Preparation and application of probiotics and fermentation products for removing dampness, tonifying blood and relieving dysmenorrhea

Abstract

The application belongs to the technical field of microorganisms, and provides a Streptococcus salivarius subsp. thermophilus HX-ST36, which has a preservation number of CGMCC NO. 32955. The Streptococcus salivarius subsp. thermophilus provided by the application can ferment Astragalus membranaceus Bunge-Angelica sinensis. In a cold coagulation and blood stasis type primary dysmenorrhea model mouse, the fermentation product thereof can significantly inhibit the writhing response, and can realize the effects of relieving dysmenorrhea and tonifying blood by reducing the levels of prostaglandin PGF2 and vasopressin ADH in uterine tissue and increasing the hemoglobin (HGB) content. The effect of the fermentation product in removing dampness is verified by improving the dampness signs of a crowd. The application further provides a probiotic preparation and a preparation method of a probiotic fermentation product. The preparation method of the probiotic preparation and the probiotic fermentation product is simple and can be produced on a large scale.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Streptococcus thermophilus subsp. saliva and its fermentation products, and probiotic preparations including Streptococcus thermophilus subsp. saliva. Background Technology

[0002] Dysmenorrhea is one of the most common gynecological symptoms, referring to lower abdominal pain, distension, backache, or other discomfort before, during, or after menstruation. Severe dysmenorrhea can affect a woman's daily life and work, leading to fatigue and difficulty concentrating. Dysmenorrhea can be divided into primary and secondary dysmenorrhea. Primary dysmenorrhea refers to dysmenorrhea without organic lesions of the reproductive organs, accounting for more than 90% of dysmenorrhea cases. The occurrence of primary dysmenorrhea is mainly related to the increased content of prostaglandins in the endometrium during menstruation. Increased levels of PGF2α and PGE2 are the main causes of primary dysmenorrhea. In addition, primary dysmenorrhea can also be affected by dampness and blood stasis. When dampness invades the body, it easily lingers in the uterus, affecting the normal flow of menstrual blood, leading to poor menstrual flow and thus causing dysmenorrhea. If a woman already has insufficient blood stasis, coupled with the increased loss of blood stasis during menstruation, it can lead to a decrease in resistance and exacerbate dysmenorrhea. Therefore, removing dampness and replenishing blood play important roles in improving dysmenorrhea symptoms.

[0003] Currently, Western medicines available for relieving menstrual cramps include nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen, aspirin, naproxen, and diclofenac. These are mostly pain relievers and do not improve a woman's overall health. Menstrual cramps tend to recur and have side effects; for example, aspirin can easily damage the gastric mucosa, severely impacting the lives and work of patients. Traditional Chinese medicine (TCM) oral medications include Wuji Baifeng Wan, Yimucao Keli, Aifu Nuangong Wan, and Danggui Buxue Tang. Danggui Buxue Tang is a classic formula created by Li Dongyuan, a physician during the Jin-Yuan period. It contains only two herbs: angelica and astragalus. Angelica has the effects of nourishing blood, regulating menstruation, promoting blood circulation, and relieving pain, while astragalus excels at tonifying qi, raising yang, and strengthening the body's defenses. The combination of these two herbs is a classic TCM approach to nourishing both qi and blood. However, these ingredients have low bioavailability, and their effectiveness often requires a certain course of treatment and long-term use to achieve the benefits of regulating the body's constitution and correcting imbalances. Probiotics can transform active ingredients that are both medicinal and edible into specific small molecules, promote the metabolism of functional active molecules, and achieve the synergistic effect of probiotics.

[0004] The reasons for the increase in active ingredients in medicinal and edible plants during probiotic fermentation can be summarized into three categories: promoting the release of active ingredients or compounds from medicinal and edible plants; generating new compounds (active ingredients are used as precursors by probiotics to synthesize new compounds, and active ingredients interact with secondary metabolites of microorganisms to form new compounds, etc.); and increasing the concentration of active ingredients. To further explore ways to enhance drug efficacy and overcome the challenges of low bioavailability and limited drug efficacy, we employ probiotic fermentation technology to achieve synergistic effects between probiotics and components of Astragalus membranaceus, Angelica sinensis, and Coix lacryma-jobi, fully leveraging the advantages of this combination to maximize the bioavailability and efficacy of the formula.

