Application of phytobacterium plantarum La10 in stable product for regulating estrogen secretion

Abstract

The invention discloses plant lactobacillus La10 and application thereof in regulating estrogen secretion stability and enhancing ovarian function, and belongs to the technical field of microorganisms. The plant lactobacillus La10 provided by the invention can effectively promote the conversion of free isoflavone (daidzein and genistein), so that the absorption and utilization of intestinal tracts are promoted; the composition has the advantages that the ovarian index and sex hormone estradiol E2 secretion level reduction caused by premature ovarian failure of rats can be obviously improved, and meanwhile, the estrogen secretion level and ovarian index up-regulation caused by mouse hysteromyoma can be improved; the irregular menstruation phenomenon of women is improved, so that the estrogen secretion disorder is regulated. The plant lactobacillus La10 disclosed by the invention has a very wide application range in preparation of functional foods and health-care foods for promoting estrogen balance and protecting female health, and is healthy and safe.

Description

Technical Field

[0001] The present invention relates to the field of microecological technology, and in particular to an application of Lactobacillus plantarum La10 (including its fermentation product) in a product for regulating the stable secretion of estrogen. Background Art

[0002] Estrogens include estradiol ( estradiol , E2), estrone, and estriol. Among them, estradiol has the largest secretion volume and the strongest activity, while estriol has the smallest activity, which is only one-two-hundredth of estradiol. These three hormones can be converted into each other in the human body and play an important regulatory role in human growth and development. The main functions of estrogen: (1) When the concentration of estrogen increases during puberty, it promotes the development of female sexual organs, endometrial hyperplasia, estrus, and menstruation; (2) At the same time, it promotes the appearance of female secondary sexual characteristics: breast development, subcutaneous fat accumulation, especially in the buttocks and breasts, pelvic widening, etc.; (3) Promote human metabolism, delay atherosclerosis, and prevent cardiovascular diseases; (4) Improve brain function, have a neuroprotective effect, and help prevent and delay Alzheimer's disease in women.

[0003] As probiotics in the human intestine, lactic acid bacteria can produce exclusive enzymes such as enzymes for organic acids, enzymes for synthesizing vitamins, and enzymes for breaking down fats and proteins. They can degrade proteins, lipids, sugars and other substances in food into a pre-digested state that is easier for the human body to absorb, giving them special biological functions.

[0004] Whether it is teenagers in adolescence, people in menopause, or even young people and the elderly, there are more or less estrogen secretion disorders. The reasons for this phenomenon may be the needs of human growth and development, ovarian dysfunction due to aging, bad living and eating habits, daily stress, anxiety, etc., which will cause serious harm to human health in the long run. When the human body lacks estrogen, it will lead to symptoms such as irregular menstruation, haggard complexion, back pain, hot flashes and night sweats, decreased libido, anxiety, palpitations, etc.; when the human body has an excess of estrogen, it will lead to symptoms such as irregular menstruation, acne, obesity, breast hyperplasia, infertility, fatigue, uterine fibroids, etc.

[0005] Therefore, how to regulate the stable secretion of estrogen and prevent and improve the occurrence and development of estrogen disorders in the human body has become an urgent problem to be solved in this field. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a plant lactobacillus La10, which can promote the transformation of soybean isoflavones from a bound state to a free state, thereby regulating the stable secretion of estrogen and helping to improve the estrogen disorder in the human body.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions: The first aspect of the present invention provides a plant lactobacillus, wherein the plant lactobacillus is plant lactobacillus ( Lactiplantibacillus plantarum )La10, deposited in Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on June 30, 2023, with the deposit number GDMCC No: 63605, and disclosed in the Chinese invention patent application with the publication date of December 22, 2023 and the publication number CN117264852A.

[0008] Plant lactobacillus is a type of Gram-positive lactobacillus, which is widely found in nature, especially distributed in various fermented foods, such as vegetables, meat, dairy products and wine. Plant lactobacillus is also present in the human gastrointestinal tract at the same time, and is a probiotic group in the human gastrointestinal tract, which has a great promoting effect on human health. Studies have shown that plant lactobacillus has multiple health effects such as regulating immunity, regulating chronic metabolic diseases, antagonizing pathogenic bacteria infection, regulating intestinal function, regulating mental and nervous functions, and can effectively regulate blood sugar, blood lipids, blood pressure balance, and has the effect of improving metabolic disorders. However, existing research has not found that plant lactobacillus has the effect of promoting soybean isoflavones to change into free state from the bound state, regulating the stable secretion of estrogen.

