Rice wine koji containing Chinese herbal medicines as well as preparation method and application of rice wine koji

Abstract

The invention discloses rice wine koji containing Chinese herbal medicines as well as a preparation method and application of the rice wine koji. The rice wine koji containing the Chinese herbal medicines is prepared from the following raw materials in parts by weight: 65 to 80 parts of rice matrix, 6.5 to 12 parts of rice wine mother koji and 12 to 18.5 parts of plant composite powder. Wherein the composite powder is prepared from the following components in parts by weight: 3.5 to 5.0 parts of flaccid knotweed herb, 2.5 to 3.5 parts of herba euphorbiae humifusae, 2.0 to 3.0 parts of sesame flower, 1.5 to 2.5 parts of honeysuckle flower, 0.5 to 1.2 parts of cortex cinnamomi, 0.3 to 0.8 part of flos caryophylli and 1.5 to 2.5 parts of couch grass. According to the technical scheme of scientific proportioning of flaccid knotweed herb, herba euphorbiae humifusae, sesame flower, honeysuckle flower, cinnamon, clove and couch grass, the functional combination of Chinese herbal medicines is optimized, the technical effects of improving the liver function and reducing lipid are achieved, meanwhile, aging thymus decline of senescence individuals can be delayed in vivo, the number of subgroups in thymus is increased, and the health care effect is achieved. The capability of the thymus for generating mature T cells is enhanced, and the number of the mature T cells in the peripheral immune organ spleen and the number of subgroups of the mature T cells are increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food fermentation and crude drug, and particularly relates to a rice wine starter containing Chinese herbal medicine as well as a preparation method and application thereof. BACKGROUND

[0002] The wine starter is the core fermentation agent of fermented food. At present, improving the fermentation efficiency and flavor quality are two main aspects of the development of the wine starter technology. Some existing technologies attempt to add Chinese herbal medicine or functional ingredients to expand the health care function of the wine starter, such as regulating metabolism, improving liver and kidney function, regulating immunity, etc. These studies have enhanced the functionality of the wine starter to some extent.

[0003] The traditional Three Gorges rice wine starter mainly depends on single polygonum hydropiper or general rhizopus, and has the problems of single flavor, ethyl acetate dominance and high rancidity rate in the late fermentation. There is a report introducing honeysuckle to inhibit bacteria, but the low-temperature fermentation characteristics of rice wine are ignored, resulting in insufficient release of plant active ingredients. In addition, local resources such as desmoschoenus transvolutatus and sesamum indicum have not been scientifically matched, and the loss rate of their flavor precursor substances (such as terpenes and phenylethanoid glycosides) in fermentation is more than 40%, which cannot reflect the Three Gorges regional characteristics. SUMMARY

[0004] The present application provides a rice wine starter containing Chinese herbal medicine as well as a preparation method and application thereof, which solves the problems of insufficient functionality, difficult balance between fermentation performance and functional ingredients, and poor stability of strain activity and functional ingredients during storage of the traditional Three Gorges wine starter.

[0005] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is as follows: A rice wine starter containing Chinese herbal medicine includes the following raw materials by weight: 65-80 parts of cooked rice matrix, 6.5-12 parts of rice wine mother starter, and 12-18.5 parts of plant composite powder. The plant composite powder is composed of the following components: 3.5-5.0 parts of lespedeza, 2.5-3.5 parts of geopelia, 2.0-3.0 parts of sesame flower, 1.5-2.5 parts of honeysuckle, 0.5-1.2 parts of cinnamon, 0.3-0.8 parts of clove, and 1.5-2.5 parts of straw. The rice substrate provides a carbon source and a rice aroma base as the main component of the wine starter, and a high content in the wine starter can ensure the energy required for microbial fermentation. The rice substrate is rich in protein and amylase, which can allow microbial fermentation and sugar production, imparting a unique flavor to the rice wine. For the selection of rice wine mother starter, the batch with the most appropriate species and proportion of microorganisms is selected by metagenomic sequencing, and batch production is carried out according to the production process of this batch, and appropriate storage conditions are ensured, which not only ensures the fermentation efficiency but also ensures the synergistic effect between the microbial populations, realizing stable fermentation function of the wine starter. The Three Gorges plant composite powder imparts specific health functions to the wine starter, and also promotes fermentation microorganisms within the scope of the present application, avoiding inhibition of the strains due to excessive addition.

[0006] The content range of lespedeza is selected based on the fact that it contains growth hormones required by various microorganisms such as rhizopus and yeast, and especially the rich flavonoid active substances contained therein can effectively help the normal growth of wine starter bacteria, and have antioxidant and anti-inflammatory functions. When the content is less than 3%, the functional effect is not obvious, and when the content is more than 5%, it may affect the activity of the fermentation strains. The terpene substances in geopelia generate citrus flavor, the chlorogenic acid in sesame flower inhibits miscellaneous bacteria and reduces the rancidity rate, the antibacterial iridoid in honeysuckle can synergistically preserve freshness, the cinnamic aldehyde in cinnamon provides warmth and spicy flavor, the eugenol in clove enhances the aftertaste of rice wine, and the use of straw is because its fiber network releases water, maintaining the fermentation humidity.

[0007] The geopelia, sesame flower, honeysuckle, cinnamon, clove, and straw must be picked in the appropriate season, and fresh, disease-free and pest-free plants, flowers, or fruits are selected, washed, and dried for use.