[0005] Comparative Example 1: Chinese patent application CN106309506B discloses a probiotic composition with menstrual regulating function and its application. The probiotics described include *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, and *Bifidobacterium longum*. The live bacteria ratio of *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, and *Bifidobacterium longum* is 1:1:2. The medicinal materials used in this solution consist of roasted licorice root, wolfberry, red dates, dried tangerine peel, dried ginger, and longan. This solution does not identify the core substances for treating dysmenorrhea and does not explain its functional effects from the perspectives of removing dampness, replenishing blood, and low bioavailability.

[0006] Comparative Example 2: Chinese patent application CN114617949B discloses a method for processing probiotic enzyme raw materials with the functions of removing dampness and relieving dysmenorrhea. It involves inoculating with Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus, fermenting jujubes and dried ginger, and assisting in the treatment of dysmenorrhea from the aspects of removing dampness and dispelling cold. However, it does not explore the core substances for treating dysmenorrhea, nor does it explain its functional effects from the perspectives of replenishing blood and low bioavailability. Summary of the Invention

[0007] The first objective of this invention is to provide a strain of Streptococcus salivarius thermophilus HX-ST36 that can improve iron bioavailability.

[0008] A second objective of this invention is to provide a probiotic preparation comprising the aforementioned Streptococcus salivarius thermophilus subsp. HX-ST36.

[0009] The third objective of this invention is to provide a fermented product that is fermented by Streptococcus salivarius, which can remove dampness, replenish blood, and relieve menstrual cramps.

[0010] This invention provides a strain of Streptococcus salivarius subsp. thermophilus, named HX-ST36, which was deposited on December 6, 2024, at the China General Microbiological Culture Collection Center (CGMCC). The depository code is CGMCC, and the address of the collection center is: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The accession number is CGMCC No. 32955. The strain was viable at the time of deposit.

[0011] The fermentation product of *Streptococcus salivarius* subsp. *thermophilus*, the fermentation product comprising... The fermentation product of *Streptococcus thermophilus* is obtained by fermenting *Angelica sinensis* and *Astragalus membranaceus* with *Streptococcus thermophilus*.

[0012] The method for preparing the fermentation product of Streptococcus salivarius includes the following steps:

[0013] Astragalus powder and Angelica powder are mixed in a weight ratio of 5:1 to obtain a compound raw material. The compound raw material is then mixed with water to obtain a 3-4 wt% fermentation raw material. 3 wt% of HX-ST36 bacterial solution is then inoculated into the fermentation raw material. After inoculation, fermentation is carried out at 37℃ for 24 hours. The fermentation liquid is then concentrated by rotary evaporation to obtain the concentrated fermentation liquid, which is then freeze-dried under vacuum.

[0014] A probiotic preparation comprising the fermentation product and a freeze-drying protectant.

[0015] The freeze-drying protectant comprises the following components by weight percentage: trehalose 5-20%, skim milk powder 15-25%, maltodextrin 1-5%, glycerol 2-5%, inulin 0.1-1%, glutamine 0.5-1%, and water as the balance.

[0016] The method for preparing the probiotic sludge includes the steps of streaking the Streptococcus salivarius thermophilus subsp. sp. and picking single bacteria into liquid MRS medium, culturing at 37°C, and after two rounds of activation, centrifuging to obtain the probiotic sludge.

[0017] The MRS culture medium comprises the following components by weight percentage: 1.0-1.5% soybean peptone, 0.5-1.5% yeast extract, 2.0-3.0% sucrose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.02-0.06% magnesium sulfate heptahydrate, 0.02-0.03% manganese sulfate monohydrate, 0.1-0.3% astragalus powder, 0.1-0.3% angelica powder, and water as the balance.

[0018] The aforementioned probiotic agent is used in the preparation of drugs that improve iron bioavailability; or

[0019] Used in the preparation of medicines for removing dampness; or

[0020] Used in the preparation of hematopoietic drugs; or

[0021] Used in the preparation of antioxidant drugs; or

[0022] It is used in the preparation of drugs to improve dysmenorrhea.

[0023] The beneficial effects of this invention are as follows:

[0024] The thermophilic subsp. salivarius provided by this invention has good probiotic functions, including gastrointestinal fluid tolerance, antibacterial ability, and ability to improve iron bioavailability.