[0009] In the present invention, the Guangdong Provincial Microecological Preparation Engineering Technology Research Center of South China Agricultural University accidentally discovered a new strain of Lactobacillus plantarum during the experiment and named it Lactobacillus plantarum ( Lactiplantibacillus plants )La10. This strain has the function of promoting the conversion of soybean isoflavones from a bound state to a free state, which is beneficial for promoting the secretion of estrogen and maintaining balance.

[0010] The second aspect of the present invention provides a probiotic preparation, wherein the probiotic preparation contains the Lactobacillus plantarum.

[0011] Furthermore, the microecological preparation includes but is not limited to bacterial suspension and fermentation broth.

[0012] The third aspect of the present invention provides a fermented product for regulating estrogen secretion, wherein the fermented product is obtained by inoculating the plant lactobacillus into a composition and fermenting the mixture; The composition comprises the following components by weight: 35-50 parts of soybeans, 10-20 parts of black sesame seeds, 10-20 parts of red dates, 5-13 parts of kudzu root, 1-6 parts of rehmannia root, 16-25 parts of monohydrate glucose and 333-360 parts of water.

[0013] Soybeans are the legumes with the highest content of soy isoflavones, which is as high as 128mg / 100g. Soy isoflavones are a natural estrogen receptor modulator. Their biologically active form is aglycones with weak estrogen-like activity. They can bind to estrogen receptor subtypes (ERα and ERβ) to exert the dual effects of estrogen and anti-estrogen. That is, when the estrogen content in the human body is high, soy isoflavones exhibit anti-estrogen activity; when the estrogen content in the human body is low, they exhibit estrogen-like activity.

[0014] Black sesame is a grain that can be used as both medicine and food. It is rich in lipids, proteins, vitamins and minerals, and has been considered a good product for health care since ancient times. The vitamin E content is extremely high, about 50.4 mg / 100 g, which can quickly promote early follicular development by regulating the sensitivity of follicles to exogenous gonadotropins, reduce follicular damage, reduce the number of follicle atresia, and help enhance ovarian function.

[0015] Insufficient Qi and blood can lead to symptoms of upper heat and lower cold, and heart-kidney disharmony, which in turn leads to disorder of the heart-kidney-uterus axis. The heart Qi does not descend and the uterine vessels are blocked. The lower cold condenses the Qi and blood in the uterine vessels, which makes the uterine vessels lose moisture and warmth, and the ovarian function decreases, which in turn affects women's menstruation and reproduction. Red dates contain a lot of iron, which can replenish Qi and blood, and help the ovaries return to a healthy state.

[0016] There are many types of active ingredients in Pueraria root, mainly including flavonoids, triterpenes and saponins, coumarins, organic acids, polysaccharides and alkaloids. Among them, flavonoids have the highest content. Most flavonoids are isoflavone derivatives, which are their characteristic components. The total isoflavone mass fraction can reach more than 8%, including puerarin, soybean flavonoids, genistein and its C- and O-glycoside derivatives (such as O-glycoside puerarin, genistein, daidzin and malonyl daidzin, etc.) More than 40 kinds of isoflavones, puerarin has phytoestrogen effect, which can regulate serum estradiol ( estradiol , E2) and estrogen receptor α ( estrogen receptor-α It can improve uterine function by reducing the expression of ER-α protein, and can play the dual role of estrogen and anti-estrogen depending on the dosage. It has a significant improvement effect on premature ovarian failure, oligomenorrhea, and various symptoms before and after menopause, and has no obvious toxicity to embryonic development.

[0017] Rehmannia root has the effects of nourishing blood and yin, replenishing essence and filling marrow. Jingyuequanshu believes that Rehmannia root is "the first pure and thick medicine among essence, blood, form and quality". Traditional Chinese medicine believes that kidney stores essence and controls reproduction, and the development of eggs is closely related to kidney. Rehmannia root is an important medicine for nourishing yin and tonifying kidney. It is often used clinically to promote the secretion of hormones in female ovaries, stimulate the growth and development of follicles and discharge, which is beneficial to delay ovarian aging.

[0018] The composition provided by the present invention contains a variety of active ingredients such as rich soy isoflavones, natural phytoestrogens such as puerarin, vitamin E, polysaccharides, etc., which not only has dual regulatory effects such as estrogen and anti-estrogen, but also can invigorate qi and blood, thereby enhancing ovarian function and delaying ovarian aging. The present invention uses soybeans, black sesame, red dates, kudzu root and Rehmannia glutinosa as fermentation raw materials in a specific ratio, and adopts plant lactobacillus La10 for in vitro fermentation, which can directly carry out biotransformation on the active ingredients therein, and convert them into free molecular forms that are easily absorbed by the human body; and the fermented product contains a large amount of active probiotics that are beneficial to the human body. After eating the fermented product, weak people with ovarian sub-health, qi and blood deficiency, ovarian function decline, etc. can absorb and utilize the nutrients therein in time, restore and enhance ovarian function; at the same time, after the active strain colonizes the intestine, it can also continue to play a role in regulating estrogen secretion and improve the situation of human estrogen disorder.

