A probiotic composition for improving menopausal symptoms and its application

Abstract

The present invention discloses a probiotic composition for improving menopausal symptoms and its application, which combines raw materials of both medicine and food with probiotics complementing each other, having a simple composition and synergistic effect, being rich in various essential nutrients and trace elements for the human body, and having the functions of strengthening the spleen and stomach, nourishing the liver and kidney, and calming the nerves, and can improve symptoms such as hot flashes and sweating, insomnia and dreaminess during menopause. The present invention can be used to improve menopausal syndrome, expanding the application field of probiotics.

Description

Technical Field

[0001] The present invention relates to a probiotic composition, and particularly to a probiotic composition for improving menopausal symptoms and its application. It belongs to the technical field of biomedical processing. Background Art

[0002] Female menopause is a spontaneous physiological process characterized by the permanent cessation of the menstrual cycle and the loss of ovarian function. Menopause is considered a sign of natural aging of the body. According to statistics, the average age of entering menopause is about 46 - 52 years old, and the age of entering menopause varies among different economic levels and lifestyles.

[0003] Female menopausal syndrome refers to a series of physical and mental symptoms caused by the decline of ovarian function and the decrease of estrogen level, mainly including depression, anxiety, sleep dysfunction, hot flashes, sweating, palpitation and restlessness, etc. In addition, with the decline of hormones, it is easier to accelerate bone loss, central abdominal fat accumulation and metabolic problems such as high blood pressure. In severe cases, it can lead to cardiovascular diseases and type 2 diabetes. Research shows that these menopausal syndromes cause a greater burden on women's physiology and psychology and will affect normal life to a certain extent.

[0004] Clinically, the treatment of menopausal syndrome mainly relies on drug treatment, generally hormone replacement therapy. This treatment method can relieve physical and mental discomfort well in the short term, but some reports point out that this method also has risks: some patients have hyperplasia of mammary epithelial cells and breast distending pain after taking the medicine; adverse reactions such as headache and poor sleep occur during the medication process; if the dosage is large, it will also cause irregular vaginal bleeding in patients. Therefore, it is urgent to find a safer and more effective treatment method for menopausal women.

[0005] Taking probiotics is a safe and effective treatment method for improving menopausal syndrome. In recent years, a large number of studies have shown that probiotics can effectively intervene in the metabolic level. Different species of Lactobacillus genera can all improve metabolic results, including glucose metabolism, lipid metabolism, visceral fat, estradiol level and LPS concentration. Therefore, the application of probiotics in improving female menopausal syndrome will effectively improve the quality of life during menopause. Summary of the Invention

[0006] The purpose of the present invention is to overcome the above-mentioned deficiencies of the prior art and provide a probiotic composition for improving menopausal symptoms and its application.

[0007] To achieve the above purpose, the present invention adopts the following technical solutions:

[0008] A probiotic composition for improving menopausal symptoms is made by mixing the following components:

[0009] Lactiplantibacillus plantarum LP115, which was deposited at the China General Microbiological Culture Collection Center on August 22, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 25576;

[0010] Lactobacillus fermentum LF26, which was deposited at the China General Microbiological Culture Collection Center on February 24, 2020. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 19431;

[0011] Lactobacillus paracasei LPC12, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 24512;

[0012] Lactobacillus rhamnosus LRH10, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 24513;

[0013] Lactobacillus delbrueckii subsp. Lactis LDL114, which was deposited at the China General Microbiological Culture Collection Center on August 22, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 25575;

[0014] Lactobacillus helveticus LA25, which was deposited at the China General Microbiological Culture Collection Center on August 22, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 25569.

[0015] Preferably, the viable cell numbers ratio of Lactiplantibacillus plantarum LP115, Lactobacillus fermentum LF26, Lactobacillus paracasei LPC12, Lactobacillus rhamnosus LRH10, Lactobacillus delbrueckii subsp. Lactis LDL114, and Lactobacillus helveticus LA25 is 3:3:3:6:1:3.

[0016] Preferably, the viable count of the composition is not less than 10 9 cfu / mL.

[0017] Use of the above probiotic composition in the preparation of a medicament for improving menopausal symptoms.

[0018] The medicament containing the above probiotic composition is made by mixing the following components by weight: 10 - 25 parts of Chinese wolfberry, 10 - 20 parts of dwarf lilyturf tuber, 10 - 18 parts of Chinese date, 10 - 18 parts of mulberry, 10 - 15 parts of poria cocos, 5 - 15 parts of smoked plum, 3 - 5 parts of rose flower, 3 - 5 parts of hawthorn fruit, 2 - 5 parts of wild jujube seed, 2 - 5 parts of liquorice root, and 2 - 5 parts of the probiotic composition.