[0025] The fermentation product provided by this invention contains core fermentation components. In mice with cold-induced blood stasis type primary dysmenorrhea, the fermentation product can significantly inhibit writhing response and can improve dysmenorrhea response by reducing the levels of prostaglandin PGF2 and angiotensin ADH in uterine tissue and increasing the content of hemoglobin (HGB).

[0026] The fermentation product of this invention has the effect of removing dampness, specifically in relieving heaviness in the body, improving digestive abnormalities, thick and greasy tongue coating, reducing secretions, improving edema, relieving abdominal pain during dysmenorrhea, and others. Detailed Implementation

[0027] Example 1

[0028] Strains screening and evaluation of probiotic functions

[0029] 1. Strain screening

[0030] Artificial gastric fluid tolerance test: The treated bacterial solution was inoculated into artificial gastric fluid at pH 3.0, and then incubated in a 37°C water bath for 3 hours. The number of viable bacteria was then measured and the survival rate was calculated.

[0031] Artificial intestinal fluid tolerance test: After treating high-survival-rate bacteria in artificial gastric fluid for 3 hours, they were inoculated into artificial intestinal fluid (containing 0.3% bile salts), placed in a 37°C water bath for 3 hours, and then the number of viable bacteria was measured and the survival rate was calculated.

[0032] Table 1. Measurement of Gastrointestinal Fluid Tolerance

[0033]

[0034] Based on the gastrointestinal fluid tolerance test, the following bacteria with a gastrointestinal fluid tolerance rate of over 90% were selected: 88T4, 110M8, BS225M4, BS683M16, HX-ST36, BS906M21, and BS11M9.

[0035] Antibacterial activity assessment: 200 μL of bacterial suspensions of Escherichia coli, Staphylococcus aureus, Shigella, and Salmonella (from Shanghai Microbiology Preservation Center) were evenly spread on blood agar plates and left to stand for 30 min. After the bacterial suspensions on the surface were fixed, 200 μL of lactic acid bacteria suspension (OD600 around 1.0) was injected into the wells using an Oxford cup. Physiological saline was used as a control. After incubation at 37℃ for 24 h, the results were observed and the diameter of the inhibition zone was measured.

[0036] Table 2 Evaluation of Antibacterial Ability

[0037]

[0038] Based on the above antibacterial ability evaluation, BS683M16, BS225M4, HX-ST36, and BS906M21 were selected.

[0039] Antioxidant capacity: Comparison of ABTS free radicals, DPPH free radicals, and O2 free radicals in the rescreened strains 2- Free radicals, OH - Free radical scavenging ability.

[0040] Table 3. Evaluation of antioxidant capacity of screened strains

[0041]

[0042] Antioxidant capacity assessment showed that BS225M4 and HX-ST36 can effectively remove ABTS, DPPH, and O. 2- OH - Free radical scavenging rate is over 80%.

[0043] Example 2

[0044] Screening of strains to improve iron bioavailability

[0045] To achieve the blood-tonifying effect, Angelica sinensis blood-tonifying decoction has a blood-tonifying effect, but its bioavailability is low. Therefore, it is necessary to screen for strains with good fermentation performance and strains that can improve the bioavailability of Angelica sinensis blood-tonifying decoction.

[0046] Evaluation of fermentation acid production performance: The strains were inoculated into MRS liquid medium and cultured at 37°C for 24 hours. After three generations of activation, the fermentation supernatant of each strain was collected, and the pH of each fermentation supernatant was measured.

[0047] Table 4 pH of fermentation supernatant

[0048]

[0049] The pH values ​​at the fermentation endpoints of the two strains showed no significant difference, indicating that both strains exhibited good acid-producing performance.

[0050] Caco-2 cell monolayer uptake model and iron bioavailability determination:

[0051] Sample preparation: Prepare an Angelica / Astragalus solution (2g Angelica / Astragalus powder dissolved in 100mL PBS). Inoculate BS225M4 and HX-ST36 lactic acid bacteria into the Angelica / Astragalus solution at an inoculum size of 3%. Fermentation temperature is 37℃, rotation speed is 100r / min, and fermentation time is 20-26h. After fermentation, inactivate all fermentation broth (100℃, 20min) and collect the inactivated liquid. Digest the inactivated bacteria in vitro with gastrointestinal fluid and collect the digested liquid.