[0008] Preferably, the rice wine mother starter contains the most appropriate species and proportion of microorganisms in the batch, as shown in Figure 1 The top three components are: Saccharomyces cerevisiae accounts for 83.12%-85.52% of all bacteria, Candida glabrata accounts for 4.88%-5.62% of all bacteria, and Lactococcus lactis accounts for 0.99%-1.56% of all bacteria. The total proportion of the three bacteria is 90.3%-91.39%, and the total proportion of the remaining various bacteria is less than 10%.

[0009] Saccharomyces cerevisiae is the core strain of wine yeast, with the advantages of short growth cycle, strong fermentation capacity, easy large-scale cultivation, and rich nutritional ingredients such as various proteins, amino acids, vitamins, and bioactive substances. The number of viable cells directly determines the fermentation efficiency and alcohol yield of the wine yeast. Its high proportion ensures the high fermentation capacity of the wine yeast, which can quickly metabolize sugar to produce alcohol during fermentation. At the same time, Saccharomyces cerevisiae can also produce a variety of aromatic substances, enriching the flavor of the wine.

[0010] Candida glabrata plays a major role in brewing by producing high-grade alcohol substances. In the rice wine mother yeast of the present invention, it only accounts for 5.62%. This appropriate proportion will not produce bitterness and irritation due to the high content of high-grade alcohol produced by it.

[0011] Lactococcus lactis is a high-quality lactic acid bacteria that can produce lactic acid through metabolism to regulate the pH of the fermentation environment, inhibit the growth of miscellaneous bacteria, maintain microbial ecological balance, and improve the acidity and taste of the wine. Such strain combination can effectively improve the fermentation efficiency of the wine yeast, optimize the fermentation environment, and give the final product excellent flavor and quality.

[0012] Preferably, the rice wine mother yeast refers to aged high-quality pepperweed wine yeast, Chu village sweet wine yeast, Hubei Chu village fermented products Co., Ltd., i.e. "old yeast", which is used as a carrier for microbial strains and is crushed into powder in advance for uniform mixing. Preferably, the pepperweed is picked from June to August, when the pepperweed plant is lush and the content of active ingredients such as pterocarpidin and flavonoids is the highest. Fresh, disease-free, and flower-bearing or unopened flower plants are selected, and the leaves and stems can be used. The pepperweed is washed and the impurities and dirt are removed, and then it is dried for use.

[0013] The preparation method of the rice wine yeast containing Chinese herbal medicine comprises the following steps: (1) Preparation of rice substrate: The rice is screened to remove impurities and broken particles, washed, soaked, and steamed; (2) Treatment of Chinese herbal medicine: The washed and dried pepperweed, doupoteng, sesame flower, honeysuckle, cassia, clove, and straw are mixed and crushed to obtain a plant composite powder; (3) Mixing: The crushed mother yeast is mixed with the cooled grain substrate, then the crushed plant composite powder is added, and pure water is gradually added and stirred until all the raw materials are uniformly mixed and the texture is moist and not clumpy; (4) Preparation of "yeast blank" by pressing the mixed raw materials, which is convenient for subsequent microbial cultivation and fermentation; (5) Yeast blank cultivation-core fermentation stage: by controlling temperature, humidity, and ventilation, to promote the reproduction of mold, yeast, and bacteria on the yeast blank, which determines the quality of the wine yeast; (6) drying matured starter, reducing moisture, obtaining dried starter containing Chinese herbal medicine. Preferably, in step (1), the soaking time is 4-6 hours, until the soaked grains can be kneaded without hard core, and then the soaked grains are steamed until there is no white core, the purpose being to gelatinize the starch for easy microbial decomposition. After steaming, spread out and cool to 30-35°C to avoid high temperature killing the microorganisms in the mother starter.

[0014] Preferably, in step (2), the cleaning method is: using a cyclone cleaning machine to clean for 5-10 min at a water flow rate of 50-70 L / min; The drying method is: using a hot air dryer to dry, setting the temperature to 50-60°C, setting the air flow rate to 1.5-2.5 m / s, and the drying time to 3-4 h, controlling the moisture content to 10-15 wt% after drying to avoid complete drying and retain active ingredients; the grinding method is: using a grinder to grind to 70-80 mesh.

[0015] Preferably, in step (3), the mixing method is: using a mixer to stir at a speed of 20-50 r / min for 5-10 min.

[0016] Preferably, in step (4), the starter forming method is: the starter forming tool is a wooden mold, square or round, with dimensions of about 10 cm x 10 cm x 5 cm, a wooden board, and a weight are used for pressing. The operation steps are as follows: fill the mixed raw materials into the wooden mold, press with hands, slightly loose in the middle and slightly tight at the edges for good ventilation; after demolding, obtain block-shaped starter, place on a bamboo mat covered with dried and sterilized straw to prevent contamination by miscellaneous bacteria, stand for 1-2 hours to slightly dry the surface to avoid sticking.

