Lactobacillus plantarum strain and method for preparing traditional chinese medicine fermented beverage

Abstract

The present application relates to the technical field of microorganism and fermentation engineering, and particularly to a lactobacillus plantarum strain and a method for preparing a traditional Chinese medicine fermented beverage. The strain has been preserved in the Guangdong Microbial Culture Collection Center on December 13, 2024, and the preservation number is CGDMCC NO: 65639. The present application provides a method for preparing a blood supplementing beverage by fermentation using the strain. The method comprises mixing a traditional Chinese medicine extract, a ginseng concentrate and a pomegranate concentrate to obtain a mixed liquid, and then inoculating lactobacillus plantarum YXLP-116 for fermentation. The fermented beverage has the effects of regulating endocrine, improving reproductive health and relieving menopausal symptoms. Experimental results show that the beverage can significantly reduce the levels of FSH and LH, and increase the level of E2, and has high safety and cell protection effects. Animal experiments further confirm the potential of the beverage in regulating endocrine.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microorganisms and fermentation engineering, and particularly relates to a lactobacillus plantarum strain and a method for preparing a traditional Chinese medicine fermentation beverage. BACKGROUND

[0002] The causes of female endocrine disorders mainly include physiological factors, nutritional factors, emotional factors and environmental factors. The physiological factors refer to the fact that women are prone to endocrine disorder symptoms as their kidney qi gradually declines with age. The nutritional factors refer to the fact that the body is prone to endocrine disorder symptoms due to nutritional deficiency caused by dietary disorders. The emotional factors refer to the fact that women are prone to endocrine disorder due to adverse emotions such as depression and irritability in daily life, which causes nervous system response, interferes with normal blood circulation, and leads to endocrine disorder. The environmental factors refer to the fact that external evils such as dampness, heat, wind and cold easily cause qi disorder and incoordination of qi and blood, and finally cause diseases. Endocrine disorders generally have the following symptoms: skin deterioration, irritable temper, gynecological diseases, obesity, infertility, breast pain, breast hyperplasia, hirsutism, premature graying, premature aging and the like.

[0003] The existing traditional Chinese medicine beverage for regulating endocrine has great room for improvement in terms of absorption rate, regulation of female endocrine, improvement of reproductive health and relief of menopausal symptoms. Lactic acid bacteria are a kind of microorganisms capable of fermenting sugars to produce lactic acid, including lactobacillus and bifidobacterium. Lactic acid bacteria are widely used in food and beverage. Lactic acid bacteria have many health benefits, including improving intestinal health, enhancing immunity, and antioxidant effects.

[0004] The present application combines lactic acid bacteria with the existing traditional Chinese medicine beverage for regulating endocrine. Through the strong metabolic capacity of lactic acid bacteria, complex components in traditional Chinese medicine are decomposed to produce active substances that are more easily absorbed. The metabolic products such as lactic acid and short-chain fatty acids can reduce the pH value of the intestinal tract, enhance the intestinal barrier function, improve the absorption rate of traditional Chinese medicine components, affect the function of the hypothalamus-pituitary-ovary axis through metabolic products, regulate the secretion of hormones such as FSH, LH and E2, inhibit the excessive secretion of FSH and LH, promote the synthesis of E2 in the ovary, regulate female endocrine, improve reproductive health, and relieve menopausal symptoms, which will have great application value. SUMMARY

[0005] The present application aims to provide a lactobacillus plantarum strain and a method for preparing a traditional Chinese medicine fermentation beverage to solve the problems in the background art.

[0006] To achieve the above object, the present application provides the following technical scheme: The present application provides a Lactiplantibacillus plantarum YXLP-116, which has been preserved in Guangdong Provincial Strategy Microorganism Culture Collection Center on December 13, 2024, with a preservation number of CGDMCC NO: 65639 and a preservation address of No. 59 Building, 5th Floor, 100 Middle Liangma Street, Guangzhou, China.

[0007] The method for preparing the traditional Chinese medicine fermented beverage by using the Lactiplantibacillus comprises the following steps:

[0008] S1, mixing the traditional Chinese medicine extract, ginseng concentrate and pomegranate concentrate juice to obtain mixed medicinal liquid; wherein the traditional Chinese medicine extract is prepared by water extraction of a traditional Chinese medicine composition comprising the following components by weight: 1-4 parts of Poria cocos, 1-4 parts of Cinnamomum cassia, 1-4 parts of Tremella fuciformis, 0.5-3 parts of Glycyrrhiza uralensis, 0.5-3 parts of Astragalus membranaceus, 0.5-3 parts of Rosa rugosa, 0.5-3 parts of Fructus Lycii and 0.1-5 parts of ginger;

[0009] The mass ratio of pomegranate concentrate juice, ginseng concentrate and Poria cocos is (5-12):(1-5):(1-4);

[0010] S2, after sterilization, inoculate the mixed medicinal liquid with Lactiplantibacillus YXLP-116 and ferment to obtain a traditional Chinese medicine fermented beverage.

[0011] Further, in S2, the inoculation amount of Lactiplantibacillus YXLP-116 is 0.2%-8%, i.e. the inoculation amount of Lactiplantibacillus YXLP-116 accounts for 0.5%-10% of the total volume of the fermentation broth.

[0012] Further, in S1, the traditional Chinese medicine composition comprises the following components by weight: 2-3 parts of Poria cocos, 2-3 parts of Cinnamomum cassia, 2-3 parts of Tremella fuciformis, 1-2 parts of Glycyrrhiza uralensis, 1-2 parts of Astragalus membranaceus, 1-2 parts of Rosa rugosa, 1-2 parts of Fructus Lycii and 2-3 parts of ginger; the mass ratio of pomegranate concentrate juice, ginseng concentrate and Poria cocos is (6-10):(2-4):(2-3).

[0013] Further, in S1, the traditional Chinese medicine composition further comprises 1-4 parts of Jujube.

[0014] Further, in S1, the traditional Chinese medicine composition further comprises 0.5-1 parts of Longan.

