Application of Lactobacillus plantarum La10 in products for regulating estrogen secretion

Abstract

The present invention discloses Lactobacillus plantarum La10 and its use in regulating estrogen secretion and enhancing ovarian function, belonging to the field of microbial technology. The Lactobacillus plantarum La10 provided by the present invention can effectively promote the conversion of free isoflavones (daidzein and genistein), thereby promoting intestinal absorption and utilization. It also has dual estrogen-like and anti-estrogen effects, specifically: it can significantly improve the decline in ovarian index and secretion of the sex hormone estradiol (E2) caused by premature ovarian failure in rats, while also improving the increase in estrogen secretion and ovarian index caused by uterine fibroids in mice. It also improves menstrual irregularities in women and regulates estrogen secretion disorders.

Description

Technical Field

[0001] The present invention relates to the field of microecological technology, and in particular to application of Lactobacillus plantarum La10 in products for regulating the stable secretion of estrogen. Background Art

[0002] Estrogens include estradiol (E2), estrone, and estriol. Among them, estradiol is secreted in the largest amount and has the strongest activity, while estriol has the lowest activity, only 2 / 100 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 are: (1) When estrogen concentration 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, and pelvic widening; (3) It promotes human metabolism, delays atherosclerosis, and prevents cardiovascular disease; (4) It improves brain function and has a neuroprotective effect, which helps 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's teenagers in adolescence, people in menopause, or even young people and the elderly, all have 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, unhealthy 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, and palpitations; 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 conversion of soy isoflavones from a bound state to a free state, thereby regulating the stable secretion of estrogen and improving the estrogen disorder of the human body.

[0007] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0008] A first aspect of the present invention provides a plant lactobacillus, which is Lactiplantibacillus plantarum La10, deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on June 30, 2023, with a deposit number of GDMCC No: 63605, and disclosed in a Chinese invention patent application with a publication date of December 22, 2023 and publication number CN117264852A.

[0009] Plant lactobacillus is a type of Gram-positive lactobacillus, widely exists in nature, and is especially distributed in all kinds of fermented foods, such as vegetables, meat, dairy products and wine etc. Plant lactobacillus is also present in the human gastrointestinal tract simultaneously, is the probiotic group in the human gastrointestinal tract, and has very large promoting effect on human health. Research shows 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, can effectively regulate blood sugar, blood fat, blood pressure balance, and has the effect of improving metabolic disorders. But existing research has not found that plant lactobacillus has the effect of promoting soy isoflavones to change into free state from bound state, regulating estrogen secretion steadily.

[0010] In this invention, the Guangdong Provincial Microecological Preparation Engineering Technology Research Center of South China Agricultural University accidentally discovered a new strain of Lactobacillus plantarum during experiments and named it Lactiplantibacillus plantarum La10. This strain has the ability to promote the conversion of soy isoflavones from bound to free forms, which helps promote balanced estrogen secretion.

[0011] The second aspect of the present invention provides a probiotic preparation, which contains the Lactobacillus plantarum.

[0012] Furthermore, the microecological preparation includes but is not limited to bacterial suspension and fermentation broth.

[0013] The third aspect of the present invention provides a fermented product for regulating estrogen secretion, wherein the fermented product is obtained by inoculating the plantarum bacteria into a composition and fermenting the mixture.

[0014] The composition comprises the following components in parts 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 prepared rehmannia root, 16-25 parts of monohydrate glucose, and 333-360 parts of water.

[0015] Soybeans are the legumes with the highest content of soy isoflavones, with a content of up to 128mg / 100g. Soy isoflavones are a natural estrogen receptor modulator. Their biologically active form is aglycone with weak estrogen-like activity. They can exert the dual effects of estrogen and anti-estrogen by binding to estrogen receptor subtypes (ERα and ERβ). 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.

[0016] Black sesame, a grain used for both medicinal and edible purposes, is rich in lipids, protein, vitamins, and minerals, and has been considered a health supplement since ancient times. It contains a high vitamin E content of approximately 50.4 mg / 100 g. This vitamin E can regulate follicles' sensitivity to exogenous gonadotropins, promoting early follicular development, minimizing follicular damage, and reducing the number of blocked follicles, thereby enhancing ovarian function.