[0017] Preferably, in step (5), the starter culture-core fermentation stage is as follows: the starter room needs to be clean, dark, and good in moisture retention, sterilized and disinfected in advance with mugwort to kill miscellaneous bacteria; Stacking and initial culture (1-3 days): stack the starter neatly, separate the layers with straw for good ventilation, the height is about 50-80 cm, the room temperature is controlled at 25-30°C, and the relative humidity is 80%-90%, this stage is the "mold growth period", the microorganisms in the starter mother begin to reproduce, white mycelium gradually appears on the surface of the starter, and the temperature slowly rises to 30-35°C; Turning and mid-term culture (4-7 days): when the center temperature of the koji base rises to 40-45℃, it is hot to the touch, and the first turning is needed. The specific method is to move the upper koji base to the lower layer, the lower layer to the upper layer, and the edge to the middle, to ensure that the temperature of each koji base is uniform to avoid local temperature exceeding 50℃, preventing the death of beneficial bacteria. After turning, continue to culture, and the mycelium gradually spreads to the inside of the koji base, the color changes from white to light yellow or light green, accompanied by the production of wine aroma or ester aroma, and the temperature is maintained at 35-40℃, and the humidity is 70%-80%. If the temperature rises again to above 45℃, the second turning is performed, a total of 2-3 times, with an interval of 2-3 days; Late maturation (8-14 days): the temperature of the koji base gradually decreases to near room temperature, the mycelium ages, the color deepens, and part of the koji base in some areas will appear brown spots, the texture becomes loose, the aroma is rich, there is no moldy smell, and the acidity is a sign of mature culture.

[0018] Preferably, in step (6), the drying and maturing koji base is dried by hot air drying: using a hot air drying machine for air drying, and setting the temperature to 40-50℃, the air flow speed to 1.5-3.0 m / s, and the air drying time to 12-24h until the moisture content is ≦12 wt%.

[0019] The storage method of the Chinese herbal medicine rice wine koji is: after drying, the rice wine koji is packed in a breathable cloth bag or a pottery jar, and stored in a cool, dry and ventilated warehouse to avoid moisture or odor pollution. It is better to use it after 3 months of storage, and the flavor of the "old koji" is more stable and the number of miscellaneous bacteria is less.

[0020] Preferably, the extraction method of the Chinese herbal medicine rice wine koji obtained by the method is: taking 0.5-1.5g of rice wine koji, crushing it into powder in a clean environment, adding 5-15 mL of deionized water, extracting in a 37℃ water bath for 25-35 minutes, centrifuging at 7000-8500 r / min for 5-15 minutes, and taking the supernatant as the Chinese herbal medicine rice wine koji extract. Further preferably, 1 g of rice wine koji is taken, crushed into powder in a clean environment, 10 mL of deionized water is added, extracted in a 37℃ water bath for 30 minutes, centrifuged at 8000 r / min for 10 minutes, and the supernatant is taken as the Chinese herbal medicine rice wine koji extract. The preparation method of the Chinese herbal medicine rice wine koji, and the application of the Chinese herbal medicine rice wine koji extract obtained by the method in the preparation of a medicine for recovering the atrophy of the thymus of a fatty liver and an aging individual.

[0021] The present application has the following beneficial effects: 1. This invention optimizes the functional combination of traditional Chinese medicines through a scientific formulation of Polygonum hydropiper, Paeonia lactiflora, sesame flowers, honeysuckle, cinnamon, cloves, and Imperata cylindrica, achieving significant effects in improving liver function and reducing fat. Compared with existing technologies that use single traditional Chinese medicines or functional ingredients to design yeast starters, this invention solves the problems of single functionality and insufficient targeting, making the yeast starter more valuable for health and more adaptable to the market.

[0022] 2. This invention optimizes the ratio of plant-based compound powder to ensure health benefits while avoiding negative impacts on the fermentation performance of the starter culture. Existing technologies often suffer from the problem of excessive addition of Chinese herbal medicines leading to inhibited microbial activity. However, this invention solves the compatibility problem between Chinese herbal medicines and fermentation microorganisms through scientific screening and formulation, thereby improving the practicality and functional stability of the starter culture.

[0023] 3. This invention employs a precise formulation of functional yeast, which not only endows the liquor with rich functionality but also optimizes its flavor characteristics. The problem of uncoordinated flavors or insignificant effects caused by unscientific formulations in existing technologies is solved in this invention through the rational design of Polygonum hydropiper and traditional Chinese medicine, thereby giving the yeast good market adaptability and commercial value. Attached Figure Description Figure 1 Metagenomic sequencing reveals the species distribution in rice wine starter; Figure 2 Metagenomic sequencing reveals the species distribution in distinctive rice wine starter; Figure 3 Effects of mother koji and special rice wine koji on liver and kidney function in a mouse model of fatty liver induced by a high-fat diet; Figure 4 Effects of mother koji and special rice wine koji on liver and kidney structure in a mouse model of fatty liver induced by a high-fat diet; Figure 5 Effects of mother koji and special rice wine koji on the thymus of aging mice; Figure 6 Effects of mother koji and special rice wine koji on the total number of thymocytes and the number of each subpopulation in aged mice; Figure 7 Effects of mother koji and special rice wine koji on the number of T cell subsets in spleen cells of aging mice: A, Effects on the number of CD3, CD4, and CD8 T cell subsets in spleen cells; B, Effects on the number of CD4 T cells in spleen cells. + Naive T, CD4 + RTE, CD8 + Naive T, CD8 + The influence of RTE cell subsets on their numbers. Detailed Implementation

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of the present application. Embodiment 1 The present embodiment provides a preparation method of rice wine koji containing Chinese herbal medicine, which comprises the following steps: (1) Preparation of rice substrate: The rice is screened to remove impurities and broken particles, washed, soaked for 5 hours, and steamed to maturity, and then 72 g of the rice is taken.

[0025] (2) Treatment of Chinese herbal medicine: 5.0 g of washed and air-dried sedum lineare, 3.0 g of desmodium, 2.5 g of sesamum indicum, 2.0 g of honeysuckle, 1.0 g of cassia, 0.5 g of clove and 2.0 g of reed are weighed, crushed to 70-80 mesh by a crusher, and fully mixed to obtain a plant composite powder; (3) Mixing: 12 g of crushed koji, grain substrate and crushed plant composite powder are taken and added to a mixer to stir at a speed of 40 r / min for 10 min. Pure water is gradually added and stirred during the stirring process until all the raw materials are uniformly mixed and moist without clumping.