[0015] Further, in S1, the water extraction process of the traditional Chinese medicine composition is as follows: mix each component contained in the traditional Chinese medicine composition with water, heat and decoct, and then filter to obtain the filtrate, which is the traditional Chinese medicine extract.

[0016] Further, in S2, the Lactobacillus plantarum YXLP-116 is fermented for 30-48 hours.

[0017] Compared with the prior art, the present application has the following advantages:

[0018] The Lactobacillus plantarum YXLP-116 (CGDMCC NO: 65639) is combined with traditional Chinese medicine to prepare a fermented beverage for regulating endocrine, which is non-toxic to various human cells and has a protective effect: the survival rate of gastric mucosa cells is 124.15% at a dose of 100.8 μg / mL (increased by 3.4% compared with the control group), the survival rate of liver cells is increased to 119.63% (106.54% for the control group), and the survival rate of nerve cells is increased from 98.56% for the control group to 115.43%, indicating that it can be safely applied to the development of functional food.

[0019] It is found through thin layer chromatography analysis that the polarity of the traditional Chinese medicine components is reduced after fermentation of the Lactobacillus plantarum YXLP-116, and is converted into small molecular substances that are easy to absorb. The lactic acid and short-chain fatty acid produced by the Lactobacillus plantarum not only optimize the intestinal pH value, but also enhance the intestinal barrier function, and significantly improve the bioavailability of the active ingredients. At the same time, the anti-inflammatory experiment shows that the fermented beverage has outstanding inhibitory effect on inflammatory factors of macrophages: at a dose of 100.8 μg / mL, NO and IL8 are decreased by 68.09% and 66.89% respectively, which is much higher than the control group (42.55%, 31.94%), and shows a dose-dependent manner, providing data support for immune regulation.

[0020] The traditional Chinese medicine fermented beverage inoculated with the Lactobacillus plantarum YXLP-116 realizes estrogen two-way regulation through the phytoestrogen of pomegranate concentrated juice, and combines the synergistic effect of multi-target of traditional Chinese medicine formula. The monarch drug (pomegranate, tremella, ginseng) mainly focuses on skin care and qi tonification, the ministerial drug (hoelen, cassia bark, ginger) strengthens the spleen and warms the meridians, and the auxiliary drug (astragalus, rose flower, wolfberry, licorice) balances the medicinal properties. Animal experiments suggest that it can improve reproductive health and menopausal symptoms by regulating the hypothalamus-pituitary-ovary axis and affecting the secretion of FSH, LH and E2 hormones, and highlights the potential of "natural replacement therapy".

[0021] In the preparation process of the Chinese medicine fermented beverage inoculated with Lactobacillus plantarum YXLP-116 of the present application, the process parameters are precisely optimized: inoculation amount 0.2% to 8%, temperature 30 to 40 DEG C, and fermentation time 30 to 48 hours, so as to ensure the activity of the strain and the stability of the product. The short-time sterilization process (103 to 118 DEG C, 8 to 20 minutes) retains the medicinal components while killing miscellaneous bacteria. The formula has strong flexibility and can add jujube, longan and the like to enhance the blood tonifying and tranquilizing functions, is suitable for skin problems, emotional fluctuations and menopausal syndrome caused by female endocrine disorders, and covers a wide range of health needs. Through the strain directional fermentation technology of the present application, the absorption efficiency and functional limitations of traditional Chinese medicine beverages are broken through, and experimental data confirm the multi-dimensional advantages of anti-inflammatory, immune regulation and endocrine balance. The natural and safe characteristics provide a new paradigm for the development of functional beverages, especially meet the needs of modern consumers for "medicinal food" products, and have significant market conversion potential. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 is a thin layer chromatography experimental result graph;

[0023] Figure 2 is a gastric mucosa cell safety experiment result graph;

[0024] Figure 3 is a liver cell safety experiment result graph;

[0025] Figure 4 is a hippocampal nerve cell safety experiment result graph;

[0026] Figure 5 is a reagent kit detection NO experiment result graph;

[0027] Figure 6 is a reagent kit detection IL8 experiment result graph. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0029] The application provides Lactiplantibacillus plantarum YXLP-116 strain, and the Lactiplantibacillus plantarum YXLP-116 provided by the application has been preserved in Guangdong Provincial Strategy Microorganism Culture Collection Center on December 13, 2024, with a preservation number of CGDMCC NO: 65639 and a preservation address of 5th Floor, Building 59, 100 Middle Liangma Street, Guangzhou, China.

[0030] The strain of the application is subjected to 16S rDNA molecular identification, and the steps are as follows:

[0031] The genome of Lactiplantibacillus plantarum YXLP-116 is extracted, the 16S rDNA sequence of the strain is obtained by amplifying the 16S rDNA sequence of the bacteria from the genomic DNA of Lactiplantibacillus plantarum YXLP-116 through primers 27F and 1492R, the amplification product is sequenced, and the 16s rDNA sequence SEQ ID NO: 1 of the strain is obtained.

[0032] SEQ ID NO: 1:

[0033]

[0034] The method for preparing the traditional Chinese medicine fermented beverage by using Lactiplantibacillus plantarum YXLP-116 comprises the following steps:

[0035] The traditional Chinese medicine extract liquid is mixed with pomegranate concentrated juice and ginseng concentrated liquid to obtain a mixed medicine liquid; the traditional Chinese medicine extract liquid is obtained by water extraction of a traditional Chinese medicine composition, and the traditional Chinese medicine composition comprises the following components in parts by weight: 1-4 parts of poria cocos, 1-4 parts of cassia bark, 1-4 parts of tremella fuciformis, 0.5-3 parts of licorice, 0.5-3 parts of astragalus, 0.5-3 parts of rose flower, 0.5-3 parts of medlar and 0.1-5 parts of ginger;

[0036] The traditional Chinese medicine fermented beverage provided by the application takes pomegranate as one of raw materials, and pomegranate contains phytoestrogens, which have a "two-way regulation" effect on the human body, that is, when the estrogen level in the body is low, they directly bind to estrogen receptors to exert estrogen effects; when the estrogen level in the body is high, they compete with estrogen to bind to estrogen receptors, thereby showing anti-estrogen effects; therefore, phytoestrogens can regulate the body's endocrine system. When the estrogen level of women is unbalanced, appropriate estrogen supplementation is crucial for women to have a beautiful and healthy life. Pomegranate also has good moisturizing, anti-wrinkle and beauty spot-removing effects. Flavonoids in pomegranate are a female hormone that can improve female skin and delay skin aging. The pomegranate concentrated juice in the application is juice with a soluble solid content of 60%-70% obtained by enzymolysis, filtration, concentration and sterilization of pomegranate pulp.