[0017] Deficiency of Qi and blood can lead to symptoms of upper heat and lower cold, a condition known as heart-kidney disharmony. This disharmony can disrupt the heart-kidney-uterus axis. Heart Qi fails to descend, blocking the uterine vessels. Lower cold congests Qi and blood in the uterine vessels, depriving them of moisture and warmth, leading to decreased ovarian function and potentially affecting menstruation and reproduction. Red dates contain a high amount of iron, which can replenish Qi and blood, helping to restore ovarian health.

[0018] Pueraria root contains a wide variety of active ingredients, primarily flavonoids, triterpenes, saponins, coumarins, organic acids, polysaccharides, and alkaloids. Flavonoids are the most abundant, and most are isoflavone derivatives, representing over 8% of the root's total mass fraction. These compounds include over 40 isoflavones, including puerarin, daidzein, genistein, and its C- and O-glycoside derivatives (e.g., O-glycoside puerarin, genistin, daidzin, and malonyldaidzin). Puerarin has phytoestrogen properties, improving uterine function by regulating serum estradiol (E2) and estrogen receptor-α (ER-α) protein expression. Depending on the dosage, it can exert both estrogen-like and anti-estrogen effects, significantly improving premature ovarian failure, oligomenorrhea, and perimenopausal symptoms, while maintaining no significant toxicity to embryonic development.

[0019] Rehmannia root (Shengdihuang) has the benefits of nourishing blood and yin, replenishing essence and filling the marrow. The Jingyue Complete Works considers it "the purest and most profound medicine among all the essence, blood, and physical qualities." Traditional Chinese Medicine believes that the kidneys store essence and govern reproduction, and egg development is closely linked to the kidneys. As a key herb for nourishing yin and tonifying the kidneys, Shengdihuang is often used clinically to promote hormone secretion in women's ovaries, stimulate the growth, development, and release of follicles, and thus help delay ovarian aging.

[0020] The composition provided by the present invention contains a variety of active ingredients such as rich soy isoflavones, puerarin and other natural plant estrogens, vitamin E, polysaccharides, etc., which not only has dual regulatory effects such as estrogen and anti-estrogen, but also can replenish qi and blood, thereby enhancing ovarian function and delaying ovarian aging. The present invention uses soybeans, black sesame, red dates, kudzu root and rehmannia root in a specific proportion as fermentation raw materials, and adopts plant lactobacillus La10 for in vitro fermentation, which can directly carry out biotransformation on the active ingredients 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 consuming 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 a timely manner, restore and enhance ovarian function; at the same time, after the active bacterial strain colonizes the intestine, it can also continue to play a role in regulating estrogen secretion and improve the situation of estrogen disorder in the human body.

[0021] In the above composition, the weight of soybeans is 35 to 50 parts, for example, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, etc. The weight of black sesame is 10 to 20 parts, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. The weight of red dates is 10 to 20 parts, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. The weight of Pueraria lobata 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 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 of glucose monohydrate 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 of water is 333 to 360 parts, for example, 333 parts, 340 parts, 345 parts, 350 parts, 355 parts, 360 parts, etc.

[0022] Furthermore, in a preferred embodiment, the composition comprises the following components in parts 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 prepared rehmannia root, 16 parts of glucose monohydrate, and 333 parts of water.

[0023] A fourth aspect of the present invention provides a method for preparing a fermented product for regulating estrogen secretion, comprising the following steps:

[0024] S1 The formula amount of soybeans, black sesame and red dates mixed, add water to the broken wall treatment; then the broken wall liquid was boiled to obtain a liquid A;

[0025] S2. The amount of the formula Pueraria and Rehmannia mixed, boiled with water; then concentrated under reduced pressure to obtain liquid B;

[0026] S3. Mix solution A and solution B, add the formula amount of glucose monohydrate to dissolve; then filter and sterilize to obtain solution C;

[0027] S4. Adding a bacterial solution of Lactobacillus plantarum La10 to the solution C for anaerobic culture; then taking the fermentation broth and freeze-drying it in vacuum to obtain the fermentation product.