[0026] (4) Koji preparation (molding): The mixed raw materials are pressed into "koji preparation" to facilitate subsequent microbial culture (fermentation). The molding tool is a wooden mold with a size of 10 cm x 10 cm x 5 cm, a wooden board and a weight. The specific operation steps are as follows: first, fill the mixed raw materials into the wooden mold and press them with hands; after demolding, the block-shaped koji preparation is obtained and placed on a bamboo mat covered with dried and sterilized straw, and left to stand for 2 hours to slightly dry the surface to avoid sticking.

[0027] (5) Koji preparation culture (core fermentation stage): The koji room is sterilized in advance by smudging with artemisia vulgaris. The koji room needs to be clean, light-proof and good in moisture retention; Stacking and initial culture for 3 days: The koji preparations are neatly stacked with straw between layers for ventilation, with a height of about 80 cm. The room temperature is controlled at 28℃ and the relative humidity is 85%. This stage is the "mold growth period", during which the microorganisms in the koji begin to reproduce and white mycelium gradually appears on the surface of the koji preparation, and the temperature slowly rises to 32℃. Turning and mid-term culture (5 days): when the center temperature of the starter rises to 40°C, the first turning is performed, i.e., the upper starter is moved to the lower layer, the lower layer is moved to the upper layer, and the edge is moved to the middle, so as to ensure that the temperature of each starter is uniform and the local temperature is prevented from exceeding 50°C to prevent the death of beneficial bacteria; after turning, the mycelium gradually spreads to the inside of the starter, the color changes from white to light yellow or light green, accompanied by the production of wine aroma or ester aroma, and the temperature is maintained at 35°C and the humidity is reduced to 75%. When the temperature rises to more than 45°C again, the second turning is performed, a total of 2 times, with an interval of 2 days; Late maturation (12 days): the temperature of the starter gradually decreases to near room temperature, the mycelium ages, the color deepens (brown spots may appear on the starter in some areas), the texture becomes loose, and the aroma is rich (without moldy smell and sour taste), indicating that the culture is mature.

[0028] (6) After fermentation is completed, a hot air circulating dryer is used, the temperature is set to 45°C, the air flow speed is 2.5 m / s, and the drying is performed until the moisture content is less than 12%. The mature starter is dried to reduce the moisture content, and dry starter is obtained.

[0029] (7) Storage: the dried starter is packed in a breathable cloth bag or a pottery jar, and stored in a cool and dry place.

[0030] Verification example (one) difference between functional starter and microorganism composition in mother starter Verification purpose: verify the comparison of the microorganism composition of the functional starter and the original starter, and evaluate the types and points of beneficial bacteria.

[0031] Experimental materials: Two batches of original mother starter samples: batch 1 was produced in June 2024, batch 2 was produced in September 2024, Chu zhai sweet starter, Hubei Chu zhai fermentation products Co., Ltd., and three batches of functional starter, which were produced in June, July and August 2025, respectively, using the method of example 1 of the application, and the samples of the local characteristic rice wine starter of the application, a total of 5 samples, each taking 5 g, were placed in sterile plastic sharp bottom centrifuge tubes and placed in dry ice.

[0032] First, the DNA of the 5 samples was extracted using the kit QIAamp PowerFecal Pro DNA Kit (Qiagen, Cat.51804) of Kajie Company, to ensure that the DNA of the 5 samples meets the requirements of metagenomic detection, and the specific method is as follows: Step 1: Sample lysis: Grind the freeze-dried yeast sample into a fine powder with a particle size ≤0.5 mm. Weigh 100 mg of yeast powder from each of the 5 samples and add it to a lysis tube pre-loaded with beads. Add 700 μL of lysis buffer. Immediately place the sample in a homogenizer and set the parameters to 30 Hz for 3 min. Pause once during the process and cool on ice for 1 min to prevent overheating.

[0033] Step 2: Enzymatic hydrolysis and incubation: Add 25 μL proteinase K; then add 50 μL enhanced lysis buffer; vortex for 10 seconds; incubate at 65°C for 10 min, then transfer to a 95°C water bath for 5 min; centrifuge at 13,000 × g for 1 min and collect the liquid from the tube wall.

[0034] Step 3: Impurity removal and DNA binding: Add 200 μL of buffer and vortex for 30 seconds; centrifuge at 13,000 × g for 5 min at 4°C; transfer approximately 600 μL of supernatant to a new tube, avoiding aspiration of precipitate; add the supernatant to the magnetic bead adsorption column; vortex for 5 min to bind DNA to the magnetic beads.

[0035] Step 4: Washing and Elution: Centrifuge the adsorption column at 6,000 × g for 1 min and discard the filtrate; add 500 μL of elution buffer, centrifuge at 6,000 × g for 1 min and discard the filtrate; add 500 μL of elution buffer; centrifuge at 13,000 × g for 1 min and discard the filtrate, repeating once; centrifuge the empty column at high speed for 2 min to completely remove ethanol; add 70 μL of AE Buffer, preheat to 70°C, and let stand for 5 min; centrifuge at 13,000 × g for 1 min and collect the DNA solution.

[0036] Metagenomic analysis was performed on five samples.