[0019] In the above composition, the weight portion of soybean is 35 to 50 parts, for example, it can be 35 parts, 36 parts, 37 parts, 38 parts, 39 parts, 40 parts, 41 parts, 42 parts, 43 parts, 44 parts, 45 parts, 46 parts, 47 parts, 48 ​​parts, 49 parts, 50 parts, etc. The weight portion of black sesame is 10 to 20 parts, for example, it can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, etc. The weight portion of red dates is 10 to 20 parts, for example, it can be 10 parts, 11 parts, 12 parts, 13 parts, 14 parts, 15 parts, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, etc. The weight portion of Puerariae Radix is ​​5 to 13 parts, for example, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, 11 parts, 12 parts, 13 parts, etc. The weight portion of Rehmannia glutinosa is 1 to 6 parts, for example, 1 part, 2 parts, 3 parts, 4 parts, 5 parts, 6 parts, etc. The weight portion of monohydrate glucose is 16 to 25 parts, for example, 16 parts, 17 parts, 18 parts, 19 parts, 20 parts, 21 parts, 22 parts, 23 parts, 24 parts, 25 parts, etc. The weight portion of water is 333 to 360 parts, for example, 333 parts, 340 parts, 345 parts, 350 parts, 355 parts, 360 parts, etc.

[0020] Furthermore, in a preferred embodiment, the composition comprises the following components by weight: 35 parts of soybeans, 20 parts of black sesame seeds, 10 parts of red dates, 13 parts of kudzu root, 6 parts of rehmannia root, 16 parts of monohydrate glucose, and 333 parts of water.

[0021] The fourth aspect of the present invention provides a method for preparing a fermented product for regulating estrogen secretion, comprising the following steps: S1. The formula amount of soybeans, black sesame and red dates mixed evenly, add water to break the wall; then the broken wall liquid was boiled to obtain a liquid A; S2. The amount of Pueraria and Rehmannia glutinosa was mixed evenly, boiled with water; then concentrated under reduced pressure to obtain liquid B; S3. Mix solution A and solution B, add the formulated amount of glucose monohydrate to dissolve; then filter and sterilize to obtain solution C; S4. Adding the bacterial liquid of Lactobacillus plantarum La10 to the liquid C for anaerobic culture; then taking the fermentation liquid, and freeze-drying it in vacuum to obtain the fermentation product.

[0022] In the above step S1, when preparing liquid A, the amount of water added is preferably 200 parts by weight, the rotation speed of the cell wall breaking treatment is preferably 28000 r / min, the cell wall breaking time is preferably 2 min, and the boiling time is preferably 20 min.

[0023] In the above step S2, when preparing liquid B, the amount of water added is preferably 133 parts by weight; after boiling, it is preferably kept at 95°C for 45 minutes. The conditions for reduced pressure concentration are preferably: under the conditions of -0.10 MPa, 70°C, and at a speed of 18 r / min.

[0024] In the above step S3, when preparing liquid C, a 100-mesh filter is preferably used for filtration; the sterilization conditions are: sterilization at 121° C. for 15 min.

[0025] Furthermore, in step S4, the amount of the bacterial solution added is 5 to 10 parts by weight, for example, 5 parts, 6 parts, 7 parts, 8 parts, 9 parts, 10 parts, etc., preferably 6 parts. The concentration of live bacteria in the bacterial solution is preferably 4×10 10 CFU / mL.

[0026] The fifth aspect of the present invention provides the use of the plant lactobacillus, the microecological preparation, and the fermentation product in the preparation of a preparation for promoting the conversion of soy isoflavones from a bound state to a free state.

[0027] The sixth aspect of the present invention provides the use of the plant lactobacillus, the microecological preparation, and the fermentation product in the preparation of a preparation for regulating estrogen secretion.

[0028] Furthermore, the plant lactobacillus includes but is not limited to live bacteria type and sterilized type.

[0029] Furthermore, the preparation includes but is not limited to food, functional food and health food.

[0030] Compared with the prior art, the present invention has the following beneficial effects: The plant lactobacillus La10 provided by the present invention can promote the transformation of soybean isoflavones from a bound state to a free state, which is beneficial to regulating the secretion of estrogen to maintain balance and improve the estrogen disorder of the human body.