[0037] The monarch drug: pomegranate has the functions of moistening the lungs and invigorating the stomach, and the lungs govern the skin and hair, so moistening the lungs can beautify the skin; tremella is neutral in nature and has the functions of nourishing yin and moistening the lungs, and beautifying the skin.

[0038] The ministerial drug: ginseng greatly supplements primordial qi and restores pulse and firmness. Poria cocos benefits water and removes dampness, invigorates the spleen and calms the heart; cassia bark is very hot in nature and can invigorate the fire and assist the yang and warm the meridians, thereby promoting blood circulation; ginger warms and invigorates the spleen and stomach. The ginseng liquid in the application refers to juice with a solid content of greater than or equal to 60% obtained by extraction and concentration of ginseng.

[0039] The assistant drug: medlar tonifies the liver and kidney and benefits essence and eyesight; astragalus and rose flower are cool in nature and tonify the spleen and stomach and reduce the heat of other medicinal materials.

[0040] The auxiliary drug: licorice tonifies the spleen and benefits qi and harmonizes all medicinal materials.

[0041] The traditional Chinese medicine fermentation beverage for regulating endocrine provided by the present application takes natural pomegranate juice as the main component, and is supplemented with traditional Chinese medicinal materials for nourishing yin and lung, tonifying qi and warming meridians to strengthen the effect of regulating endocrine. The heat of the medicinal materials is adjusted by using pueraria, so that the qi and blood are considered, and the medicinal materials are neither dry nor hot. The medicinal materials can also nourish liver and kidney, so that the prescription can not only strengthen the spleen and stomach, the acquired constitution, but also nourish the liver and kidney, the congenital essence, which is suitable for women of various constitutions. The lung governs hair, and the lung and large intestine are related to the surface. The prescription can not only moisten the lung and tonify the large intestine, but also produce and transform qi and blood, and moisten the skin, which is a good product for beauty and skin care. Therefore, the traditional Chinese medicine fermentation beverage provided by the present application has the effects of regulating endocrine, nourishing yin and moisturizing, and beautifying and skin care, and is easy to absorb. Moreover, after being inoculated with lactobacillus plantarum YXLP-116 and fermented, the beverage is easy to be absorbed, and the effect, taste and safety of the beverage after being inoculated with lactobacillus plantarum YXLP-116 and fermented can be further improved.

[0042] In the present application, the water extraction process preferably comprises the following steps:

[0043] Each component of the traditional Chinese medicine composition is mixed with water, heated and filtered after decocting. In the present application, the mass ratio of the traditional Chinese medicine composition to water is preferably 1: (3-14), and more preferably 1: (10-14). In the present application, the temperature of decocting is preferably 60-100 DEG C, 75-95 DEG C, and most preferably 90-95 DEG C. The extraction time is preferably 0.5-3 h, 1.5-3.0 h, and 2.5-3.0 h.

[0044] In the present application, the temperature of vacuum concentration is preferably 60 DEG C, and the pressure of vacuum concentration is ≤-0.095 MPa.

[0045] After obtaining the traditional Chinese medicine extract, the traditional Chinese medicine extract is mixed with pomegranate concentrated juice and ginseng concentrated liquid to obtain a mixed medicinal liquid. In the present application, the mixed amount of the traditional Chinese medicine extract is based on the mass of poria cocos. The mass ratio of the pomegranate concentrated juice, the ginseng concentrated liquid and the poria cocos is (5-12):(1-5):(1-4), and more preferably (6-10):(2-4):(2-3).

[0046] The mixed medicinal liquid is sterilized and inoculated with lactobacillus plantarum YXLP-116 for lactobacillus plantarum YXLP-116 fermentation. In the present application, the sterilization can kill bacteria and prevent the growth of bacteria during the fermentation process to destroy the medicinal properties. In the present application, the sterilization temperature is 103-118 DEG C, and more preferably 110-115 DEG C. The sterilization time is 8-20 min, and more preferably 10-15 min. Under the sterilization conditions, the sterilization is complete and the content of active substances in the mixed medicinal liquid is not affected.

[0047] In the present application, the Lactobacillus plantarum YXLP-116 strain is preferably activated before inoculation, and the activation preferably uses MRS liquid medium. In the present application, the inoculation amount of the Lactobacillus plantarum YXLP-116 is preferably independently 0.5% to 10%, the fermentation temperature of the Lactobacillus plantarum YXLP-116 is 30°C to 40°C, and the Lactobacillus plantarum YXLP-116 can maintain a high activity during fermentation; the fermentation time is preferably 30 h to 48 h, which ensures sufficient fermentation.

[0048] After the Lactobacillus plantarum YXLP-116 fermentation, the present application preferably homogenizes and filters the obtained fermentation liquor to obtain a traditional Chinese medicine fermented beverage. In the embodiments of the present application, the homogenization is performed in a high-shear homogenizer, the rotation speed of the homogenization is preferably 10,000 rpm, and the homogenization time is preferably 2 min; the filtration uses a plate-and-frame filter, and the pore size of the plate-and-frame filter is preferably 10 microns.