[0037] The results show that the top three microorganisms in the original mother koji sample were: Saccharomyces cerevisiae (Saccharomyces cerevisiae) Saccharomyces cerevisiae 83.12%~85.52%, Candida glabrata ( Nakaseomyces glabratus ) 4.88% ~ 5.62%, Lactococcus lactis ( Lactococcus lactis 0.99%–1.56%, with the fourth and fifth most common pathogens being Klebsiella pneumoniae (Klebsiella pneumoniae). Klebsiella pneumoniae 0.68%–0.75% and Staphylococcus aureus ( Staphylococcus aureus 0.45%~0.48% (see) Figure 1 (Table 1) Table 1. Percentage of each species in rice wine starter.

[0038] The first five components of the functional rice wine starter prepared using this invention are brewing yeast ( Saccharomyces cerevisiae 7.44%–29.03%, Lactobacillus weisseri ( Weissella confusa 12.46%~13.51%, Candida glabrata ( Nakaseomyces glabratus 6.63%–10.52% of *Saccharomyces rudela* (Yeast) Saccharomycodes ludwigii 2.35%–3.60%, *Hylocereus gracilis* ( Sporisorium graminicola The predominant microbial community in the specialty rice wine starter is 0.68%–0.75%, consisting of probiotics or non-pathogenic microorganisms. The proportion of some conditionally pathogenic bacteria in the original mother starter is significantly reduced or undetectable (see [link to original text]). Figure 2 (Table 2).

[0039] Table 2. Percentage of each species in the specialty rice wine starter

[0040] Verification Example (II): Comparison of the effects of functional yeast on improving liver function and reducing fat. Verification objective: To verify the effects of the microbial composition of Example 1 of the present invention and the original mother culture on improving liver function and reducing fat, and to evaluate its impact on liver function and fatty liver.

[0041] Experimental animals: C57BL / 6J mice, female, 6-8 weeks old, 20 mice in total.

[0042] Grouping, modeling, and drug administration: All mice were randomly divided into four groups: control group, mother group 1, Example 1 group 0.5, and Example 1 group 1.0; with five mice in each group. All mice received a high-fat diet for four weeks. Model validation: In normal mice, the liver lobule structure was intact and the outline was clearly visible. The hepatic sinusoids were normal, and the hepatic cords were arranged neatly in a radial pattern with the central vein as the center. The hepatocyte cytoplasm was uniform, polygonal, and without degeneration. In the model group, vacuolar fatty degeneration and structural damage were clearly visible in the liver parenchyma.

[0043] The extraction method of the mother koji extract stock solution is as follows: take 1 g of rice wine mother koji, crush it into powder in a clean environment, add 10 mL of deionized water, extract in a 37 ℃ water bath for 30 minutes, centrifuge at 8000 r / min for 10 minutes, and take the supernatant to obtain the mother koji extract stock solution with a concentration of 1 g / mL. a. Control group: Normal diet for 4 weeks, followed by gavage administration of an equal amount of purified water in the 5th week.

[0044] b. Mother koji group 1: 1 g / mL of the original extract of mother koji was administered by gavage. c. Example 1 Group 0.5: 0.5 g / mL of original starter extract solution (0.5 mL of starter extract solution + 0.5 mL of pure water) was administered by gavage. d. Example 1 Group 1.0: 1 g / mL of original starter extract solution was administered by gavage.

[0045] The mice were administered by gavage once a day, 10 mL / kg of body weight each time, for 7 consecutive days.

[0046] Detection index combination 1: Alanine aminotransferase (ALT): After 7 days of gavage treatment, the mice were taken peripheral blood into non-anticoagulant tubes one day apart, and the mice were executed by cervical dislocation; the blood in the non-anticoagulant tube was placed at room temperature, and after the blood coagulated, it was centrifuged at 3000 r / min for 10 min, and the upper serum was sucked into 1.5 mL EP tubes for detection of ALT.

[0047] Aspartate aminotransferase (AST): The serum sample for detection was collected as above Urea: The serum sample for detection was collected as above Creatinine (Crea): The serum sample for detection was collected as above Experimental environment: The laboratory temperature was controlled at 20±1℃, and the light cycle was 12 hours of light and dark alternation.

[0048] Extract preparation: 1 g of mother starter and Example 1 starter were taken, extracted with 10 mL of deionized water in a 37℃ water bath for 30 minutes, centrifuged at 8000 r / min for 10 minutes, and the supernatant was taken.

[0049] The experimental results show that Example 1 group has a significant effect on improving the liver function of the high-fat-fed-induced fatty liver model in mice, and the ALT of the 0.5 g / mL and 1 g / mL groups is significantly lower than that of the control group (P<0.01, P<0.001), tending to be normal level 50.8 ± 16.8 IU / L; the ALT of the mother starter 1 group has no statistical difference compared with the control group (P>0.05). p <0.01, p <0.001). Figure 3

[0050] The AST of the 0.5 g / mL and 1 g / mL groups of Example 1 is significantly lower than that of the control group (P<0.05, P<0.05), tending to be normal level 131.5 ± 22.9 IU / L; the AST of the mother starter 1 group has no statistical difference compared with the control group (P>0.05). p <0.05, p <0.05). Figure 3 .​​

[0051] The experimental results show that the two indicators Urea and Crea of kidney function of the high-fat feeding induced fatty liver model mice are not significantly changed after the treatment of Example 1 group and mother liquor 1, p >0.05( Figure 3 ).

