, 1g maltodextrin, 100mL water, 115°C, sterilization for 15min; the protective agent 1 and the protective agent 2 after high-temperature sterilization are cooled to room temperature and mixed in a volume ratio of 1:1 to obtain the protective agent.

4. The preparation method according to claim 1, characterized in that: The black beans are from Shenchi black beans in Xinzhou, Shanxi.

5. The preparation method according to claim 1, characterized in that: The aged vinegar pulp aged for 8 years has the following contents: total acid ≥6.5g / 100mL, non-volatile acid ≥2.5g / 100mL, amino nitrogen ≥0.30g / 100mL, total ester ≥3.0g / 100mL, reducing sugar ≥2.5g / 100mL, soluble solids ≥15g / 100mL, flavonoids ≥200mg / 100g, and ligustrazine ≥90mg / L.

6. A freeze-dried black bean powder fermented with double bacteria and soaked in vinegar, rich in nattokinase, gamma-aminobutyric acid and active probiotics, prepared by the method described in any one of claims 1 to 5.

7. The double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, γ-aminobutyric acid and active probiotics according to claim 6, characterized in that: The double-bacteria fermented vinegar-soaked black bean freeze-dried powder rich in nattokinase, γ-aminobutyric acid and active probiotics is black purple, and the nattokinase is ≥16000U / g, the γ-aminobutyric acid is ≥200mg / 100g, the isoflavone aglycone is ≥90mg / L, the proanthocyanidins are ≥2000mg / 100g, and the probiotics are ≥10 7 cfu / g.

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