[0031] The present invention uses Lactobacillus plantarum La10 to ferment a composition including soybeans, black sesame seeds, red dates, kudzu root and Rehmannia glutinosa, which can directly bioconvert the active ingredients into free molecular forms that are easily absorbed by the human body, and the obtained fermented products contain rich active probiotics. After eating the fermented products, weak people with ovarian sub-health, qi and blood deficiency, ovarian function decline, etc. can absorb and utilize the nutrients in them in time to restore and enhance ovarian function; at the same time, after the active strains colonize the intestines, they can continue to play a role in regulating estrogen secretion and improve the estrogen disorder of the human body. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0033] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0034] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are all conventional methods, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0035] Strain screening: Cut fresh kelp into pieces, take an appropriate amount and culture it in MRS liquid medium at 37℃ for 48h, take appropriate bacterial liquid and spread it on MRS plate (add 0.05% cysteine ​​hydrochloride and 100 mg / L mupirocin), and culture it in 37℃ anaerobic workstation for 72h. After a single colony grows, pick a colony to continue streaking and purification, and finally obtain pure culture. The pure culture is identified by third-generation 16S amplicon sequencing analysis, and the strain is determined to be Lactobacillus plantarum, which is named La10.

[0036] Gene sequence identification results:

[0037] Test Example 1: Soybean Isoflavone Conversion Experiment Test 1. Fermentation broth preparation (1) Stock solution: Wash dry, insect-free, and non-corrupted soybeans with clean water, drain the water, weigh 30 parts, add 150 parts of purified water, break the soybean wall at 28,000 r / min for 2 min, weigh, boil for 20 min, weigh, make up the weight with purified water, add 10 parts of glucose monohydrate, stir to dissolve, sterilize at 121°C for 15 min, and cool to 37°C.

[0038] (2) Bacterial liquid: After thawing the freeze-dried tube of Lactobacillus plantarum La10, take 0.4 μL of the bacterial liquid and spread it on MRS solid medium. Incubate anaerobically at 37°C for 48 h. Pick 2 loops of colonies and add them to 10 mL of liquid C. Incubate anaerobically at 37°C for 18 h. Adjust the viable bacterial count to 4 × 10 10 CFU / mL.

[0039] (3) Fermentation broth A: Take 0 parts of the bacterial solution and add it to the original solution, mix well, incubate anaerobically at 37℃ for 18 hours, and then refrigerate at 4℃ for 10 hours.

[0040] (4) Fermentation liquid B: Take 3 portions of bacterial liquid and add them to the original liquid, mix well, incubate anaerobically at 37℃ for 18 hours, and then refrigerate at 4℃ for 10 hours.

[0041] (5) Fermentation broth C: Take 6 portions of the bacterial solution and add them to the original solution. Mix well and incubate anaerobically at 37°C for 18 h. Then, refrigerate at 4°C for 10 h.

[0042] (6) Fermentation broth D: Take 9 portions of the bacterial solution and add them to the original solution. Mix well and incubate anaerobically at 37°C for 18 h. Then, refrigerate at 4°C for 10 h.

[0043] (7) Postbiotic E: Fermentation broth C was heat-inactivated by keeping it at 65°C for 45 min and then at 85°C for 15 min.

[0044] (8) The original solution was freeze-dried in vacuum (freeze-drying parameters were -80°C pre-freezing for 12 h and -70°C drying for 24 h), crushed to 80 mesh using a grinder, and sieved. The resulting product was sample F.

[0045] (9) After the fermentation broths A to D were freeze-dried in vacuum (freeze-drying parameters were -80°C pre-freezing for 12 h and -70°C drying for 24 h), they were crushed to 80 mesh using a grinder and sieved. The resulting products were fermentation products A to D.

[0046] (10) After vacuum freeze-drying (freeze-drying parameters are -80°C pre-freezing for 12 h and -70°C drying for 24 h), postbiotic E is crushed to 80 mesh using a grinder and sieved. The resulting product is postbiotic product E.

[0047] 2. According to GB / T 26625 Grain and Oil Inspection - Determination of Soybean Isoflavones - High Performance Liquid Chromatography, the soy isoflavones content of sample F, fermented products A to D, and postbiotic product E were tested. The results are shown in Table 1.

[0048] 3. Results and analysis

[0049] Daidzein and genistin are bound soy isoflavones, while daidzein and genistein are free soy isoflavones. Since the absorption rate and amount of free soy isoflavones in the human intestine are much greater than those in the bound soy isoflavones, soy isoflavones can only be effectively absorbed and utilized in the human intestine to play a role in regulating estrogen if they are converted from bound to free.

[0050] It can be seen from Table 1 that the sum of the contents of daidzein and genistin in sample F is much greater than the sum of the contents of daidzein and genistein, indicating that before fermentation, soy isoflavones mainly exist in a bound state.