[0052] Group design: Angelica A group: BS225M4 + Angelica solution; Angelica B group: HX-ST36 + Angelica solution; Astragalus A group: BS225M + Astragalus solution; Astragalus B group: HX-ST36 + Astragalus solution; The positive control group was 15ml of a mixture of FeSO4 containing 10μg of iron and vitamin C containing 1mmol / L.

[0053] Uptake model construction: Caco-2 cells were maintained in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% non-essential amino acids (NEAA) and cultured at 37°C and 5% CO2. The cells were then incubated at a rate of 50,000 cells / cm². 2Cells were seeded at a density in 6-well plates. The culture medium was changed every 2-3 days after seeding. Cell uptake experiments were performed after 13 days of culture (cells had differentiated into a monolayer uptake model). Before each experiment, the DMEM medium was replaced with MEM medium (containing 1% non-essential amino acids but excluding fetal bovine serum (FBS)). The inactivated liquid and MEM medium were mixed at a 1:2 ratio and cultured at 37°C. After 13 days of culture, the culture medium in each well was aspirated, the cells were washed twice with MEM, 1 mL of MEM nutrient solution was added, and the cells were cultured in Transwell plates. Digested liquid was added to the upper chamber, and the reaction was shaken for 2 hours. The upper chamber was removed, and 1 mL of MEM nutrient solution was added to each well. Cells were cultured for another day, and then the cells were collected. The MEM medium was aspirated, and the cell monolayer was washed sequentially with 2 mL of washing buffer and 2 mL of iron removal solution. 2 mL of ultrapure water was added, and the cells were scraped off with a cell scraper. The liquid in the wells was collected and the cells were sonicated at 4°C. The protein content in cell sonication fluid was detected using a BCA kit, and the ferritin content in cells was detected using a ferritin assay kit. Iron bioavailability was calculated as: ferritin content in cells (ng) / protein content in cells (mg).

[0054] Table 5. Protein content in Caco-2 cells

[0055]

[0056] Protein content (mg / mL) by group: Angelica A group 0.51±0.05, Angelica B group 0.52±0.02, Astragalus A group 0.48±0.03, Astragalus B group 0.42±0.01, Positive control group (FeSO4+Vitamin C) 0.38±0.02.

[0057] There was no significant difference in protein content in the cells of each experimental group, indicating that the number of cells in the experimental groups was relatively balanced.

[0058] Table 6 Iron bioavailability in different groups

[0059]

[0060] This shows that the iron bioavailability of Angelica sinensis group B and Astragalus membranaceus group B was not significantly different from that of the positive control group. It is evident that the raw materials fermented with HX-ST36 can better promote the production of metabolites and enhance iron bioavailability.

[0061] Example 3

[0062] Screening strain identification results and sources

[0063] Table 7. Identification results and sources of screened strains

[0064]

[0065] The 16S DNA sequence of Streptococcus salivarius subsp. HX-ST36 is shown below:

[0066]

[0067] Example 4

[0068] Preparation method of probiotic agents

[0069] Probiotic fermentation culture: After streaking the probiotics, single bacteria are picked and transferred to liquid MRS, cultured at 37°C, and after two rounds of activation, centrifuged to obtain probiotic sludge; the culture medium includes the following components by weight percentage: soybean peptone 1.0-1.5%, yeast extract 0.5-1.5%, sucrose 2.0-3.0%, Tween 0.05-0.15%, dipotassium hydrogen phosphate heptahydrate 0.1-0.2%, sodium acetate trihydrate 0.4-0.6%, magnesium sulfate heptahydrate 0.02-0.06%, manganese sulfate monohydrate 0.02-0.03%, astragalus powder 0.1-0.3%, angelica powder 0.1-0.3%, and distilled water balance;

[0070] Preparation of probiotic freeze-dried powder: The probiotic slurry is mixed evenly with a freeze-drying protectant and freeze-dried to obtain probiotic freeze-dried powder; the freeze-drying protectant includes the following components by weight percentage: trehalose 5-20%, skim milk powder 15-25%, maltodextrin 1-5%, glycerol 2-5%, inulin 0.1-1%, glutamine 0.5-1%, and water as the balance.

[0071] The probiotic is Streptococcus salivarius thermophilus subsp. HX-ST36.