[0052] Detection index combination 2: The pathological changes of liver (Liver), take the middle part of the liver connected to the gallbladder, cut half and put it into a 5 mL test tube containing 4 mL of 4 % paraformaldehyde, follow the routine procedure for subsequent treatment and HE staining. The pathological changes of kidney (Kidney), take the left kidney, cut half and put it into a 5 mL test tube containing 4 mL of 4 % paraformaldehyde, follow the routine procedure for subsequent treatment and HE staining. The experimental results show that the original structure disorder and fatification are significantly improved after the treatment of 0.5 g / mL and 1 g / mL groups of Example 1 on the high-fat feeding induced fatty liver mouse model, and the kidney structure has no obvious change Figure 4 ).

[0053] From the mechanism, the scientifically matched Chinese herbal medicine can more effectively regulate the internal environment of the body by adjusting the types and proportions of bacteria in the wine starter, especially the oral direct gavage can inoculate the probiotics in the wine starter extract into the digestive tract, play the conditioning role of probiotics, regulate the metabolism of the liver through the gut-liver axis, and improve the phenotype of fatty liver; since the kidney function is within the normal range, no obvious change is observed after the treatment of the extract of Example 1, which indicates from another aspect that the extract of Example 1 has no side effect on kidney function.

[0054] Verification example (three) recovery effect of functional wine starter on atrophy of thymus of aging individuals Verification purpose: verify the recovery effect of the microbial composition of Example 1 and the original starter mother liquor on the atrophy of thymus of aging individuals, evaluate the effect of increasing the thymus index of the aged individuals and the influence on various T cell subgroups of the thymus.

[0055] Experimental animals: C57BL / 6J mice, female, 17 w, a total of 16.

[0056] Grouping, administration: All mice were randomly divided into 4 groups, namely control group, mother liquor group 1, Example 1 group 0.5, and Example 1 group 1.0; 4 mice in each group.

[0057] The extraction method of the mother koji extract stock solution is as follows: 1 g of rice koji is crushed into powder in a clean environment, 10 mL of deionized water is added, and extraction is performed in a 37 °C water bath for 30 minutes. Centrifugation is performed at 8000 r / min for 10 minutes, and the supernatant is obtained to obtain the mother koji extract stock solution with a concentration of 1 g / mL. a. Control group: intragastrically administered with the same volume of deionized water to 18 m (18 months old).

[0058] b. Mother koji group 1: intragastrically administered with the mother koji extract stock solution 1 g / mL. c. Example 1 group 0.5: intragastrically administered with the koji extract stock solution 0.5 g / mL (0.5 mL of koji extract stock solution + 0.5 mL of pure water). d. Example 1 group 1.0: intragastrically administered with the koji extract stock solution 1 g / mL.

[0059] Intragastric administration was performed once every 2 days, and each time 10 mL / kg of body weight was administered, and the administration was continuously performed for 14 times.

[0060] Detection index combination 1: Thymus index: after 14 times of intragastric administration of mice, the mice were weighed with an interval of one day, expressed in grams, and then the mice were euthanized by cervical dislocation, the thymus of the mice was obtained, and the thymus weight was weighed, expressed in milligrams; the thymus index of mice was calculated according to the following formula: thymus index of mice = thymus weight (mg) / body weight (g).

[0061] Spleen index: the mice were treated as in detection index combination 2: The mouse thymus was placed in a plastic sterile culture dish containing 2 mL DMEM, cut into 1 mm3tissue fragments with a sharp scalpel, collected the tissue and liquid in a 5 mL sterile centrifuge tube, constant volume to 5 mL, inverted gently up and down for 3 min to mix thoroughly, to obtain a single cell suspension. Take 10 μL of thymocyte suspension in a 1.5 mL EP tube containing 90 μL PBS, then count for the first time. Take 300 μL of thymocyte suspension in a 96-well plate with an automatic sample gun, centrifuge at 300 g for 5 min, discard the supernatant. Respectively add 20 μL of antibody (antibody combination as follows: Lin-APC (1:20 dilution), C-kit (CD117)-PE (1:100 dilution), CD4-APC / cy7 (1:50 dilution), CD8-BV510 (1:100 dilution), CD44-Percp / Cy5.5 (1:50 dilution), CD25-FITC (1:50 dilution), CD16 / 32 FcR (1:50 dilution, used to block FcR to avoid non-specific binding of antibodies)) prepared with PBS containing 2% BSA, mix the antibody and cells thoroughly, incubate at 4 ℃ for 30 min, wash with PBS and run the sample on a flow cytometer. After the above operation, the thymus suspension tube was naturally placed for 1 min until the tissue precipitated, then 5 mL tube was added with DMEM constant volume to 4 mL, inverted gently up and down for 3 min to mix thoroughly, and the remaining thymocytes were obtained. Take 10 μL of cell suspension into 90 μL of PBS for the second count. According to the results of two cell counts, calculate the total number of thymocytes in the thymus of each mouse, and according to the proportion of each subpopulation of cells detected by flow cytometry, calculate the change in the number of thymocytes and each subpopulation of cells.