[0019] The preparation method of the above medicament is as follows: first, crush other components except the probiotic composition according to the formula amount through a 60 - 100 - mesh sieve and mix them evenly to form a mixture, then mix the mixture evenly with the probiotic composition according to the formula amount, and then fill it.

[0020] Advantages of the present invention:

[0021] In the present invention, the raw materials of medicine and food supplement each other, the composition is simple and has a synergistic effect, is rich in a variety of essential nutrients and trace elements for the human body, has the functions of strengthening the spleen and nourishing the stomach, nourishing the liver and kidney, and calming the mind, and can improve symptoms such as hot flashes and sweating, insomnia and dreaminess during menopause. The present invention can be used to improve menopausal syndrome and expands the application field of probiotics.

[0022] The raw materials of medicine and food are as follows:

[0023] Chinese wolfberry is recorded in "Shennong's Herbal Classic": "Chinese wolfberry mainly treats internal evils, heat - induced thirst, rheumatism with general pain. Taking it for a long time can strengthen the tendons and bones, make the body light and not old, and resist cold and heat". Chinese wolfberry is flat in nature and sweet in taste, nourishes the liver and nourishes blood, can clear the lung and improve eyesight, and is suitable for treating symptoms such as deficiency of liver and kidney yin, dizziness and vertigo. Modern medical research shows that Chinese wolfberry contains rich protein, carotene, vitamins, crude fiber, unsaturated fatty acids, amino acids and trace elements required by the human body, and has good effects of promoting immunity, reducing blood sugar, antioxidation, delaying aging, etc.

[0024] Dwarf lilyturf tuber, the summary of its efficacy in "New Compilation of Materia Medica": "It purges the latent fire in the lung, clears the heat pathogen in the stomach, tonifies the fatigue injury of the heart qi, stops vomiting of hematemesis patients, benefits essence and strengthens yin, relieves restlessness and quenches thirst, improves complexion, makes the skin smooth. It has a miraculous effect in removing deficiency heat, is particularly effective in relieving lung dryness, and has great merits in relieving cough". Dwarf lilyturf tuber is sweet, slightly bitter, slightly cold in nature, and belongs to the stomach, lung and heart meridians, and has the effects of nourishing yin and moistening the lung, promoting the production of body fluid to quench thirst, and clearing away heart - fire to remove vexation. Modern pharmacological research shows that dwarf lilyturf tuber mainly contains ophiopogonin, steroidal saponins, alkaloids, sitosterol, glucose, amino acids, vitamins, etc., and has the effects of anti - fatigue, scavenging free radicals, improving cellular immune function and reducing blood sugar.

[0025] Red dates were listed as top-grade medicinal materials in "Shennong Ben Cao Jing": "It soothes the middle energizer and nourishes the spleen, assisting the twelve regular meridians. It balances the stomach qi, opens the nine orifices, supplements qi deficiency, fluid deficiency, and deficiencies in the body." There are many expositions in "Treatise on Febrile Diseases", "Synopsis of the Golden Chamber", and "Compendium of Materia Medica": It has the effects of tonifying the middle qi and nourishing the blood to soothe the mind, and is applicable to middle qi deficiency, blood deficiency syndrome, hysteria syndrome, etc. Modern medical research shows that red dates integrate medicine, food, and tonics, have the effect of nourishing the blood, beautifying the face and body, can prevent and treat cardiovascular diseases, and have the effects of enhancing immunity, anti-aging, anti-tumor, anti-allergy, and detoxifying and protecting the liver.

[0026] Mulberries were first recorded in "Tang Materia Medica" for medicinal use. Mulberries are sweet in taste and cold in nature, entering the heart, liver, and kidney meridians, nourishing yin and enriching the blood, calming the mind and soothing the nerves, reducing blood pressure and relieving thirst, and having the effects of treating yin deficiency with less body fluid, insomnia, etc. Modern medical clinical practice has proved that mulberries have significant curative effects on reducing lipid levels, alleviating neurasthenia, arteriosclerosis, sexual dysfunction, deafness and blurred vision, premature whitening of hair, internal heat with thirst, blood deficiency and constipation, and rheumatic joint pain.