[0049] Example 1: 1 kg of Poria cocos, 4 kg of Cinnamomum cassia, 1 kg of Tremella fuciformis, 3 kg of Glycyrrhiza uralensis, 0.5 kg of Astragalus membranaceus, 0.5 kg of Rosa rugosa, 1 kg of Canarium album, 0.1 kg of ginger, 4 kg of Jujube, and 0.5 kg of Lycium barbarum are weighed to a total of 15.1 kg. The above-mentioned raw materials are placed in 45.3 kg of water and decocted at 60°C for 3 h, and the medicinal liquor is obtained by filtration, and then the medicinal liquor is concentrated under vacuum to obtain a traditional Chinese medicine extract;

[0050] The obtained traditional Chinese medicine extract is mixed with 12 kg of pomegranate concentrate juice with a soluble solid mass percentage of 60% and 1 kg of ginseng concentrate liquid with a solid mass percentage of 60% to obtain a mixed medicinal liquor.

[0051] The obtained mixed medicinal liquor is placed in a fermentation tank and sterilized at 103°C for 20 min.

[0052] After the Lactobacillus plantarum YXLP-116 is activated, it is inoculated into the sterilized mixed medicinal liquor at an inoculation amount of 2%, the fermentation temperature is controlled at 30°C, and the fermentation is performed for 48 h to obtain an endocrine-regulating traditional Chinese medicine fermented beverage.

[0053] Example 2: 4 kg of Poria cocos, 1 kg of Cinnamomum cassia, 4 kg of Tremella fuciformis, 0.5 kg of Glycyrrhiza uralensis, 3 kg of Astragalus membranaceus, 3 kg of Rosa rugosa, 0.5 kg of Canarium album, 5 kg of ginger, 1 kg of Jujube, and 3 kg of Lycium barbarum are weighed to a total of 22 kg. The above-mentioned raw materials are placed in 220 kg of water and decocted at 100°C for 0.5 h, and the medicinal liquor is obtained by filtration, and then the medicinal liquor is concentrated to obtain a traditional Chinese medicine extract;

[0054] The obtained traditional Chinese medicine extract is mixed with 5 kg of pomegranate concentrate juice with a soluble solid mass percentage of 70% and 5 kg of ginseng concentrate liquid with a solid mass percentage of 65% to obtain a mixed medicinal liquor.

[0055] The obtained mixed medicinal liquid is placed in a fermentation tank and sterilized at 118°C for 8 minutes;

[0056] After the Lactobacillus plantarum YXLP-116 is activated, it is inoculated into the sterilized mixed medicinal liquid at an inoculation amount of 0.5%, the fermentation temperature is controlled at 40°C, and the fermentation is performed for 30 hours to obtain the endocrine-regulating traditional Chinese medicine fermented beverage.

[0057] In Example 3, 2 kg of Poria cocos, 3 kg of Cinnamomum cassia, 2 kg of Tremella fuciformis, 2 kg of Glycyrrhiza uralensis, 1 kg of Astragalus membranaceus, 1 kg of Rosa rugosa, 0.8 kg of Canarium album, 2 kg of Zingiber officinale, 3 kg of Ziziphus jujuba, and 2 kg of Lycium barbarum are weighed, totaling 17.8 kg. The above-mentioned raw materials are placed in 89 kg of water and decocted at 70°C for 2 hours, and the medicinal liquid is obtained by filtration, and then the medicinal liquid is concentrated to obtain a traditional Chinese medicine extract;

[0058] The obtained traditional Chinese medicine extract is mixed with 6 kg of pomegranate concentrated juice with a soluble solid mass percentage of 65% and 2 kg of ginseng concentrated liquid with a solid mass percentage of 70% to obtain a mixed medicinal liquid.

[0059] The obtained mixed medicinal liquid is placed in a fermentation tank and sterilized at 110°C for 12 minutes;

[0060] After the Lactobacillus plantarum YXLP-116 is activated, it is inoculated into the sterilized mixed medicinal liquid at an inoculation amount of 2%, the fermentation temperature is controlled at 35°C, and the fermentation is performed for 45 hours to obtain the endocrine-regulating traditional Chinese medicine fermented beverage.

[0061] In Example 4, 3 kg of Poria cocos, 2 kg of Cinnamomum cassia, 3 kg of Tremella fuciformis, 1 kg of Glycyrrhiza uralensis, 2 kg of Astragalus membranaceus, 2 kg of Rosa rugosa, 0.6 kg of Canarium album, 3 kg of Zingiber officinale, 2 kg of Ziziphus jujuba, and 1 kg of Lycium barbarum are weighed, totaling 17.6 kg. The above-mentioned raw materials are placed in 140.8 kg of water and decocted at 80°C for 1.5 hours, and the medicinal liquid is obtained by filtration, which is a traditional Chinese medicine extract;

[0062] The obtained traditional Chinese medicine extract is mixed with 10 kg of pomegranate concentrated juice with a soluble solid mass percentage of 68% and 4 kg of ginseng concentrated liquid with a solid mass percentage of 75% to obtain a mixed medicinal liquid.

[0063] The obtained mixed medicinal liquid is placed in a fermentation tank and sterilized at 115°C for 15 minutes;

[0064] After each bacterium is activated, it is inoculated into the sterilized mixed medicinal liquid at an inoculation amount of 2%, the fermentation temperature is controlled at 35°C, and the fermentation is performed for 40 hours to obtain the endocrine-regulating traditional Chinese medicine fermented beverage.

[0065] Example 5, 2.5 kg of Poria cocos, 2.5 kg of Cinnamomum cassia, 2.5 kg of Tremella fuciformis, 1.5 kg of Glycyrrhiza uralensis, 1.5 kg of Astragalus membranaceus, 1.5 kg of Rosa rugosa, 0.8 kg of Canarium album, 2.5 kg of Zingiber officinale, 2.5 kg of Ziziphus jujuba and 1.5 kg of Lycium barbarum, a total of 17.8 kg, are weighed. The above raw materials are placed in 89 kg of water and decocted at 90℃ for 1 h, filtered to obtain a medicinal liquid, and then the medicinal liquid is concentrated to obtain a traditional Chinese medicine extract;

[0066] The obtained traditional Chinese medicine extract is mixed with 8 kg of pomegranate concentrated juice with a soluble solid mass percentage of 62% and 3 kg of ginseng concentrated liquid with a solid mass percentage of 70% to obtain a mixed medicinal liquid;

[0067] The obtained mixed medicinal liquid is placed in a fermentation tank. Sterilization is performed at 108℃ for 16 min;

[0068] After activation, Lactobacillus plantarum YXLP-116 is inoculated into the sterilized mixed medicinal liquid at an inoculation amount of 10%, the fermentation temperature is controlled at 35℃, and fermentation is performed for 36 h to obtain an endocrine-regulating traditional Chinese medicine fermented beverage.