[0051] Compared with sample F, the contents of daidzein, genistin, daidzein and genistein in fermented product A were slightly reduced, but there was basically no significant change, indicating that the bacterial solution of Lactobacillus plantarum La10 was not added for fermentation and the soy isoflavones were not converted into a free state.

[0052] Compared with sample F, the content of daidzein and genistin in fermented products B to D was significantly reduced, and the content of daidzein and genistein was significantly increased, indicating that after fermentation with the bacterial solution of Lactobacillus plantarum La10, soy isoflavones gradually changed from a bound state to a free state. Among them, the content of daidzein and genistin in group D was the lowest, and the content of daidzein and genistein was the highest, and there was no significant difference with group C, indicating that the amount of bacterial solution added by Lactobacillus plantarum La10 in group C made the fermentation process reach a saturated state, and the amount of bacterial solution added by group C could fully transform the soy isoflavones in the stock solution from a bound state to a free state. After human consumption, it can be efficiently absorbed and utilized, which is beneficial to regulate the balance of estrogen secretion.

[0053] Compared with group C, the contents of daidzein, genistin, daidzein and genistein in postbiotic product E were slightly reduced, but there was basically no significant change, indicating that after heat inactivation treatment, the soy isoflavones in the fermented products still exist mainly in the free state, which is also beneficial to regulating the balance of estrogen secretion after human consumption. Example 1

[0054] This embodiment provides a method for preparing a fermented product for regulating the stable secretion of estrogen, comprising the following steps: S1. Select dry, insect-free, non-corrupted and non-deteriorated soybeans and red dates, and select qualified quality black sesame seeds, kudzu root, rehmannia root and monohydrate glucose.

[0055] S2. Wash soybeans, red dates, black sesame, kudzu root and Rehmannia root with clean water, drain, remove the core of red dates and take out the flesh, cut the kudzu root into 3×3 cm blocks, weigh 35 parts of soybeans, 20 parts of black sesame, 10 parts of red dates, 13 parts of kudzu root and 6 parts of Rehmannia root.

[0056] S3, Liquid A: After soybeans, black sesame seeds and red dates are evenly mixed, add 200 parts of purified water, break the wall at 28000r / min for 2min, weigh, boil for 20min, weigh, make up the weight with purified water, and stir evenly.

[0057] S4, Liquid B: After Pueraria root and Rehmannia root are evenly mixed, 133 parts of purified water are added, weighed, boiled, maintained at 95℃ for 45min, and concentrated under reduced pressure at -0.10MPa, 70℃, and 18r / min to the original weight.

[0058] S5. Liquid C: Mix Liquid A and Liquid B evenly, add 16 parts of glucose monohydrate and stir to dissolve, filter through a 100-mesh filter, take the filtrate, sterilize at 121°C for 15 min, and cool to 37°C.

[0059] S6. Bacterial solution: After thawing the freeze-dried storage tube of Lactobacillus plantarum La10, take 0.4 μL of bacterial solution and spread it on MRS solid medium. Incubate anaerobically at 37°C for 48 h. Pick 2 loops of colonies and add them to 10 mL of C solution. Incubate anaerobically at 37°C for 18 h. Adjust the viable bacterial count to 4×10 10 CFU / mL.

[0060] S7, fermentation liquid: take 6 portions of bacterial liquid and add them into liquid C, mix well, culture anaerobically at 37℃ for 18h, and then refrigerate at 4℃ for 10h.

[0061] S8. After the fermentation liquid is freeze-dried in vacuum (freeze-drying parameters are -80°C pre-freezing for 12 hours and -70°C drying for 24 hours), it is crushed into 80 meshes using a grinder and sieved. The resulting product is the fermentation product.

[0062] Test Example 2: Ovarian dysfunction model rat test

[0063] 1. Sample preparation Weigh 5 g of the fermented product prepared in Example 1, add 50 mL, stir and dissolve, as Group 1; weigh 10 g of the fermented product prepared in Example 1, add 50 mL, stir and dissolve, as Group 2; weigh 15 g of the fermented product prepared in Example 1, add 50 mL, stir and dissolve, as Group 3.

[0064] 2. Conjugated estrogen solution: Grind the conjugated estrogen tablets into powder, dissolve in purified water to prepare a solution with a concentration of 0.075 mg / mL, and store in a refrigerator at 4°C for later use.

[0065] 3. Ten-week-old female SD rats were randomly divided into 6 groups, 8 rats in each group, numbered as group A to group F. After one week of adaptive feeding, rats in group B to group F were intraperitoneally injected with cyclophosphamide (60 mg / kg) for 1 d + cyclophosphamide (10 mg / kg) for 14 d to establish the induced premature ovarian failure model. Group A was intraperitoneally injected with an equal amount of normal saline. During the feeding period, all rats had free access to food and water.