[0027] Poria cocos is one of the medicinal edible fungi that were first included in the "Chinese Pharmacopoeia". It is mild in nature and sweet in taste, belonging to the heart, lung, spleen, and kidney meridians. It can be used to treat edema with less urine, phlegm retention with dizziness and palpitations, spleen deficiency with less food intake, loose stools and diarrhea, restlessness of the mind, and palpitations and insomnia. Modern medical clinical practice has proved that Poria cocos has the effects of strengthening the spleen and stomach, removing dampness and calming the nerves, enhancing immunity, and anti-cancer.

[0028] Semen Ziziphi Spinosae belongs to the raw materials of both medicine and food. "Wild jujube" was first recorded in "Shennong Ben Cao Jing", and then "Semen Ziziphi Spinosae" appeared in "Ben Cao Cheng Ya Ban Jie". Semen Ziziphi Spinosae has the effects of nourishing the liver, calming the nerves, and stopping sweating, and is mainly used to treat symptoms such as vexation and insomnia, palpitations and dreaminess, excessive sweating due to physical weakness, and thirst due to fluid impairment. The various pharmacological components such as alkaloids, saponins, and flavonoids contained in Semen Ziziphi Spinosae have significant effects on improving sleep, regulating the cardiovascular system and immune system.

[0029] Licorice has a sweet taste and benefits the spleen, belonging to the spleen, stomach, heart, and lung meridians, and has the effects of tonifying the heart and spleen, relieving spasm and pain, regulating the properties of drugs, purging fire and detoxifying, moistening the lungs and relieving cough, etc. There are many prescriptions with licorice: Tonifying qi and strengthening the middle, generating qi and blood, such as Zhigancao Decoction in "Treatise on Febrile Diseases"; Tonifying both qi and blood, such as Guipi Decoction in "Jisheng Fang" and Bazhen Decoction in "Orthopedic and Traumatological Classic". Modern research and analysis show that the main components of licorice are triterpenoids and flavonoids, and also contain alkaloids, polysaccharides, coumarins, amino acids, and trace elements. In addition to the effects of tonifying the spleen and qi, moistening the lungs and relieving cough, clearing heat and detoxifying, and regulating various drugs, licorice is also used for symptoms such as sore throat, spleen and stomach cold deficiency, peptic ulcer, and poisoning.

[0030] Currently, the health effects of multi-strain probiotic combinations have received extensive attention. Research has found that the effects of multi-strains are influenced by factors such as the compatible strains and the target population. When targeting Helicobacter pylori infection, no significant improvement was found using a single probiotic strain or a 4-strain mixture, while the infection was significantly alleviated by a 2-strain mixture. For certain physiological abnormalities (such as irritable bowel syndrome, antibiotic-associated diarrhea, atopic dermatitis, etc.), multi-strain mixtures did not show more significant effects than single strains. However, in some diseases (such as necrotizing enterocolitis in children, Helicobacter infection), probiotic combination interventions have better efficacy.

[0031] Research has shown that the reasons for multi-strains to exert health effects may be as follows:

[0032] 1) Multi-strain probiotic preparations can decompose and transform more nutrients (such as increasing the diversity of digestive enzymes) and create a better microecological environment (anaerobic, suitable pH) to better maintain the diversity of the human gut microecology.

[0033] 2) There is cross-feeding behavior among the strains, which then plays a synergistic role. Lactobacillus and Bifidobacterium can synergistically enhance the formation of butyrate. The nutritional interaction between Bifidobacterium and Lactobacillus can promote the formation of acetate, butyrate, propionate, and formate in the intestine. There is also a potential intestinal symbiotic relationship between the same genus of bacteria. The mucin cross-feeding triggered by Bifidobacterium bifidum can promote the growth and metabolite accumulation of Bifidobacterium breve.

[0034] 3) Strain combination enriches its potential physiological regulatory mechanisms. Compared with single probiotics, the combined use of Lactobacillus helveticus and Bifidobacterium longum is more effective in regulating the negative feedback of glucocorticoids on the HPA (hypothalamic-pituitary-adrenal axis) axis, and thus shows more significant effects in alleviating stress-induced visceral pain. Probiotic combinations have a synergistic effect in alleviating toll-like receptor 3 (TLR3)-related inflammation and maintaining cell homeostasis compared with single strains. Research based on the VSL#3 microecological preparation has proved that multi-strain combination can increase the intestinal adhesion of various target strains, thereby promoting the interaction between the strains and host cells.