[0028] 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 minutes, and the boiling time is preferably 20 minutes.

[0029] In step S2, when preparing solution B, the amount of water added is preferably 133 parts by weight; after boiling, the solution is preferably maintained at 95°C for 45 minutes. The reduced pressure concentration conditions are preferably: -0.10 MPa, 70°C, and a speed of 18 rpm.

[0030] 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 minutes.

[0031] 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 viable bacteria in the bacterial solution is preferably 4×10 10 CFU / mL.

[0032] The fifth aspect of the present invention provides the use of the Lactobacillus plantarum, the probiotic 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.

[0033] The sixth aspect of the present invention provides the use of the Lactobacillus plantarum, the probiotic preparation, and the fermentation product in preparing a preparation for regulating estrogen secretion.

[0034] Furthermore, the Lactobacillus plantarum includes but is not limited to live bacteria and sterilized bacteria.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The Lactobacillus plantarum La10 provided by the present invention can promote the conversion of soy isoflavones from a bound state to a free state, thereby helping to regulate estrogen secretion and maintain balance, thereby improving estrogen disorders in the human body.

[0037] 2. This invention utilizes Lactobacillus plantarum La10 to ferment a composition containing soybeans, black sesame seeds, red dates, kudzu root, and Rehmannia root. This fermentation process directly converts the active ingredients into free molecules that are easily absorbed by the human body. The resulting fermented product is rich in active probiotics. This fermented product can be used by individuals with ovarian sub-health, Qi and blood deficiency, or ovarian dysfunction, enabling them to absorb and utilize the nutrients, restoring and enhancing ovarian function. Furthermore, after colonizing the intestines, the active strain can continuously regulate estrogen secretion, improving estrogen imbalances. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to specific examples so that those skilled in the art can better understand the present invention and implement it, but the examples are not intended to limit the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0040] Unless otherwise specified, the experimental methods used in the following examples and comparative examples are conventional methods, and the materials and reagents used are all commercially available unless otherwise specified.

[0041] Strain screening:

[0042] Fresh kelp was minced and an appropriate amount was cultured in MRS liquid medium at 37°C for 48 hours. An appropriate amount of the bacterial suspension was then spread onto MRS plates (supplemented with 0.05% cysteine ​​hydrochloride and 100 mg / L mupirocin) and incubated at 37°C in an anaerobic workstation for 72 hours. After a single colony grew, one was streaked and purified to obtain a pure culture. This pure culture was then identified by third-generation 16S amplicon sequencing, confirming that the strain was Lactobacillus plantarum and designated La10.

[0043] Gene sequence identification results:

[0044]

[0045] Test Example 1: Soybean Isoflavone Conversion Experiment Test

[0046] 1. Fermentation broth preparation

[0047] (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 minutes, weigh, boil for 20 minutes, weigh, make up the weight with purified water, add 10 parts of glucose monohydrate, stir to dissolve, sterilize at 121℃ for 15 minutes, and cool to 37℃.

[0048] (2) Bacterial solution: After thawing the freeze-dried 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 liquid C. Incubate anaerobically at 37°C for 18 h. Adjust the viable bacterial count to 4 × 10 10 CFU / mL.

[0049] (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.

[0050] (4) Fermentation liquid B: Take 3 parts of the 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.

[0051] (5) Fermentation broth C: Take 6 portions of bacterial solution and add them to the original solution, mix well, incubate anaerobically at 37℃ for 18 hours, and then refrigerate at 4℃ for 10 hours.

[0052] (6) Fermentation broth D: Take 9 portions of bacterial solution and add them to the original solution, mix well, incubate anaerobically at 37℃ for 18 hours, and then refrigerate at 4℃ for 10 hours.

[0053] (7) Postbiotic E: Fermentation broth C was heat-inactivated, i.e., kept at 65°C for 45 min and 85°C for 15 min.

[0054] (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.

[0055] (9) After the fermentation broths A to D were freeze-dried in a 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.