[0072] Example 5

[0073] Preparation method of fermented medicinal and edible raw materials: Raw material preparation: Astragalus powder and Angelica powder are compounded in a 5:1 ratio to obtain the medicinal and edible compound raw material. Preparation of raw materials before fermentation: 3-4% of the compound raw material is prepared using purified water (30-40ml of medicinal and edible fermentation raw material is added to 1000ml of ultrapure water), and then concentrated by rotary evaporation to obtain the pre-fermentation concentrate (10ml), which is used for subsequent animal experiments; Preparation of raw materials after fermentation: 3-4% of the compound raw material is prepared using purified water (30-40ml of medicinal and edible fermentation raw material is added to 1000ml of ultrapure water), mixed evenly, and then inoculated with 3% HX-ST36 bacterial solution. After inoculation, fermentation is carried out at 37℃ for 24h, and then the fermentation liquid is concentrated by rotary evaporation to obtain the post-fermentation concentrate (10ml), which is used for subsequent animal experiments.

[0074] Post-fermentation extract: After fermentation, take 10 mL of the post-fermentation bacterial solution, add 10 mL of 80% ethanol, heat in a water bath to 45°C, shake repeatedly for more than 20 minutes, collect the supernatant after ethanol soaking, which is the post-fermentation extract. After lyophilization, reconstitute with 10 mL of PBS for subsequent animal experiments.

[0075] Example 6

[0076] Extraction and detection of active ingredients after fermentation of probiotics into food-medicine homology formulations:

[0077] 10 mL of the fermented bacterial broth was drawn up using a sterile syringe, and 10 mL of 80% ethanol was added. The mixture was heated to 45°C in a water bath. The ethanol-soaked supernatant was collected and loaded onto an AB-8 macroporous adsorption resin column at a flow rate of 1.0 BV / h for adsorption. Sugars and some flavanones were removed with 5% ethanol solution, and then eluted with 70% ethanol aqueous solution at a flow rate of 2.0 BV / h for 4 BV. The eluent was collected. The collected eluent was analyzed by HPLC to detect the effective components. The same components were combined and concentrated under vacuum. The concentrate was collected, dissolved in DMSO, and loaded onto a DAC dynamic axial compression system. After HPLC analysis, the target products were combined and freeze-dried under vacuum to obtain the pure compound.

[0078] Take 0.1 mg of the above compound and dilute to 1 mL in a volumetric flask. Filter through a 0.22 μm filter membrane and then perform ultra-high performance liquid chromatography (UHPLC) for detection. The chromatographic conditions used were as follows: a C18 column, with double-distilled water (ddH2O) containing 2‰ formic acid as mobile phase A (aqueous phase) and chromatographic grade methanol as mobile phase B (organic phase). Gradient elution and separation of the adsorbed active components were performed. Elution conditions were: 0 min, 5% (phase B); 1 min, 15% (phase B); 5.0 min, 65%; 6.0 min, 70%; 9.0 min, 90%; 15 min, 15%; 18 min, 5%. Flow rate: 1.2 mL / min; column temperature: 50 °C; single injection volume: 1 μL; two injections per sample to reduce error; detection wavelength: 330 nm. The substance with the highest retention time of 8.58 min extracted using the above method has the highest abundance and is likely its core active ingredient. This substance was diluted to 50 mg / mL and used as the active ingredient group after fermentation for subsequent animal experiments.

[0079] Its spectral data are as follows:

[0080] 1H NMR (300MHz, CD3CN, δ, ppm): 9.72(1H in CHO), 7.12(1H in 1-ethylene), 7.02(1H in 1-benzene), 6.77(1Hin 1-benzene), 5.43(1H in 1-ethylene), 5.42(1H in1-ethylene), 3.54-3.285(2H in methine), 3.14(1H in methine), 2.85(2H inmethylene), 2.75(2H in methylene), 2.61(1H in methine), 2.48(1H in methine), 2.15(2H in methylene), 1.665(2H inmethylene), 1.63(3H in methyl), 1.07(1H in secamine), 0.83(3H in methyl)Protocol The C-13 NMR Prediction (Lib = S) shows the following values: 201.5, 183.0, 150.8, 142.3, 133.3, 129.5, 129.3, 125.3, 123.7, 122.0, 119.9, 74.3, 59.9, 52.9, 51.2, 41.6, 37.7, 24.6, 24.3, 17.6, 13.1. Based on the above data, the structure of this active ingredient is as follows:

[0081] .