[0035] Generally speaking, the health regulatory effect of probiotics does not show an obvious dependence on the number of strain combinations, but is affected by various complex factors (such as probiotic strains, differences in dietary patterns, characteristics of the target population, intervention time, etc.). Whether it is a probiotic combination with enhanced multi-strain synergy or a single probiotic strain, its health effect mainly depends on in vitro tests, evidence-based medical evidence, etc., rather than just the number of combined strains. Description of the Drawings

[0036] Figure 1 Experiment on Prolonging the Sleep Time of Sodium Pentobarbital

[0037] Figure 2 Comparison of Fat Mass of Rats in Each Group

[0038] Preservation Information

[0039] Classification and Naming: Lactiplantibacillus plantarum LP115

[0040] Latin Scientific Name: Lactiplantibacillus plantarum

[0041] Name of the Preservation Unit: General Microbiology Center, China National Culture Collection Center

[0042] Address of the Preservation Unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0043] Preservation Date: August 22, 2022

[0044] Preservation Number: CGMCC NO.25576

[0045] Classification and Naming: Lactobacillus fermentum LF26

[0046] Latin Scientific Name: Lactobacillus fermentum

[0047] Name of the Preservation Unit: General Microbiology Center, China National Culture Collection Center

[0048] Address of the Preservation Unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0049] Preservation Date: February 24, 2020

[0050] Preservation Number: CGMCC NO.19431

[0051] Classification and Naming: Lactobacillus paracasei LPC12

[0052] Latin Scientific Name: Lactobacillus paracasei

[0053] Name of the Preservation Unit: General Microbiology Center, China National Culture Collection Center

[0054] Address of the Preservation Unit: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0055] Preservation Date: March 11, 2022

[0056] Preservation Number: CGMCC NO.24512

[0057] Classification name: Lactobacillus rhamnosus LRH10

[0058] Latin scientific name: Lactobacillus rhamnosus

[0059] Name of the depositary institution: General Microbiology Center, China Committee for Culture Collection of Microorganisms

[0060] Address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0061] Date of deposit: March 11, 2022

[0062] Deposit number: CGMCC NO.24513

[0063] Classification name: Lactobacillus delbrueckii subsp. Lactis LDL114

[0064] Latin scientific name: Lactobacillus delbrueckii subsp. Lactis

[0065] Name of the depositary institution: General Microbiology Center, China Committee for Culture Collection of Microorganisms

[0066] Address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0067] Date of deposit: August 22, 2022

[0068] Deposit number: CGMCC NO.25575

[0069] Classification name: Lactobacillus helveticus LA25

[0070] Latin scientific name: Lactobacillus helveticus

[0071] Name of the depositary institution: General Microbiology Center, China Committee for Culture Collection of Microorganisms

[0072] Address of the depositary institution: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing

[0073] Date of deposit: August 22, 2022

[0074] Deposit number: CGMCC NO.25569 Detailed implementation manners

[0075] The present invention will be further described below in conjunction with embodiments. It should be noted that the following description is only for explaining the present invention and does not limit its content.

[0076] Application embodiments

[0077] A probiotic composition for improving menopausal symptoms, which is prepared by mixing the following components:

[0078] Lactiplantibacillus plantarum LP115* (formerly known as Lactobacillus plantarum LP115), which was deposited on August 22, 2022, at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 25576;

[0079] Lactobacillus fermentum LF26* (also known as Limosilactobacillus fermentum LF26), which was deposited on February 24, 2020, at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 19431;

[0080] Lactobacillus paracasei LPC12* (also known as Lacticaseibacillus paracasei LPC12), which was deposited on March 11, 2022, at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 24512

[0081] Lactobacillus rhamnosus LRH10* (also known as Lacticaseibacillus rhamnosus LRH10), which was deposited on March 11, 2022, at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 24513;

[0082] Lactobacillus delbrueckii subsp. Lactis LDL114, which was deposited on August 22, 2022, at the China General Microbiological Culture Collection Center, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number being CGMCC NO. 25575;

[0083] Lactobacillus helveticus LA25, which was deposited at the China General Microbiological Culture Collection Center on August 22, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 25569.

[0084] (Note: "*" is adjusted according to the requirements of the Announcement of the National Health Commission "Announcement on the Update of the List of Strains That Can Be Used in Foods and the List of Strains That Can Be Used in Infant Foods (No. 4, 2022)")

[0085] The viable count ratio of Lactiplantibacillus plantarum LP115, Lactobacillus fermentum LF26, Lactobacillus paracasei LPC12, Lactobacillus rhamnosus LRH10, Lactobacillus delbrueckii subsp. lactis LDL114, and Lactobacillus helveticus LA25 is 3:3:3:6:1:3.