[0056] (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.

[0057] 2. The soy isoflavone content of sample F, fermented products A to D, and postbiotic product E was tested in accordance with GB / T 26625 Inspection of Cereals and Oils - Determination of Soybean Isoflavone Content - High Performance Liquid Chromatography Method. The results are shown in Table 1.

[0058] 3. Results and Analysis

[0059]

[0060] Daidzein and genistin are bound soy isoflavones, while daidzein and genistein are free soy isoflavones. Because the human intestine absorbs free soy isoflavones at a much higher rate and in greater quantities than bound soy isoflavones, soy isoflavones must be converted from bound to free form to be effectively absorbed and utilized in the intestine and exert their estrogen-regulating effects.

[0061] It can be seen from Table 1 that the total content of daidzein and genistin in sample F is much greater than the total content of daidzein and genistein, indicating that before fermentation, soy isoflavones mainly exist in a bound state.

[0062] 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 fermentation was not carried out with the bacterial solution of Lactobacillus plantarum La10, and the soy isoflavones were not converted into a free state.

[0063] Compared with sample F, fermented products B to D showed significantly lower levels of daidzein and genistin, while significantly higher levels of daidzein and genistein. This suggests that fermentation with the Lactobacillus plantarum La10 culture medium gradually converts soy isoflavones from bound to free forms. Group D had the lowest levels of daidzein and genistin, while the highest levels of daidzein and genistein, with no significant differences from Group C. This suggests that the amount of Lactobacillus plantarum La10 culture medium added to Group C saturated the fermentation process, allowing the isoflavones in the original solution to be fully converted from bound to free forms. After consumption by the human body, they can be efficiently absorbed and utilized, helping to regulate estrogen secretion balance.

[0064] Compared with group C, the content of daidzein, genistin, daidzein and genistein in postbiotic product E was slightly reduced, but there was basically no significant change, indicating that after heat inactivation treatment, the soy isoflavones in the fermented product still mainly exist in the free form, which is also beneficial to regulating the balance of estrogen secretion after human consumption. Example 1

[0065] This embodiment provides a method for preparing a fermented product for regulating the stable secretion of estrogen, comprising the following steps:

[0066] S1. Select dry, insect-free, and non-corrupted soybeans and red dates, and select qualified black sesame seeds, kudzu root, rehmannia root, and monohydrate glucose.

[0067] S2. Wash soybeans, red dates, black sesame seeds, kudzu root, and Rehmannia root with clean water and drain. Remove the core of the red dates and cut the kudzu root into 3×3 cm pieces. Weigh 35 parts of soybeans, 20 parts of black sesame seeds, 10 parts of red dates, 13 parts of kudzu root, and 6 parts of Rehmannia root.

[0068] S3. Liquid A: After mixing soybeans, black sesame seeds and red dates evenly, add 200 parts of purified water, break the wall at 28000r / min for 2 minutes, weigh, boil for 20 minutes, weigh, make up the weight with purified water, and stir evenly.

[0069] S4, Liquid B: Mix Puerariae Radix and Rehmanniae Preparata evenly, add 133 parts of purified water, weigh, boil, maintain at 95℃ for 45min, and concentrate under reduced pressure at -0.10MPa, 70℃, and 18r / min to the original weight.

[0070] 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.

[0071] S6. Bacterial solution: After thawing the freeze-dried tube of Lactobacillus plantarum La10, 0.4 μL of bacterial solution was spread on MRS solid medium and incubated anaerobically at 37°C for 48 h. Two loops of colonies were picked and added to 10 mL of solution C and incubated anaerobically at 37°C for 18 h. The viable bacterial count was adjusted to 4 × 10 10 CFU / mL.

[0072] S7, Fermentation broth: Take 6 portions of bacterial solution and add them to solution C, mix well, incubate anaerobically at 37°C for 18 hours, and then refrigerate at 4°C for 10 hours.

[0073] 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 mesh using a grinder and sieved. The resulting product is the fermentation product.