[0062] Detection index combination 3: Collect mouse spleen and place in a plastic dish containing 2 mL DMEM, crush the spleen with the end of a syringe to release the spleen cells, carefully discard the splenic capsule, collect the single cell suspension of the spleen into a 5 mL EP tube, then centrifuge at 300 g for 5 min, discard the supernatant. Add 500 μL red blood cell lysis solution to lyse the red blood cells for 10 min at room temperature, then add 500 μL PBS to stop the lysis, then centrifuge at 300 g for 5 min, discard the supernatant. Add 5 mL DMEM to resuspend the cell pellet, mix well, then take 10 μL and add to a 1.5 mL EP tube containing 190 μL PBS for counting. Take 300 μL of the remaining single cell suspension of the spleen and add to a 96-well plate, centrifuge at 350 g for 5 min, discard the supernatant. Then add 20 μL of antibody prepared with 2% BSA in PBS (antibody combinations are as follows: CD3e-percpcy5.5 (1:50 dilution), CD4-APC / cy7 (1:50 dilution), CD8-BV510 (1:100 dilution), CD44-BV421 (1:50 dilution), CD45RB-PE (1:100 dilution), CD62L-APC (1:50 dilution), PD1-PE-cy7 (1:100 dilution), CD16 / 32 FcR (1:50 dilution, used to block FcR to avoid non-specific binding of antibodies)), incubate at 4°C for 45 min. After washing with PBS, flow cytometry is performed to detect the proportion of each subpopulation of cells, and the total number of spleen lymphocytes is combined to calculate the changes in the number of T lymphocytes and each subpopulation of cells in the spleen.

[0063] Run the sample on the flow cytometer to detect the changes in the number of each subpopulation of T lymphocytes in the peripheral blood of the mice.

[0064] The experimental results show that after the 18-month-old aging mice are treated with the 0.5 g / mL and 1 g / mL groups of Example 1, the original atrophied thymus has a weight of <20 mg, while the mother group 1, the Example 1 group 0.5, and the Example 1 group 1.0 can all increase the weight of the atrophied thymus to 20 mg or more, and compared with the mother group 1, the Example 1 group 0.5 and the Example 1 group 1.0 have a more significant effect of increasing the thymus weight of the elderly individuals. Figure 5 At the same time, the spleen index does not increase, indicating that only the thymus weight is increased, without the risk of increasing the spleen.

[0065] The experimental results show that the Example 1 group has a significant effect in promoting the recovery of the atrophied thymus of the aging mice, and the thymocyte subpopulations (double positive cells (DP), CD8 single positive cells (CD8 SP), and CD4 single positive cells (CD4 SP)) of the 1 g / mL group are all significantly higher than those of the control group.p <0.0001, p <0.05, p <0.05); the number of double-positive thymocytes in the 0.5 g / mL group was also significantly increased compared with the control group ( p <0.0001); while in the mother group 1, compared with the control group, only the number of double-positive thymocytes was increased ( p <0.01); while other subgroups showed no statistically significant difference ( Figure 6 ).

[0066] Experimental results showed that after treatment with Example 1 and Mother Curd 1, the levels of various subsets of T lymphocytes in the spleen of aged mice (CD3-positive T cells (CD3+)) were significantly reduced. + T cells, CD4-positive T cells (CD4) + T), CD8-positive T cells (CD8) + T), CD4-positive naive T cells (CD4) + Naive T cells, CD4-positive recently migrated T cells (CD4+) + RTE), CD8-positive naive T cells (CD8) + Naive T cells, CD8-positive recently migrated T cells CD8 + All showed a promoting trend in RTE. Among them, the 1 g / mL group in Example 1, compared with the control group, significantly increased the number of T cell subsets ( p <0.05); compared with the control group, the 0.5 g / mL group significantly increased the number of CD4-positive naive T cells ( p <0.05); while compared with the control group, there was no statistically significant change in the number of each subgroup. This indicates that Example 1 can more significantly increase the number of total T cells in the spleen of aging individuals, and the increase in the number of each subgroup is more pronounced ( Figure 7 ).

[0067] From a mechanistic perspective, scientifically formulated traditional Chinese medicine can more effectively regulate the internal environment of the body by adjusting the types and proportions of bacteria in the yeast. In particular, direct oral gavage can directly inoculate the probiotics in the yeast extract into the digestive tract, exerting the conditioning effect of probiotics and regulating liver metabolism through the gut-liver axis, thus improving the phenotype of fatty liver. Since the kidney function itself was within the normal range, no significant changes were observed after treatment with the extract of Example 1, which, from another perspective, demonstrates that the extract of Example 1 has no side effects on kidney function.

[0068] From another aspect, the scientifically proportioned Chinese herbal medicine regulates the internal environment of the body by regulating the types and proportions of various probiotics in the wine starter, reduces the inflammatory level of the body, and reduces the inflammatory level of the thymus of the aging individual, so as to improve the internal environment of the thymus, enhance the function of the thymus nurturing cells, and nurture more thymus cells to develop, differentiate, and mature, which is manifested as more various thymus cell subgroups, increased thymus volume and weight; at the same time, more mature T cells are output from the thymus, and the number of T cells transferred to the periphery such as the spleen increases, and the number of various T cell subgroups in the spleen also significantly increases.

[0069] The above is based on the ideal embodiments of the present application, and through the above description, relevant personnel can make various changes and modifications without deviating from the technical idea of the present application. The technical scope of the present application is not limited to the content of the specification, and must be determined according to the scope of the claims.

Claims

1. A rice wine koji containing Chinese herbal medicines, characterized by comprising, The raw materials include, by weight, 65-80 parts of a cooked rice base, 6.5-12 parts of rice wine mother koji, and 12-18.5 parts of a plant composite powder; The plant composite powder is composed of 3.5-5.0 parts of lespedeza hispida, 2.5-3.5 parts of desmos chinensis, 2.0-3.0 parts of sesamum indicum, 1.5-2.5 parts of honeysuckle, 0.5-1.2 parts of cinnamomum cassia, 0.3-0.8 parts of syzygium aromaticum, and 1.5-2.5 parts of thatch.