[0086] The viable count of this composition is not less than 10 9 CFU.

[0087] Example 1:

[0088] 25 parts of wolfberry, 10 parts of Ophiopogon japonicus, 15 parts of red date, 13 parts of mulberry, 12 parts of Poria cocos, 5 parts of smoked plum, 3 parts of rose, 3 parts of hawthorn, 5 parts of Ziziphus jujuba var. spinosa, 5 parts of licorice, and 4 parts of probiotic composition (reference example).

[0089] Specific production process:

[0090] S1. Weigh each component according to the proportion and set aside for use;

[0091] S2. Mix wolfberry, Ophiopogon japonicus, red date, mulberry, Poria cocos, smoked plum, rose, hawthorn, Ziziphus jujuba var. spinosa, and licorice to obtain a traditional Chinese medicine mixture. The traditional Chinese medicine mixture is mixed with water at a mass ratio of 1:20, heated to boiling over high heat, and kept boiling over low heat for 30 minutes. After filtering to remove the residue, it is dried and pulverized through a 100-mesh sieve to make an extract powder;

[0092] S3. Mix the extract powder in S2 with the probiotic composition and fill it into containers.

[0093] Example 2:

[0094] 15 parts of wolfberry, 13 parts of Ophiopogon japonicus, 10 parts of red date, 18 parts of mulberry, 10 parts of Poria cocos, 15 parts of smoked plum, 4 parts of rose, 4 parts of hawthorn, 2 parts of Ziziphus jujuba var. spinosa, 4 parts of licorice, and 5 parts of probiotic composition (reference example).

[0095] Specific production process:

[0096] S1. Weigh each component according to the proportion for later use;

[0097] S2. Mix wolfberry, ophiopogon root, red date, mulberry, poria cocos, smoked plum, rose, hawthorn, wild jujube seed, and liquorice to obtain a traditional Chinese medicine mixture. Mix the traditional Chinese medicine mixture with water at a mass ratio of 1:30, heat it to boiling over high heat, maintain boiling over low heat for 30 minutes, filter to remove residues, dry, and then pulverize and sieve through a 60-mesh sieve to make an extract powder.

[0098] S3. Mix the extract powder in S2 with the probiotic composition and fill it into containers.

[0099] Example 3:

[0100] 10 parts of wolfberry, 20 parts of ophiopogon root, 18 parts of red date, 10 parts of mulberry, 15 parts of poria cocos, 10 parts of smoked plum, 5 parts of rose, 5 parts of hawthorn, 3 parts of wild jujube seed, 2 parts of liquorice, 2 parts of probiotic composition (reference example).

[0101] Specific production process:

[0102] S1. Weigh each component according to the proportion for later use;

[0103] S2. Mix wolfberry, ophiopogon root, red date, mulberry, poria cocos, smoked plum, rose, hawthorn, wild jujube seed, and liquorice to obtain a traditional Chinese medicine mixture. Mix the traditional Chinese medicine mixture with water at a mass ratio of 1:25, heat it to boiling over high heat, maintain boiling over low heat for 25 minutes, filter to remove residues, dry, and then pulverize and sieve through an 80-mesh sieve to make an extract powder.

[0104] S3. Mix the extract powder in S2 with the probiotic composition and fill it into containers.

[0105] Experimental Example 1

[0106] 1.1 Animal experiment

[0107] Ovariectomize (OVX) 10-week-old female rats weighing 200 - 250 g to establish a menopause model. Before OVX, randomly conduct intestinal microbiota normalization feeding management for 4 - 6 weeks. After normalization, the rats are randomly divided into 5 groups, with 10 rats in each group. The specific grouping is shown in Table 1.

[0108] Among them, PBS is phosphate buffer solution, which is prepared by the following method: Weigh 8.0 g of NaCl, 0.2 g of KCl, 1.44 g of Na 2 HPO 4 , 0.24 g of KH 2 PO 4 Dissolve in 800 mL of distilled water, adjust the solution to 7.4 with HCl, and finally make up the volume to 1 L with distilled water to obtain it.