[0074] Test Example 2: Ovarian dysfunction model rat test

[0075] 1. Sample preparation

[0076] Weigh 5 g of the fermented product prepared in Example 1, add 50 mL, and stir to dissolve, as Group 1; weigh 10 g of the fermented product prepared in Example 1, add 50 mL, and stir to dissolve, as Group 2; weigh 15 g of the fermented product prepared in Example 1, add 50 mL, and stir to dissolve, as Group 3.

[0077] 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 until use.

[0078] 3. Ten-week-old female Sprague-Dawley rats were randomly divided into six groups of eight rats each, designated A through F. After one week of adaptive feeding, rats in Groups B through F were intraperitoneally injected with cyclophosphamide (60 mg / kg) for one day followed by cyclophosphamide (10 mg / kg) for 14 days to establish an induced premature ovarian failure model. Group A received an intraperitoneal injection of an equal volume of normal saline. During the feeding period, all rats had free access to food and water.

[0079] 4. After modeling, all rats were gavaged with drugs. The specific administration method is shown in Table 2, and the gavage was continued for 28 days.

[0080]

[0081] 5. Record rat body weight changes weekly. After the final dose, rats were fasted for 12 hours and anesthetized with isoflurane inhalation. Blood was collected from the abdominal aorta into a coagulant tube and centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected and the serum estradiol (E2) concentration was measured using a rat serum estradiol ELISA kit. The average group results are shown in Table 3. The ovaries were quickly removed, all adipose tissue removed, and then washed with saline, blotted dry with filter paper, and weighed on an analytical balance. The ovarian index was calculated and the average group results are shown in Table 3.

[0082]

[0083] 6. Results and Analysis

[0084]

[0085] 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.

[0086] As shown in Table 3, compared with group A, the ovarian index and serum E2 content of group B rats were significantly reduced, indicating that the cyclophosphamide-induced premature ovarian failure model rats were successfully established.

[0087] Compared to Group B, the ovarian index and serum E2 levels of rats in Groups D through F were all increased, indicating that the fermented product of Example 1 has a regulatory effect on estrogen secretion in mice, promoting estrogen secretion and improving the symptoms of premature ovarian failure. Among them, the ovarian index and serum E2 levels of rats in Group F were the highest, and were close to those in Group A, indicating that Group 3 is most effective in regulating estrogen secretion in rats with premature ovarian failure.

[0088] Test Example 3: Uterine fibroids model mouse experiment

[0089] 1. Sample preparation

[0090] Weigh 5 g of the fermented product prepared in Example 1, add 50 mL, and stir to dissolve, as Group 1; weigh 10 g of the fermented product prepared in Example 1, add 50 mL, and stir to dissolve, as Group 2; weigh 15 g of the fermented product prepared in Example 1, add 50 mL, and stir to dissolve, as Group 3.

[0091] 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.

[0092] 3. Eight-week-old, female, ICR mice were randomly divided into six groups of eight mice each, designated G through L. After one week of adaptive feeding, mice in groups H through L were injected intramuscularly with estradiol benzoate (2 ml of estradiol benzoate injection solution mixed with 2 ml of Tween 80, then diluted with normal saline to 100 ml) once daily for 15 consecutive days at a dose of 0.1 ml / 10 g to establish an induced uterine fibroid model. Group G received an equal volume of normal saline intramuscularly. During this period, all mice had free access to food and water.

[0093] 4. After modeling, all mice were gavaged and administrated with the specific administration method shown in Table 4 for 21 consecutive days.

[0094]

[0095] 5. Mouse body weight changes were recorded weekly. After the last administration, mice were fasted for 12 hours and then anesthetized with isoflurane inhalation. Blood was collected from the abdominal aorta into a coagulant tube and centrifuged at 3000 rpm for 15 minutes at 4°C. The supernatant was collected and the mouse serum estradiol (E2) concentration was measured using a mouse estradiol ELISA kit. The average group results are shown in Table 5. The ovaries were quickly removed, all adipose tissue removed, and then washed with saline, blotted dry with filter paper, and weighed on an analytical balance. The ovarian index was calculated and the average group results are shown in Table 5.