[0066] 4. After modeling, all rats were gavaged with drugs. The specific administration method was shown in Table 2, and the drugs were continuously gavaged for 28 days.

[0067]

[0068] 5. Record the changes in rat body weight every week. After the last administration, the rats were fasted for 12 hours and anesthetized with isoflurane inhalation. Blood was collected from the abdominal aorta into a coagulation tube and centrifuged at 3000 r / min for 15 min at 4°C. The supernatant was taken and the concentration of estradiol (E2) in rat serum was detected using a rat serum estradiol Elisa detection kit. The average group was calculated and the results are shown in Table 3. The ovaries were quickly removed, all adipose tissue was removed, and then washed with saline, dried with filter paper, and weighed on an analytical balance. The ovarian index was calculated and the average group was calculated. The results are shown in Table 3.

[0069]

[0070] 6. Results and Analysis

[0071] The ovarian index is an indicator that directly reflects the status of the ovaries. Premature ovarian failure will cause a decrease in serum E2 levels, which is also the most common diagnostic indicator of premature ovarian failure.

[0072] As shown in Table 3, compared with group A, the ovarian index and serum E2 content of rats in group B were significantly reduced, indicating that the cyclophosphamide-induced premature ovarian failure model rats were successfully established.

[0073] Compared with group B, the ovarian index and serum E2 content of rats in groups D to F were all increased, indicating that the fermented product of Example 1 has a regulatory effect on the estrogen secretion of mice, can promote estrogen secretion, and improve the symptoms of premature ovarian failure. Among them, the ovarian index and serum E2 content of rats in group F were the highest, and were relatively close to those of group A, indicating that group 3 had the best effect on regulating estrogen secretion in rats with premature ovarian failure.

[0074] Test Example 3: Uterine fibroids model mouse experiment

[0075] 1. Sample preparation Weigh 5 g of the fermented product prepared in Example 1, add 50 mL, stir and dissolve, as Group 1; weigh 10 g of the fermented product prepared in Example 1, add 50 mL, stir and dissolve, as Group 2; weigh 15 g of the fermented product prepared in Example 1, add 50 mL, stir and dissolve, as Group 3.

[0076] 2. Guizhi Fuling solution: Take out the inner capsule powder of Guizhi Fuling capsule, dissolve it with purified water to prepare a solution with a concentration of 0.47g / mL, and store it in a refrigerator at 4℃ for later use.

[0077] 3. 8-week-old female ICR mice were randomly divided into 6 groups, 8 mice in each group, numbered as G to L. After one week of adaptive feeding, mice in groups H to L were injected intramuscularly with estradiol benzoate (2 ml of estradiol benzoate injection was mixed with 2 ml of Tween 80, and then mixed with normal saline to make 100 ml) once a day for 15 consecutive days, with each injection volume of 0.1 ml / 10 g, to establish an induced uterine fibroids model. Group G was injected intramuscularly with an equal amount of normal saline. During the feeding period, all mice had free access to food and water.

[0078] 4. After modeling, all mice were gavaged with drugs. The specific administration method is shown in Table 4, and gavage was continued for 21 days.

[0079]

[0080] 5. Record the weight changes of mice every week. After the last administration, mice were fasted for 12 hours and anesthetized with isoflurane inhalation. Blood was collected from the abdominal aorta into a coagulation tube and centrifuged at 3000 r / min for 15 min at 4°C. The supernatant was taken and the concentration of estradiol (E2) in the mouse serum was detected using a mouse estradiol Elisa detection kit. The average group was calculated and the results are shown in Table 5. The ovaries were quickly removed, all adipose tissue was removed, and then washed with physiological saline, dried with filter paper, and weighed on an analytical balance. The ovarian index was calculated and the average group was calculated. The results are shown in Table 5.

[0081] 6. Results and Analysis

[0082] As shown in Table 5, compared with group G, the ovarian index and serum E2 content of mice in group H were significantly increased, indicating that the uterine fibroids model mouse model induced by estradiol benzoate was successfully established.

[0083] Compared with group H, the ovarian index and serum E2 content of mice in groups J to L were all reduced, indicating that the fermented product of Example 1 has a regulatory effect on the estrogen secretion of mice, can reduce estrogen secretion, and improve the symptoms of estrogen secretion disorder caused by uterine fibroids. The ovarian index and serum E2 content of mice in group L were the lowest, and were relatively close to those of group G, indicating that group 3 had the best effect on regulating the estrogen secretion of mice with uterine fibroids and had an anti-estrogen effect.