[0109] Table 1 Grouping of animal experiment

[0110]

[0111]

[0112] During the OVX surgery, anesthesia was performed with isoflurane at a mass concentration of 2%. In the OVX group, two ovaries of the rats were excised, while in the sham-operated group, the ovaries of the rats were kept intact. The experiment started one week after OVX, and the rats were gavaged three times a week. The experiment lasted for 16 weeks. After the experiment, all the rats were sacrificed for data analysis. In the above groups, all samples were dissolved in PBS except for 17β-estradiol, which was dissolved in physiological saline containing 1% (mass concentration) dimethyl sulfoxide (DMSO).

[0113] 1.2 Experimental methods

[0114] 1.2.1 Sleep improvement experiment

[0115] The sleep improvement experiment was carried out referring to the part of the evaluation specification for sleep improvement function in the "Technical Specification for the Inspection and Evaluation of Health Foods". All experiments were carried out in a suitable temperature and quiet environment.

[0116] 1.2.1.1 Direct sleep experiment

[0117] After each gavage, it was judged whether the rats in each group showed direct sleep by whether the righting reflex disappeared. The righting reflex means that if the rat is placed in the dorsal recumbent position, it can immediately right its body position. If the rat cannot right its body position for more than 30 - 60 s, it is considered that the righting reflex has disappeared and the sleep state begins. When the rat resumes the righting reflex, it is considered that its sleep state ends.

[0118] 1.2.1.2 Experiment on prolonging the sleeping time of sodium pentobarbital

[0119] Before the formal experiment, a preliminary experiment was carried out to determine the intraperitoneal injection dose of sodium pentobarbital. The dose at which all the rats fell asleep after intraperitoneal injection and the sleep duration was appropriate was used as the standard, and this dose was used for the formal experiment. 30 minutes after the last gavage of the rats, sleep was induced by intraperitoneal injection of sodium pentobarbital, and the sleeping time of the rats was recorded as the time from the disappearance of the righting reflex to its reappearance. It was observed whether the sleeping time of sodium pentobarbital in each group was prolonged.

[0120] 1.2.2 Influence on endocrine levels

[0121] The endocrine levels were mainly analyzed by serum estradiol. After the 16-week experiment, blood samples were collected from the abdominal aorta of the rats, centrifuged at 4°C and 1000 rmp for 15 minutes to separate the serum. A coated counting kit was used to measure the serum estradiol level.

[0122] 1.2.3 Measurement of fat content and bone mineral density in rats

[0123] The fat mass and bone mineral density were measured by dual x-ray absorptiometry. The rats were anesthetized with a constant flow of 2% isoflurane in an oxygen / nitrous oxide mixture. After calibration, each rat was placed in the prone position on a platform and scanned whole body. The rats were tested for fat content 16 weeks after OVX and scanned at 8, 12, and 16 weeks for bone mineral density testing.

[0124] 1.3 Results and Analysis

[0125] 1.3.1 Evaluation of Sleep Improvement Effect

[0126] 1.3.1.1 Direct Sleep Experiment

[0127] The experimental results showed that none of the five experimental groups of rats had sleep phenomena and could move normally, indicating that the test substances in each group had no direct hypnotic effect on the rats.

[0128] 1.3.1.2 Experiment on Prolonging the Sleep Time of Sodium Pentobarbital

[0129] Through preliminary experiments, the intraperitoneal injection dose of sodium pentobarbital that could make all rats fall asleep and have a moderate sleep duration was determined to be 50 mg / kg. As Figure 1 shown, compared with OVX control experimental group 2, experimental groups 4, 5, 6, and 7 significantly prolonged the sleep duration induced by sodium pentobarbital. Experimental group 4 had a better effect on prolonging the sleep time, approaching the pre-menopausal state level (experimental group 1). It indicated that the mixture of probiotic composition + traditional Chinese medicine could effectively prolong the sleep time of rats,

[0130] 1.3.2 Evaluation of Regulating Endocrine Levels

[0131] The serum estradiol levels of rats in each group are shown in Table 2.

[0132] Table 2 Comparison of Serum Estradiol Levels of Rats in Each Group

[0133] Group Estradiol Concentration (pg / mL) Experimental Group 1 84.5±9.6 Experimental Group 2 54.2±10.7 Experimental Group 3 117.9±11.4 Experimental Group 4 82.6±9.1 Experimental Group 5 69.8±8.8 Experimental Group 6 73.7±8.3 Experimental Group 7 75.6±6.8

[0134] As can be seen from Table 2, the estradiol level of OVX rats 16 weeks after surgery decreased compared with experimental group 1. Among them, the estradiol level of OVX rats in experimental group 3 directly supplemented with 17β-estradiol increased significantly, exceeding the normal level; the estradiol levels of OVX rats in experimental groups 4, 5, 6, and 7 also increased after treatment, approaching the normal estradiol level of experimental group 1. Among them, experimental group 4 was closer to experimental group 1 (i.e., the pre-menopausal state level). The results showed that supplementing the mixture of probiotic composition + traditional Chinese medicine was beneficial to the recovery of endocrine levels during menopause, and experimental group 4 had the best effect.