[0096] 6. Results and Analysis

[0097]

[0098] 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 induced by estradiol benzoate was successfully established in mice.

[0099] Compared with Group H, the ovarian index and serum E2 levels of mice in Groups J to L were all reduced, indicating that the fermented product of Example 1 has a regulatory effect on estrogen secretion in mice, can reduce estrogen secretion, and improve the symptoms of estrogen secretion disorders caused by uterine fibroids. The ovarian index and serum E2 levels of mice in Group L were the lowest, and were relatively close to those in Group G, indicating that Group 3 has the best effect on regulating estrogen secretion in mice with uterine fibroids and has an anti-estrogenic effect.

[0100] Comparative Examples 1 to 7

[0101] 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.

[0102] Example 2

[0103] This embodiment provides a method for preparing a solid preparation, comprising the following steps:

[0104] After thawing the freeze-dried tube of S1 and 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 transferred to 10 mL of MRS liquid medium and cultured anaerobically at 37°C for 18 h. The viable cell count was adjusted to 4 × 10 10 CFU / mL, and the activated bacterial solution is obtained.

[0105] S2. Take 6 portions of activated bacterial solution and add them to MRS liquid culture medium, mix well, culture anaerobically at 37°C for 18 hours, centrifuge, take the precipitate, and mix well with freeze-dried protective agent to obtain bacterial solution No. 1.

[0106] After the bacterial liquid S3 and No. 1 were vacuum freeze-dried (freeze-drying parameters were -80°C pre-freezing for 12 hours and -70°C drying for 24 hours), they were crushed to 80 mesh using a grinder and sieved. The resulting product was the live bacterial type Lactobacillus plantarum La10 bacterial powder.

[0107] 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

[0108] This embodiment provides a method for preparing a solid preparation, comprising the following steps:

[0109] After thawing the freeze-dried tube of S1 and 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 transferred to 10 mL of MRS liquid medium and cultured anaerobically at 37°C for 18 h. The viable cell count was adjusted to 4 × 10 10 CFU / mL, and the activated bacterial solution is obtained.

[0110] S2. Take 6 portions of activated bacterial solution and add them to MRS liquid culture medium, mix well, culture anaerobically at 37°C for 18 hours, centrifuge, take the precipitate, and mix well with freeze-dried protective agent to obtain bacterial solution No. 1.

[0111] After the bacterial liquid S3 and No. 1 were vacuum freeze-dried (freeze-drying parameters were -80°C pre-freezing for 12 hours and -70°C drying for 24 hours), they were crushed to 80 mesh using a grinder and sieved. The resulting product was Lactobacillus plantarum La10 bacterial powder.

[0112] S4. Keep the Lactobacillus plantarum La10 powder at 65° C. for 50 min and at 78° C. for 18 min, grind it into 80 mesh with a grinder, and sieve it. The obtained product is the inactivated Lactobacillus plantarum La10 powder.

[0113] S5. Take 10 parts of inactivated Lactobacillus plantarum La10 powder and mix evenly with 90 parts of oligofructose to obtain solid preparation No. 2.

[0114] Test Case 4: Crowd Test

[0115] 1. Recruit 80 female volunteers aged 25 to 40 years with irregular menstruation for 2 to 4 months and a BMI of 21 to 24 kg / m2. 2 of people.

[0116] Diagnostic criteria: Meet the relevant standards of "Research on Traditional Chinese Medicine Syndrome Differentiation and Treatment of Gynecological Menstrual Disorders".

[0117] (1) The menstrual cycle is shortened by more than 7 days, or even occurs once every 15 days, or the menstrual cycle is prolonged by more than 35 days, with irregular menstruation such as menstruation being advanced or delayed by more than 7 days;

[0118] (2) Menstrual volume increases significantly (>80 mL) or decreases (<20 mL), or even spotting and then stops;

[0119] (3) Or symptoms such as abdominal pain, dizziness, fatigue, anemia, soreness of waist and knees, breast tenderness, amenorrhea, and irritability during menstruation may occur;

[0120] (4) The main symptoms are premature menstruation, menorrhagia, prolonged menstruation, and light menstrual color. The secondary symptoms are pale face, dry mouth, chills and sweating, fatigue, shortness of voice and low breath, dizziness, and stringy pulse.