[0084] Comparative Examples 1 to 7

[0085] The preparation methods of the fermented products of Comparative Examples 1 to 7 are the same as those of the examples, except that the composition ratios of the fermented products are different. The specific composition ratios of the fermentation raw materials are shown in Table 6.

[0086]

[0087] Example 2 This embodiment provides a method for preparing a solid preparation, comprising the following steps: S1. After thawing the freeze-dried storage tube of Lactobacillus plantarum La10, 0.4 μL of bacterial solution was spread on MRS solid medium and cultured anaerobically at 37°C for 48 h. Two loops of colonies were picked and added to 10 mL of MRS liquid medium and cultured anaerobically at 37°C for 18 h. The number of viable bacteria was adjusted to 4 × 10 10 CFU / mL, and the activated bacterial solution is obtained.

[0088] S2. Take 6 portions of activated bacterial solution and add them into MRS liquid culture medium, mix them evenly, culture them anaerobically at 37°C for 18 hours, centrifuge them, take the precipitate, and mix it evenly with the freeze-dried protective agent to obtain bacterial solution No. 1.

[0089] S3, No. 1 bacterial solution was vacuum freeze-dried (freeze-drying parameters were -80°C pre-freezing for 12 hours and -70°C drying for 24 hours), crushed to 80 mesh with a grinder, and sieved. The obtained product was the live bacteria type Lactobacillus plantarum La10 bacterial powder.

[0090] S4. Take 10 parts of live bacteria type Lactobacillus plantarum La10 powder and mix them evenly with 90 parts of oligofructose to obtain solid preparation No. 1. Example 3

[0091] This embodiment provides a method for preparing a solid preparation, comprising the following steps: S1. After thawing the freeze-dried storage tube of Lactobacillus plantarum La10, 0.4 μL of bacterial solution was spread on MRS solid medium and cultured anaerobically at 37°C for 48 h. Two loops of colonies were picked and added to 10 mL of MRS liquid medium and cultured anaerobically at 37°C for 18 h. The number of viable bacteria was adjusted to 4 × 10 10 CFU / mL, and the activated bacterial solution is obtained.

[0092] S2. Take 6 portions of activated bacterial solution and add them into MRS liquid culture medium, mix them evenly, culture them anaerobically at 37°C for 18 hours, centrifuge them, take the precipitate, and mix it evenly with the freeze-dried protective agent to obtain bacterial solution No. 1.

[0093] S3, No. 1 bacterial solution was vacuum freeze-dried (freeze-drying parameters were -80°C pre-freezing for 12 h, -70°C drying for 24 h), crushed to 80 mesh using a grinder, and sieved, and the obtained product was Lactobacillus plantarum La10 bacterial powder.

[0094] S4. Keep the Lactobacillus plantarum La10 powder at 65° C. for 50 min and at 78° C. for 18 min, then grind it into 80 meshes with a grinder, and sieve it. The obtained product is the inactivated Lactobacillus plantarum La10 powder.

[0095] S5. Take 10 parts of inactivated Lactobacillus plantarum La10 powder and mix them evenly with 90 parts of oligofructose to obtain solid preparation No. 2.

[0096] Test case 4: Crowd test

[0097] 1. Recruit 80 female volunteers aged 25 to 40, all with irregular menstruation lasting 2 to 4 months and BMI index 21 to 24 kg / m 2 of people.

[0098] Diagnostic criteria: Meet the relevant standards of "Research on Traditional Chinese Medicine Differentiation and Treatment of Gynecological Menstrual Disorders".

[0099] (1) The menstrual cycle is shortened by more than 7 days, or even once every 15 days, or the menstrual cycle is prolonged by more than 35 days, showing irregular menstruation such as early or delayed menstruation by more than 7 days; (2) Menstrual flow increases significantly (>80 mL) or decreases (<20 mL), or even spotting and then stopping; (3) Or symptoms such as abdominal pain, dizziness, fatigue, anemia, soreness of waist and knees, breast tenderness, amenorrhea, and irritability during menstruation may occur; (4) The main symptoms are premature menstruation, menorrhagia, prolonged menstruation, and light menstrual blood. Secondary symptoms are pale face, dry mouth, chills, excessive sweating, fatigue, shortness of voice, dizziness, and a stringy pulse.

[0100] Inclusion criteria: (1) aged 25-40 years, with complete clinical data, good general condition, no serious underlying diseases, no serious organ lesions, and able to tolerate routine examinations and treatment; (2) clear consciousness and able to accurately describe the condition; (3) no treatment for menstrual disorders caused by qi deficiency and blood stasis; (4) patients and their families were aware of the study and agreed.