[0069] Example 6, an endocrine-regulating traditional Chinese medicine fermented beverage is prepared according to the scheme of Example 5, except that the inoculation amount of Lactobacillus plantarum YXLP-116 in this example is 1%.

[0070] Example 7, an endocrine-regulating traditional Chinese medicine fermented beverage is prepared according to the scheme of Example 5, except that the inoculation amount of Lactobacillus plantarum YXLP-116 in this example is 3%.

[0071] Example 8, an endocrine-regulating traditional Chinese medicine fermented beverage is prepared according to the scheme of Example 5, except that the inoculation amount of Lactobacillus plantarum YXLP-116 in this example is 7%.

[0072] Example 9, an endocrine-regulating traditional Chinese medicine fermented beverage is prepared according to the scheme of Example 5, except that the inoculation amount of Lactobacillus plantarum YXLP-116 in this example is 0.5%.

[0073] Example 10, an endocrine-regulating traditional Chinese medicine fermented beverage is prepared according to the scheme of Example 5, except that the inoculation amount of Lactobacillus plantarum YXLP-116 in this example is 8%.

[0074] Example 11, an endocrine-regulating traditional Chinese medicine fermented beverage is prepared according to the scheme of Example 5, except that the inoculation amount of Lactobacillus plantarum YXLP-116 in this example is 6%.

[0075] Example 12, an endocrine-regulating traditional Chinese medicine fermented beverage is prepared according to the scheme of Example 5, except that the inoculation amount of Lactobacillus plantarum YXLP-116 in this example is 2%.

[0076] The present application detects the change of the polarity of the components in the fermentation liquor by thin layer chromatography, and verifies the change of the effective components of the Chinese herbal medicine after the inoculation of Lactobacillus plantarum YXLP-116 fermentation.

[0077] (I) Thin layer chromatography experiment:

[0078] 1) Control solution: Ginsenoside Rg1, Re and Rb1 were taken as the control, and methanol was added to prepare a mixed solution containing 1.3 mg of the control per 1 mL.

[0079] 2) Sample processing: The fermentation liquor obtained by the above technical solution was extracted according to the fermentation liquor and n-butanol = 4:1 (volume ratio), and the n-butanol phase was taken after layering.

[0080] The pomegranate ginseng endocrine regulating traditional Chinese medicine fermented beverage prepared in Example 1 was taken as the experimental group, and samples were taken at 0h, 8h, 24h, 32h, 40h and 48h for analysis;

[0081] 3) Silica gel plate: purchased finished product silica gel plate: 50*100mm (40 pieces / box), Qingdao Shenghai Fine Silica Gel Chemical Co., Ltd.;

[0082] 4) Developing agent: 10 mL of a solvent system composed of water, methanol and chloroform in a volume ratio of 0.2:4:6 was poured into the developing tank as the developing agent.

[0083] 5) Sample application: A pencil was gently drawn a line about 1 cm from the bottom of the point plate to mark the sample application site, and a capillary tube was used to apply the sample and the control.

[0084] 6) Development: Place the silica gel plate after sample application into the developing tank, the developing agent should not submerge the previous pencil line, and let the solvent develop upward about 80%-90% of the length of the thin plate.

[0085] 7) Take out the silica gel plate from the developing tank and immediately mark the leading position reached by the solvent with a pencil.

[0086] 8) Observation: Let the solvent on the silica gel plate evaporate, and place the silica gel plate into a container filled with iodine vapor for color development.

[0087] 9) Make TLC marks and calculate the Rf value of each spot.

[0088] The results are shown in Figure 1 .

[0089] From Figure 1 it can be seen that after inoculation of Lactobacillus plantarum YXLP-116 fermentation, the points representing the components in the fermentation liquor move up, and are converted into molecules with smaller polarity, thereby improving the bioabsorption rate of the effective components of Chinese herbal medicine.

[0090] (B) Cell experiment

[0091] Sample preparation:

[0092] Post-treatment of fermentation liquor: the bacteria in the fermentation liquor were broken and homogenized by using a homogenizer to obtain a homogenized mixture, and the obtained homogenized mixture was taken as the detection object.

[0093] The mixed medicinal liquid obtained before fermentation of the Lactobacillus plantarum YXLP-116 of Example 5 was taken as the control group. The pomegranate ginseng endocrine-regulating traditional Chinese medicine fermented beverage prepared in Example 5 was taken as the experimental group.

[0094] Cell safety test:

[0095] 1. Plating:

[0096] The well-cultured human normal gastric mucosa cells GES-1, liver cells LO2, and hippocampal nerve cells HT22 were subjected to microscopic examination (cell density 70%-80%), enzyme digestion, centrifugation, and aspiration of supernatant. 30 mL of DMEM+FBS+AB was added to the centrifuge tube, and the cell concentration was controlled to be 2.0*10 5 6 / mL by 100-fold microscopic counting and dilution. One 96-well plate was taken, and 0.1 mL of cell suspension was added per well. The cover was covered, and the plate was placed in a carbon dioxide incubator for culture at 37°C and 5% for 24 h.