2. The rice wine koji containing Chinese herbal medicine according to claim 1, characterized by: The rice wine mother koji refers to aged high-quality lespedeza hispida wine koji, i.e., "old koji", which is used as a carrier of microbial strains and is crushed into powder in advance to facilitate uniform mixing.

3. The rice wine koji containing Chinese herbal medicine according to claim 1, characterized by: The lespedeza hispida is picked in June-August, and fresh, pest-free plants with flowers or not fully bloomed are selected, washed, and dried for use.

4. The method for preparing the rice wine starter containing Chinese herbal medicines according to any one of claims 1 to 3, characterized in that, The preparation method includes the following steps: (1) Preparation of the rice base: the rice is screened to remove impurities and broken particles, washed, soaked, and cooked; (2) Treatment of Chinese herbal medicines: the washed and dried lespedeza hispida, desmos chinensis, sesamum indicum, honeysuckle, cinnamomum cassia, syzygium aromaticum, and thatch are mixed and crushed to obtain a plant composite powder; (3) Mixing: the crushed mother koji is mixed with the cooled grain base, the crushed plant composite powder is added, and pure water is gradually added and stirred until all the raw materials are uniformly mixed and moistened without clumping; (4) Koji preparation: the mixed raw materials are pressed into a koji block; (5) Koji block cultivation: the temperature, humidity, and ventilation are controlled to promote the reproduction of microorganisms on the koji block; (6) Drying of the matured koji block to reduce the moisture content and obtain a dried wine koji containing Chinese herbal medicines.

5. The method for preparing rice wine starter containing traditional Chinese medicine according to claim 4, characterized in that, In step (1), the soaking time is 4-6 hours until the soaked grain can be crushed without a hard core, and then the soaked grain is cooked until there is no white core. After cooking, the grain is spread out and cooled to 30-35℃; In step (2), the washing method is as follows: the grain is washed in a cyclone washing machine at a water flow rate of 50-70 L / min for 5-10 min; The drying method is as follows: the grain is dried in a hot air dryer at a temperature of 50-60℃ and an air flow rate of 1.5-2.5 m / s for 3-4 h, and the moisture content after drying is controlled to be 10-15 wt%; The crushing method is as follows: the grain is crushed to 70-80 mesh using a crusher.

6. The method for preparing rice wine starter containing traditional Chinese medicine according to claim 4, characterized in that, In step (3), the mixing method is as follows: the mixed raw materials are stirred in a mixer at a speed of 20-50 r / min for 5-10 min; In step (4), the koji preparation method is as follows: the koji preparation tool is a wooden mold, which is square or circular with dimensions of 10 cm x 10 cm x 5 cm, a wooden board, and a weight. The operation steps are as follows: the mixed raw materials are filled into the wooden mold, and the mold is pressed with hands. After demolding, the block-shaped koji block is obtained and placed on a bamboo mat covered with straw for 1-2 h.

7. The method for preparing rice wine starter containing traditional Chinese medicine according to claim 4, characterized in that, In step (5), the koji block cultivation is as follows: Stacking and initial culture: the koji is stacked neatly, and the layers are separated by straw to facilitate ventilation. The height is 50-80 cm, the room temperature is controlled at 25-30℃, and the relative humidity is 80%-90%. This stage is the "mold growth period", during which the microorganisms in the koji mother start to reproduce, and white mycelium gradually appears on the surface of the koji. The temperature slowly rises to 30-35℃; Koji turning and mid-term culture: when the temperature at the center of the koji rises to 40-45℃, the first koji turning is needed. The specific method is to move the upper koji to the lower layer, the lower koji to the upper layer, and the edge to the middle, to ensure uniform temperature of each koji. After turning, continue to culture, and the mycelium gradually spreads to the inside of the koji. The color changes from white to light yellow or light green, accompanied by the production of wine aroma or ester aroma. The temperature is maintained at 35-40℃, and the humidity is 70%-80%. If the temperature rises again to above 45℃, the second koji turning is performed, and a total of 2-3 times of turning is performed, with an interval of 2-3 days; Late maturation: the temperature of the koji gradually decreases to near room temperature, the mycelium ages, the color deepens, the texture becomes loose, and the aroma is rich, indicating that the culture is mature.

8. The method for preparing rice wine starter containing traditional Chinese medicine according to claim 4, characterized in that, In step (6), the drying method of the mature koji is hot air drying: using a hot air dryer to dry, and setting the temperature to 40-50℃, the air flow speed to 1.5-3.0 m / s, and the drying time to 12-24 h, until the moisture content is ≦12 wt%.

9. The method for preparing the rice wine starter containing Chinese herbal medicine according to any one of claims 1-8, characterized in that, The extraction method of the rice wine koji containing Chinese herbal medicine obtained by the method is: taking 0.5-1.5 g of rice wine koji, crushing it into powder in a clean environment, adding 5-15 mL of deionized water, extracting in a 37℃ water bath for 25-35 minutes, centrifuging at 7000-8500 r / min for 5-15 minutes, and taking the supernatant as the Chinese herbal medicine rice wine koji extract; preferably, taking 1 g of rice wine koji, crushing it into powder in a clean environment, adding 10 mL of deionized water, extracting in a 37℃ water bath for 30 minutes, centrifuging at 8000 r / min for 10 minutes, and taking the supernatant as the Chinese herbal medicine rice wine koji extract.

10. The method for preparing rice wine starter containing traditional Chinese medicine according to claim 9, characterized in that, The Chinese herbal medicine rice wine koji extract obtained by the method is used in the preparation of a drug for recovering thymus atrophy in fatty liver and aging individuals.

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