[0121] 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 irregularities due to qi deficiency and blood stasis; (4) patients and their families were aware of the study and agreed.

[0122] Exclusion criteria: (1) Allergy to drugs used in the combined 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 from the study before the end of the study or refusal to cooperate with the study.

[0123] 2. Volunteers were randomly divided into 10 groups, 8 in each group, as shown in Table 7. Each group consumed the corresponding fermented product, 2 g each time, three times a day, half an hour after meals, mixed with 100 mL of 37°C warm water, for 60 days.

[0124] Table 7 Volunteer grouping

[0125]

[0126] 3. On the 61st day, the number of volunteers with marked effect, improvement, and ineffectiveness was counted. The results are shown in Table 8.

[0127] The improvement effect was evaluated based on the New Edition of Traditional Chinese Medicine Gynecology. A marked effect was defined as the disappearance of symptoms (such as abdominal pain and backache), the return of abnormal menstruation to normal, and no recurrence within three months. Improvement was defined as improvement if clinical symptoms were significantly alleviated and abnormal menstruation improved, but recurred within three months. Any failure to meet these criteria was considered ineffective.

[0128] 4. At 8:00 a.m. on the first and 61st days, 5 mL of cubital venous blood was drawn from the volunteers while they were fasting. After centrifugation for 15 min (4°C, 3000 r / min), the supernatant was collected and the serum estradiol (E2) content was detected using the estradiol ELISA kit. The average values ​​were calculated and shown in Table 8.

[0129] 5. Results and Analysis

[0130]

[0131] As shown in Table 8, the fermented products in different groups have different degrees of improvement on the volunteers' menstrual irregular symptoms. Among Groups A to H, Group H has the largest number of significantly effective patients, indicating that the fermented product in 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.

[0132] In terms of estrogen content, compared with the first day, the estrogen content in the serum of volunteers increased significantly after consuming the fermented products for 60 days, indicating that the fermented products in 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 most effective in regulating the balance of estrogen secretion.

[0133] In summary, the Lactobacillus plantarum La10 and its fermentation product provided by the present invention can promote the conversion of soy isoflavones from a bound state to a free state, thereby regulating the stable secretion of estrogen and improving the estrogen disorder in the human body.

[0134] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A fermentation product of Lactobacillus plantarum for regulating estrogen secretion, characterized in that: The plant lactobacillus is plant lactobacillus ( Lactiplantibacillus plantarum ) La10, whose deposit number is GDMCC No: 63605; The fermented product is obtained by inoculating Lactobacillus plantarum La10 into a composition and fermenting the composition; wherein the composition comprises the following components in parts 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 glucose monohydrate and 333 parts of water.

2. The fermentation product of Lactobacillus plantarum for regulating estrogen secretion according to claim 1, characterized in that: The method for preparing the fermented product comprises the following steps: S1. The formula amount of soybeans, black sesame seeds and red dates are mixed evenly, and water is added to break the wall; the broken wall liquid is then boiled to obtain liquid A; S2. The formula amount of Pueraria root and Rehmannia glutinosa was mixed evenly, boiled with water; then concentrated under reduced pressure to obtain solution B; S3. Mix solution A and solution B, add the formula amount of glucose monohydrate to dissolve; then filter and sterilize to obtain solution C; S4. The bacterial solution of Lactobacillus plantarum La10 was added to the C solution for anaerobic culture; the fermentation broth was then freeze-dried under vacuum to obtain the fermentation product; In step S4, the amount of the bacterial solution added is 5 to 10 parts by weight, and the concentration of live bacteria in the bacterial solution is 4×10 10 CFU / mL.

3. Use of the fermented product of Lactobacillus plantarum for regulating estrogen secretion according to any one of claims 1 to 2 in the preparation of a product for improving the symptoms of premature ovarian failure or improving the symptoms of estrogen secretion disorder caused by uterine fibroids.

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