[0097] 2. Drug addition:

[0098] Drug preparation: the drug liquids of the control group and the experimental group were vacuum concentrated to dryness, 0.5 g of drug dry matter was weighed, and a solution with a concentration of 50 mg / mL was prepared by using 9.5 mL of DMEM+FBS+AB (prepared by 500 mL of DMEM, 50 mL of FBS, and 5 mL of AB) according to the dose of 37.8 ug / ml, 75.5 ug / ml, and 150.9 ug / ml. The drug was added to the experimental wells of the 96-well plate, and blank control wells and negative control wells were left. Blank control: DMEM+FBS+AB was added to the well without adding test sample and lipopolysaccharide; negative control: DMEM+FBS+AB was added, and lipopolysaccharide was added without adding test sample.

[0099] After drug addition, the 96-well plate was covered and placed in a carbon dioxide incubator for culture at 37°C and 5% for 24 h.

[0100] 3. Detection:

[0101] MTT method was used for detection.

[0102] The results are shown in Figures 2-4 and Tables 1-3, wherein Figure 2The experimental results of the safety of gastric mucosal cells (P<0.05), Figure 2 The curves 1-3 are the experimental results of the safety of gastric mucosal cells of the experimental group, the control group and the blank control group, respectively, Figure 3 The experimental results of the safety of hepatocytes (P<0.01), Figure 3 The curves 1-3 are the experimental results of the safety of hepatocytes of the experimental group, the control group and the blank control group, respectively, Figure 4 The experimental results of the safety of hippocampal nerve cells (P<0.01), Figure 4 The curves 1-3 are the experimental results of the safety of hippocampal nerve cells of the experimental group, the control group and the blank control group, respectively.

[0103] Table 1 The safety test results of gastric mucosal cells of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 5

[0104]

[0105] Table 2 The safety test results of hepatocytes of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 5

[0106]

[0107] Table 3 The safety test results of hippocampal nerve cells of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 5

[0108]

[0109] The safety performance of the fermented beverage obtained was tested according to the above detection method, and the results are shown in Tables 4-6.

[0110] Table 4 The safety test results of gastric mucosal cells of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 1

[0111]

[0112] Table 5 The safety test results of hepatocytes of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 1

[0113]

[0114] Table 6 The safety test results of hippocampal nerve cells of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 1

[0115]

[0116] The mixed liquid obtained before fermentation without inoculation with Lactobacillus plantarum YXLP-116 in Example 2 was used as the control group, and the pomegranate and ginseng fermented traditional Chinese medicine beverage for regulating endocrine function prepared in Example 2 was used as the experimental group. The safety performance of the obtained fermented beverage was tested according to the above detection method, and the results are shown in Tables 7 to 9.

[0117] Table 7. Safety test results of gastric mucosal cells of the traditional Chinese medicine fermented beverage for regulating endocrine function in Example 2.

[0118]

[0119] Table 8. Hepatocyte safety test results of the traditional Chinese medicine fermented beverage for regulating endocrine function in Example 2.

[0120]

[0121] Table 9. Safety test results of hippocampal nerve cells in the fermented herbal beverage for regulating endocrine function in Example 2.

[0122]

[0123] The mixed medicinal liquid obtained before fermentation without inoculation with Lactobacillus plantarum YXLP-116 in Example 4 was used as the control group, and the pomegranate and ginseng fermented traditional Chinese medicine beverage for regulating endocrine function prepared in Example 4 was used as the experimental group. The safety performance of the obtained fermented beverage was tested according to the above detection method, and the results are shown in Tables 10-12.

[0124] Table 10. Results of the gastric mucosal cell safety test of the traditional Chinese medicine fermented beverage for regulating endocrine function in Example 4.

[0125]

[0126] Table 11. Hepatocellular safety test results of the traditional Chinese medicine fermented beverage for regulating endocrine function in Example 4.

[0127]

[0128] Table 12. Safety test results of hippocampal nerve cells in the fermented herbal beverage for regulating endocrine function in Example 4.

[0129]

[0130] Depend on Figures 2-4 As can be seen from the results in Tables 1-12, within the dosage range of 159.6 ug / ml, the safety of pomegranate ginseng traditional Chinese medicine fermented with Lactobacillus plantarum YXLP-116 to gastric mucosal cells, hepatocytes, and hippocampal nerve cells was improved. This indicates that the traditional Chinese medicine fermented beverage for regulating endocrine function provided by this invention has improved safety due to the fermentation process.

[0131] The present application selects RAW264.7 macrophages to verify the endocrine regulation effect of the product by reducing inflammatory factors (NO, IL8) to improve immunity.

[0132] Cell efficacy experiment steps:

[0133] 1. Plating:

[0134] The cultured human normal macrophage RAW264.7 was observed under a microscope (cell density 70%-80%), enzyme digestion, centrifugation, and supernatant aspiration. 30 mL of DMEM+FBS+AB (prepared by mixing 500 mL of DMEM, 50 mL of FBS, and 5 mL of AB) was added to the centrifuge tube, and the cell concentration was controlled to be 2.0*10 5 6 / mL by 100 times microscopic counting and dilution. Take 1 24-well plate, add 1 mL of cell suspension per well. Cover the cover, and put it into a carbon dioxide incubator for culture, 37℃, 5%, 24h.

[0135] 2. Drug addition:

[0136] Prepare the drug. The drug solution of the control group and the experimental group was vacuum concentrated to dryness, 0.5 g of drug dry was weighed, and 9.5 mL of DMEM+FBS+AB was prepared into a 50 mg / mL solution. The drug concentration was designed according to the doses of 37.8 ug / ml, 75.5 ug / ml, and 150.9 ug / ml. Add the drug to the experimental wells of the 24-well plate, and leave blank control wells and negative control wells. Blank control: add DMEM+FBS+AB wells without adding test samples and lipopolysaccharide;

[0137] Negative control: add DMEM+FBS+AB wells, add lipopolysaccharide, and do not add test sample wells;

[0138] After adding the drug, cover the 24-well plate and put it into a carbon dioxide incubator for culture, 37℃, 5%, 3h.

[0139] Add 5 μl of LPS solution with a concentration of 1 mg / mL to the cultured 24-well plate, and mix gently.

[0140] Put it into a carbon dioxide incubator for culture, 37℃, 5%, 24h.

[0141] 3. Detection:

[0142] Use the ELISA kit to detect NO and IL8.