[0135] 1.3.3 Evaluation of Improving Body Fat and Bone Mineral Density

[0136] The fat mass of rats is shown in Figure 2 , as can be seen from the figure, compared with experimental group 1 without surgery, the fat content in experimental group 2 (OVX rats) increased significantly. Compared with experimental group 2, the use of estradiol and experimental groups 4, 5, 6, and 7 had a certain therapeutic effect on preventing the increase in fat content, and experimental group 4 had the best effect.

[0137] The results of the bone mineral density of the femur in rats are shown in Table 3.

[0138] Table 3 Comparison of bone mineral density levels in rats of each group

[0139]

[0140] As can be seen from Table 3, the bone mineral density of OVX rats decreased significantly at 8 weeks after surgery. Compared with experimental group 2, the bone mineral density of OVX rats in experimental groups 3, 4, 5, 6, and 7 increased significantly. The data at 12 weeks and 16 weeks showed that the OVX rats in experimental group 4 showed the highest bone mineral density.

[0141] Experimental Example 2

[0142] 2.1 Human experiment

[0143] The selection criteria for participants in the human experiment are as follows:

[0144] (1) Women aged between 45 and 55 years old. The main reason for choosing this age group is that the average age of entering menopause is about 46 - 52 years old, and the age of entering menopause varies with different economic levels and lifestyles;

[0145] (2) Patients without menopause index and several mild menopause symptoms, and evaluated as 26 to 50 points by the simplified menopause index (SMI);

[0146] (3) People with normal serum estradiol levels;

[0147] (4) People with normal menstrual cycles (the menstrual cycle interval is 3 - 7 days, and the menstrual cycle interval is 25 - 38 days);

[0148] (5) People with a body mass index (BMI) between 18.6 and 30.

[0149] A total of 80 volunteers meeting the above requirements were randomly divided into two groups, with 40 in each group, and an experiment was conducted for 12 weeks. Among them, the placebo group took a starch packet, 2 g / bar, 1 bar per day; the experimental group took the probiotic powder packet prepared in Example 2, 2 g / bar (the viable bacteria count is not less than 10 9CFU / strip), take 1 strip daily. Have a normal diet during the experiment and avoid taking other antibiotic drugs. Conduct a follow-up after the 12-week experiment and record the results according to the scoring criteria in Table 5.

[0150] 2.1.1 Basic information statistics

[0151] A total of 67 volunteers finally completed the experiment. According to the grouping situation, the basic information of 30 volunteers in each group, a total of 60 volunteers, was statistically analyzed, as shown in Table 4.

[0152] Table 4 Basic information statistics of volunteers

[0153] Parameter Placebo Group (n = 30) Experimental Group (n = 30) P Value Age 48.5±0.5 49.5±0.5 0.19 BMI 22.5±0.4 22.6±0.4 0.90 Body Fat Percentage 29.4±1.0 28.6±1.1 0.93 Menstrual Cycle / Day 28.2±0.6 28.6±0.5 0.64 Menstrual Period / Day 6.1±0.2 5.8±0.1 0.34 Estradiol Level / pg / mL 65.4±11.4 59.1±9.6 0.67 SMI Score 46.2±1.7 46.7±1.9 0.83

[0154] During the entire experiment process, no adverse events occurred to any volunteers, including those who did not complete the entire experiment process.

[0155] 2.1.2 Scoring criteria

[0156] The scoring criteria for improving the menopausal syndrome of volunteers are mainly based on the Kupperman modified score, as shown in Table 5 for details.

[0157] Table 5 Scoring criteria

[0158]

[0159]

[0160] The curative effect is judged in three grades: (1) Marked effect: The symptom score decreases by more than 70% after treatment; (2) Effective: The symptom score decreases by 30%-69% after treatment; (3) Ineffective: The symptom score decreases by less than 30% after treatment.

[0161] Effective rate = (Marked effect + Effective) / n × 100%.