[0101] Exclusion criteria: (1) Allergy to the drugs used in the study, history of severe gynecological diseases, history of vascular and nerve damage, malignant tumors or other congenital diseases; ② The use of immunosuppressants and other immune system diseases; ③ Withdrawal or refusal to cooperate with the study before the end of the study.

[0102] 2. The volunteers were randomly divided into 10 groups, 8 people in each group, and the grouping is shown in Table 7. Each group consumed the corresponding fermented product, half an hour after meals, 3 times a day, 2g each time, mixed with 37℃, 100mL warm water and drank for 60 days.

[0103]

[0104] 3. On the 61st day, the number of volunteers with marked efficacy, improvement, and ineffectiveness were counted. The results are shown in Table 8.

[0105] The improvement effect was evaluated based on the New Edition of TCM Gynecology. The patient's symptoms (abdominal pain, backache, etc.) disappeared, abnormal menstruation returned to normal, and there was no recurrence within 3 months, which was considered as markedly effective; clinical symptoms were significantly relieved, abnormal menstruation was significantly improved, but recurred within 3 months, which was considered as improvement; those that did not meet the above criteria were considered as ineffective.

[0106] 4. At 8:00 a.m. on the 1st day and the 61st day, 5 mL of cubital venous blood was drawn from the volunteers under fasting conditions. After centrifugation for 15 min (4°C, 3000 r / min), the supernatant was collected and the serum estradiol (E2) content was tested using the estradiol Elisa test kit. The average value was calculated and the results are shown in Table 8.

[0107] 5. Results and Analysis

[0108] As shown in Table 8, different groups of fermented products have different degrees of improvement on the volunteers' menstrual irregular symptoms. Among groups A to H, group H has the largest number of effective patients, indicating that the fermented product of Example 1 has the best effect on improving menstrual irregularities and regulating the balance of estrogen secretion, and can improve the disorder of estrogen secretion.

[0109] In terms of estrogen content, compared with the first day, the estrogen content in the serum of the volunteers increased significantly after consuming the fermented products for 60 days, indicating that the fermented products of different groups are effective in regulating estrogen secretion. The solid preparation containing live / inactivated Lactobacillus plantarum La10 is also effective in regulating estrogen secretion. Among them, the estrogen content of the volunteers in Group H was the highest on the 61st day, which further illustrates that the fermented product of Example 1 is best effective in regulating the balance of estrogen secretion.

[0110] In summary, the Lactobacillus plantarum La10 and its fermented product provided by the present invention can promote the transformation of soybean isoflavones from a bound state to a free state, thereby regulating the stable secretion of estrogen, which is beneficial to improving the estrogen disorder in the human body.

[0111] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. Use of Lactobacillus plantarum or its fermented product in products for promoting the conversion of soy isoflavones from bound state to free state and / or regulating the stable secretion of estrogen, characterized in that: The Lactobacillus plantarum is Lactobacillus plantarum La10, and its preservation number is GDMCC No: 63605.

2. The use according to claim 1, characterized in that: The Lactobacillus plantarum La10 is a live bacteria type or a sterilized type.

3. The use according to claim 1, characterized in that: The products include food, functional food and health food.

4. The use according to claim 1, characterized in that: The fermented product is obtained by inoculating Lactobacillus plantarum La10 into the composition and fermenting it; The composition comprises the following components by weight: 35-50 parts of soybeans, 10-20 parts of black sesame seeds, 10-20 parts of red dates, 5-13 parts of kudzu root, 1-6 parts of rehmannia root, 16-25 parts of monohydrate glucose and 333-360 parts of water.

5. The use according to claim 4, characterized in that: The composition comprises the following components by weight: 35 parts of soybeans, 20 parts of black sesame seeds, 10 parts of red dates, 13 parts of kudzu root, 6 parts of rehmannia root, 16 parts of monohydrate glucose and 333 parts of water.

6. The use according to claim 4, characterized in that: The method for preparing the fermented product comprises the following steps: S1. The formula amount of soybeans, black sesame and red dates mixed evenly, add water to break the wall; then the broken wall liquid was boiled to obtain a liquid A; S2. The amount of Pueraria and Rehmannia glutinosa was mixed evenly, boiled with water; then concentrated under reduced pressure to obtain liquid B; S3. Mix solution A and solution B, add the formulated amount of glucose monohydrate to dissolve; then filter and sterilize to obtain solution C; S4. Adding the bacterial liquid of Lactobacillus plantarum La10 to the liquid C for anaerobic culture; then taking the fermentation liquid, and freeze-drying it in vacuum to obtain the fermentation product.

7. The use according to claim 6, characterized in that: In step S4, the amount of the bacterial solution added is 5-10 parts by weight, and the concentration of live bacteria in the bacterial solution is 4×10 10 CFU / mL.

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