[0143] The results are shown in Figures 5-6 and Tables 13-14, Figure 5For the experimental results of the kit for detecting NO, curves 1-4 are the NO detection results of the blank control, the experimental group, the control group, and the negative control, respectively, Figure 6 For the experimental results of the kit for detecting IL8, curves 1-4 are the IL8 detection results of the blank control, the experimental group, the control group, and the negative control, respectively.

[0144] Table 13: Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 5 for reducing NO

[0145]

[0146] Table 14: Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 5 for reducing IL8

[0147]

[0148] The mixed medicinal liquid obtained before fermentation of the un-inoculated Lactobacillus plantarum YXLP-116 of Example 1 was used as the control group, and the endocrine-regulating traditional Chinese medicine fermented beverage of pomegranate and ginseng prepared in Example 1 was used as the experimental group. The efficacy of the obtained fermented beverage was detected according to the above-mentioned detection method, and the results are shown in Tables 15-16.

[0149] Table 15: Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 1 for reducing NO

[0150]

[0151] Table 16: Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 1 for reducing IL8

[0152]

[0153] The mixed medicinal liquid obtained before fermentation of the un-inoculated Lactobacillus plantarum YXLP-116 of Example 2 was used as the control group, and the endocrine-regulating traditional Chinese medicine fermented beverage of pomegranate and ginseng prepared in Example 2 was used as the experimental group. The efficacy of the obtained fermented beverage was detected according to the above-mentioned detection method, and the results are shown in Tables 17-18.

[0154] Table 17: Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 4 for reducing NO

[0155]

[0156] Table 18: Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 4 for reducing IL8

[0157]

[0158] The mixed medicinal liquid obtained before fermentation of the Lactobacillus plantarum YXLP-116 of Example 4 is used as a control group, and the pomegranate ginseng endocrine-regulating traditional Chinese medicine fermented beverage prepared in Example 4 is used as an experimental group. The efficacy of the obtained fermented beverage is detected according to the above detection method, and the results are shown in Tables 19-20.

[0159] Table 19 Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 4 on reducing NO

[0160]

[0161] Table 20 Test results of the endocrine-regulating traditional Chinese medicine fermented beverage of Example 4 on reducing IL8

[0162]

[0163] From Figure 5 As can be seen from Tables 13, 15, 17 and 19, the efficacy of the medicinal liquid fermented by inoculating the Lactobacillus plantarum YXLP-116 on reducing NO is enhanced.

[0164] From Figure 6 As can be seen from Tables 14, 16, 18 and 20, the efficacy of the medicinal liquid fermented by inoculating the Lactobacillus plantarum YXLP-116 on reducing IL8 is enhanced.

[0165] Therefore, the endocrine-regulating traditional Chinese medicine fermented beverage provided by the present application has enhanced endocrine-regulating efficacy due to fermentation.

[0166] (Three) animal experiments;

[0167] Experimental Example 1, modeling: 60 were randomly selected for modeling, and the remaining 10 were used as a normal group. The Kafali modeling method was used to establish a female rat model. The 60 female rats for modeling were given free access to granular rat feed and water, and each female rat was given daily intragastrical administration of letrozole (1 mg / kg·d, in 1% carboxymethyl cellulose (CMC), for a total of 21 days), and the normal group was given intragastrical administration of the same volume of 1% carboxymethyl cellulose. After 22 days of modeling, blood was collected by tail clipping, and the serum estradiol (E2) level of the female rats was detected. If the level is lower than that of the normal group (P<0.05), it indicates that the modeling is successful.

[0168] Animal grouping and administration method: 60 female rats with successful modeling were randomly divided into administration group and control group (30 rats in each group), wherein the administration group was divided into small, medium and large dose groups (10 rats in each group), and the control group was divided into small, medium and large dose groups (10 rats in each group); drug selection: the control group took the mixed drug solution fermented by the plant lactobacillus YXLP-116 in example 1 as the drug solution for administration. The administration group carried out post-treatment on the fermentation broth obtained by inoculating plant lactobacillus YXLP-116 in example 1 for 35h: the bacteria were broken and homogenized by using a homogenizer to obtain a homogenized mixed drug solution as the drug solution for administration.

[0169] Dose of administration: the daily drug dose of female rats (g / kg) = the daily drug dose of human (g / kg) × 6.3, the gavage dose of the administration group from small to large was 0.5, 1, 2 times of the daily drug dose of female rats, and the final administration dose of the small, medium and large dose groups of the administration group and the control group was 2ml, 4ml and 8ml respectively, once a day for 21 days. After the last administration, the female rats were fasted for 12h, the serum of each group of female rats was collected by heart puncture, and the content of FSH (follicle stimulating hormone), LH (luteinizing hormone) and E2 (estradiol) in the serum was determined by using a kit, and the results are shown in table 21.

[0170] Table 21: detection results of serum hormone content of female rats in each group (n=10)

[0171]

[0172] Note: compared with the control group, *P<0.05, P<0.01

[0173] In this experiment, Kafali modeling method was used, and endocrine disorder model was established by gavage of letrozole (1 mg / kg·d, for 21 days) to female rats. After successful modeling, the serum estradiol (E2) level of female rats decreased significantly (P<0.05), which met the model standard. 30 female rats with successful modeling were randomly divided into small, medium and large dose groups, 10 rats in each group. The administration dose was 0.5, 1 and 2 times of the daily drug dose of female rats, once a day for 21 days.

[0174] As can be seen from Table 21, the small-dose group: the contents of FSH and LH in the serum of female rats are lower than those of the model group, but not significant; the content of E2 is significantly increased (P<0.05). The medium-dose group: the content of LH in the serum of female rats is significantly lower than that of the model group (P<0.05), and the contents of FSH and E2 are extremely significantly different from those of the model group (P<0.01). The large-dose group: the contents of FSH, LH and E2 in the serum of female rats are extremely significantly different from those of the model group (P<0.01). The contents of FSH and LH in the serum of female rats of the small-dose group are lower than those of the control group, but not significant, and the content of E2 is significantly increased (P<0.05); the content of LH in the serum of female rats of the medium-dose group is significantly lower than that of the control group (P<0.05), and the contents of FSH and E2 are extremely significantly different from those of the control group (P<0.01); the contents of FSH, LH and E2 in the serum of female rats of the large-dose group are extremely significantly different from those of the control group (P<0.01).