[0162] 2.1.3 Experimental results

[0163] The symptom scores of the two groups before and after the experiment were statistically analyzed, as shown in Table 6 for details; the results of the experimental effective rate are shown in Table 7 for details.

[0164] Table 6 Comparison of symptom scores of each group before and after the experiment

[0165]

[0166] Note: Compared with before the experiment, *P < 0.05, **P < 0.01.

[0167] Table 7 Comparison of experimental effective rates of each group

[0168] Group Marked Effect (cases) Effective (cases) Ineffective (cases) Effective Rate Placebo Group 3 8 19 36.67％ Experimental Group 9 17 4 86.67％

[0169] During the experiment, the symptoms of the volunteers in the experimental group improved significantly. Typical menopausal syndrome such as hot flashes, sweating, insomnia, and mood swings were effectively improved, and the effective rate reached 86.67%. No adverse reactions were found in the overall population during the experiment. Therefore, the probiotic composition and its application of the present invention have significant effects on improving menopausal syndrome, are convenient and safe to use, and have very good development prospects for helping women through this physiological stage.

[0170] Although the specific implementation manners of the present invention are described above, they are not limitations on the protection scope of the present invention. Based on the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. Use of a probiotic composition in the preparation of a medicament for improving menopausal symptoms; The probiotic composition is prepared by mixing the following components: Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) LP115, which was deposited at the China General Microbiological Culture Collection Center on August 22, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 25576; Lactobacillus fermentum ( Lactobacillus fermentum ) LF26 ， This strain was deposited at the China General Microbiological Culture Collection Center on February 24, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 19431; Lactobacillus paracasei ( Lactobacillus paracasei ), strain LPC12, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, at the address No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO. 24513 Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), strain LRH10, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 24517; Lactobacillus delbrueckii subsp. lactis ( Lactobacillus delbrueckii subsp. Lactis ), strain LDL114, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 25575; Lactobacillus helveticus ( Lactobacillus helveticus ) LA25, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 25569; The mass ratio of Lactiplantibacillus plantarum LP115, Lactobacillus fermentum LF26, Lactobacillus paracasei LPC12, Lactobacillus rhamnosus LRH10, Lactobacillus delbrueckii subsp. Lactis LDL114, and Lactobacillus helveticus LA25 is 3:3:3:6:1:3; The viable count of the composition is not less than 10 9 CFU.

2. A medicament, characterized in that by weight, it is prepared from the following components: 10-25 parts of Chinese wolfberry, 10-20 parts of Ophiopogon japonicus, 10-18 parts of red dates, 10-18 parts of mulberries, 10-15 parts of Poria cocos, 5-15 parts of smoked plums, 3-5 parts of rose flowers, 3-5 parts of hawthorns, 2-5 parts of wild jujube seeds, 2-5 parts of liquorice, and 2-5 parts of the probiotic composition; The probiotic composition is prepared by mixing the following components: Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ) LP115, which was deposited at the China General Microbiological Culture Collection Center on August 22, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 25576; Lactobacillus fermentum ( Lactobacillus fermentum ) LF26 ， This strain was deposited at the China General Microbiological Culture Collection Center on February 24, 2022. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number is CGMCC NO. 19431; Lactobacillus paracasei ( Lactobacillus paracasei ), strain LPC12, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, at the address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO. 24513 Lactobacillus rhamnosus ( Lactobacillus rhamnosus ), strain LRH10, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 24517; Lactobacillus delbrueckii subsp. lactis ( Lactobacillus delbrueckii subsp. Lactis ), strain LDL114, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number CGMCC NO. 25575; Lactobacillus helveticus ( Lactobacillus helveticus ) LA25, which was deposited at the China General Microbiological Culture Collection Center on March 11, 2022, at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC NO. 25569; The mass ratio of Lactiplantibacillus plantarum LP115, Lactobacillus fermentum LF26, Lactobacillus paracasei LPC12, Lactobacillus rhamnosus LRH10, Lactobacillus delbrueckii subsp. Lactis LDL114, and Lactobacillus helveticus LA25 is 3:3:3:6:1:3; The viable count of the composition is not less than 10 9 CFU.

3. Preparation method of the medicament according to claim 2, characterized in that first, other components except the probiotic composition, with a total amount of 100-120 g, are subjected to water extraction with boiling water at a ratio of medicinal materials to water of 1:20-1:30 for 20-30 min. After filtering to remove residues, they are dried and then pulverized and passed through a 60-100 mesh sieve to prepare an extract powder. Then, the extract powder is mixed evenly with the probiotic composition and filled.

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