[0175] FSH (follicle-stimulating hormone) is one of the important hormones that regulate the female reproductive system, and its main function is to promote the development and maturation of follicles. High FSH levels can cause ovarian hyperstimulation, leading to irregular menstruation, ovarian cysts and other problems. As can be seen from Table 21, the fermented beverage of traditional Chinese medicine inoculated with Lactobacillus plantarum YXLP-116 of the present application can reduce FSH levels, helping to regulate the menstrual cycle of women and reduce the risk of ovarian hyperstimulation, thereby improving the reproductive health of women.

[0176] For women with polycystic ovary syndrome (PCOS) or irregular menstruation caused by high FSH levels, the fermented beverage of traditional Chinese medicine inoculated with Lactobacillus plantarum YXLP-116 of the present application has been shown through rat experiments to have a certain effect on reducing FSH (follicle-stimulating hormone) levels, helping to restore normal menstrual cycles and ovulation function.

[0177] LH (luteinizing hormone) is another important reproductive hormone, and its main function is to promote ovulation and the formation of corpus luteum. High LH levels can cause corpus luteum hyperfunction, leading to irregular menstruation, endometrial thickening and other problems. The fermented beverage of traditional Chinese medicine inoculated with Lactobacillus plantarum YXLP-116 of the present application can reduce LH levels, helping to regulate corpus luteum function and reduce the risk of corpus luteum hyperstimulation, thereby improving the reproductive health of women. For women with irregular menstruation, corpus luteum hyperfunction and other problems caused by high LH levels, the fermented beverage of traditional Chinese medicine inoculated with Lactobacillus plantarum YXLP-116 of the present application can help restore normal ovulation and corpus luteum function.

[0178] E2 (estradiol) is the main estrogen in women's body, which plays an important role in maintaining women's reproductive system function, skin health, bone health, etc. Low E2 levels can lead to menopausal symptoms such as hot flashes, night sweats, mood swings, dry skin, osteoporosis, etc. The Chinese medicine fermented beverage inoculated with Lactobacillus plantarum YXLP-116 of the present application can increase E2 levels, help alleviate menopausal symptoms, and improve women's overall health. For menopausal symptoms caused by low E2 levels, the Chinese medicine fermented beverage inoculated with Lactobacillus plantarum YXLP-116 of the present application may have certain auxiliary therapeutic effects, helping to relieve symptoms such as hot flashes, night sweats, mood swings, etc., while also having certain protective effects on skin and bone health.

[0179] The Chinese medicine fermented beverage inoculated with Lactobacillus plantarum YXLP-116 of the present application can simultaneously regulate the levels of FSH, LH and E2, helping to restore the balance of women's endocrine system and improve various symptoms caused by endocrine disorders. By regulating the levels of FSH and LH, the Chinese medicine fermented beverage inoculated with Lactobacillus plantarum YXLP-116 of the present application helps to improve women's reproductive health and reduce the occurrence of menstrual disorders, ovarian cysts, and hyperfunction of corpus luteum. By increasing E2 levels, the Chinese medicine fermented beverage inoculated with Lactobacillus plantarum YXLP-116 of the present application helps to alleviate menopausal symptoms and improve women's quality of life. According to the experimental results, the Chinese medicine fermented beverage inoculated with Lactobacillus plantarum YXLP-116 of the present application has high safety while regulating hormone levels, and has not shown obvious adverse reactions to cells and animal models.

[0180] The present application is not limited to the details of the above-described exemplary embodiments, but can be implemented in other specific forms without departing from the spirit or scope of the present application, which is apparent to those skilled in the art. Therefore, the embodiments of the present application are exemplary and non-limiting. The scope of the present application is defined by the appended claims, not the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present application. Any reference signs in the claims should not be considered as limiting the claims to which they relate.

Claims

1. A method for preparing a traditional Chinese medicine fermented beverage by using Lactiplantibacillus plantarum YXLP-116, characterized in that, The Lactiplantibacillus plantarum YXLP-116 has a preservation number of CGMCC NO: 65639. The method comprises the following steps: S1, mixing a traditional Chinese medicine extract, a ginseng concentrate and a pomegranate concentrate to obtain a mixed medicinal liquid; wherein the traditional Chinese medicine extract is prepared by water extraction of a traditional Chinese medicine composition comprising the following components in parts by weight: 1-4 parts of Poria cocos, 1-4 parts of Cinnamomum cassia, 1-4 parts of Tremella fuciformis, 0.5-3 parts of Glycyrrhiza uralensis, 0.5-3 parts of Astragalus membranaceus, 0.5-3 parts of Rosa rugosa, 0.5-3 parts of Lycium chinense and 0.1-5 parts of Zingiber officinale, 1-4 parts of Fructus Jujubae, and 0.5-1 parts of Canarium album; The mass ratio of the pomegranate concentrate, the ginseng concentrate and Poria cocos is (5-12):(1-5):(1-4); S2, sterilizing the mixed medicinal liquid and inoculating Lactiplantibacillus plantarum YXLP-116 to obtain a traditional Chinese medicine fermented beverage.

2. The method of claim 1, wherein: In S2, the inoculation amount of Lactiplantibacillus plantarum YXLP-116 is 0.5%-10% of the total volume of the fermentation liquid.

3. The method of claim 1, wherein: In S1, the water extraction process of the traditional Chinese medicine composition is as follows: mixing each component of the traditional Chinese medicine composition with water, heating and decocting, and then filtering to obtain a filtrate, which is the traditional Chinese medicine extract.

4. The method of claim 1, wherein: In S2, the fermentation time of the Lactiplantibacillus plantarum YXLP-116 is 30-48 hours.

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