[0082] Example 7

[0083] animal experiments

[0084] Animal Experiment Design: The model animal was the Wistar rat, female, 7-8 weeks old, weighing 200-220g. After one week of acclimatization feeding, 70 rats were randomly divided into 7 groups (n=10 per group): normal control group, model group, ibuprofen positive control group (POS), pre-fermentation compound raw material group (B-AP group), post-fermentation compound raw material group (A-AP group), post-fermentation extract, post-fermentation active ingredient dilution, and probiotic group. The normal control group received no treatment. The model group was established using a combination of ice water stimulation and injection of estradiol benzoate and oxytocin to create a rat model of primary dysmenorrhea due to cold stagnation and blood stasis. Rats were subjected to cold stimulation at a fixed time each day in an ice-water mixture at (0±1)℃ (the water level should be sufficient to immerse the rat's hind limbs and lower abdomen). This procedure was performed once daily for 20 minutes each time, for 12 consecutive days. Meanwhile, rats were given subcutaneous injections of 0.4 mg / rat of estradiol benzoate daily, and 0.8 mg / rat on day 1 and day 12 (the first and last injection doses were doubled to ensure stable blood drug concentration). One hour after the last injection of estradiol benzoate, oxytocin 2 IU / rat was injected intraperitoneally.

[0085] Rats in the control and model groups were administered physiological saline by gavage;

[0086] Ibuprofen positive control group (POS): Ibuprofen was administered orally at a dose of 0.2 g / kg / day starting from day 1 for 12 consecutive days;

[0087] Pre-fermentation group (B-AP group): Starting from day one, patients were given 1 ml / kg / day of pre-fermentation concentrated raw material orally.

[0088] Post-fermentation group (A-AP group): Orally administered 1 ml / kg / day of post-fermentation concentrated raw material starting from day 1;

[0089] Fermentation extract group (T-AP group): 1 ml / kg / day of fermentation extract was administered orally starting from day 1;

[0090] Probiotic group (PRO group): Starting from day one, oral administration of 0.1 billion CFU / kg / day of probiotics;

[0091] Post-fermentation active ingredient dilution (AI-AP): Raw material after oral administration of 1 ml / kg / day of fermented active ingredient starting from day one;

[0092] Evaluation indicators:

[0093] ① Observe the number of writhing responses, the incidence of writhing, and the rate of writhing inhibition. Number of writhing responses: Record the number of writhing responses in each group of mice within 30 minutes after intraperitoneal injection of oxytocin.

[0094] Incidence of writhing = (Number of animals exhibiting writhing in each group / Total number of experimental animals in each group) × 100%

[0095] Writhing inhibition rate = [(Number of writhing responses in the model group - Number of writhing responses in the drug-treated group) ÷ Number of writhing responses in the model group] × 100%

[0096] ② ELISA kit for detecting prostaglandin PGF2 and angiotensin (ADH) levels in uterine tissue

[0097] ③ Complete blood count: Red blood cells (RBC), White blood cells (WBC), Hemoglobin (HGB)

[0098] Table 8. Effects of different groups on writhing response

[0099]

[0100] Compared with the model group, the number of writhing responses in rats was significantly reduced after probiotic fermentation, significantly decreasing the incidence of writhing and increasing the writhing inhibition rate. In particular, the writhing inhibition rate of the fermented extract group and the active ingredient group reached 60% or more.

[0101] Table 9. Key gene levels in uterine tissue

[0102]

[0103] After modeling, the levels of PGF2 and ADH in the rat uterine tissue were significantly increased. Treatment with the compound raw materials reduced both PGF2 and ADH levels, with the fermented group showing better results than the unfermented group, nearly restoring them to normal levels. Probiotics also showed some inhibitory effect. Furthermore, the fermented extract group also significantly reduced PGF2 and ADH levels, indicating that the extract contains key core components. Compared to the fermented extract group, the active ingredient group showed no significant difference in reducing PGF2 and ADH levels, suggesting that its active ingredient is the core substance that reduces the levels of key genes in the uterine tissue.

[0104] Table 10 Blood routine analysis of different groups

[0105]

[0106] Blood routine analysis results showed that the RBC, HGB, and WBC counts in the model group were significantly lower than those in the control group. After intervention, compared with the model group, the pre-fermentation, post-fermentation, and extract groups all showed increased red blood cell levels, decreased white blood cell levels, and increased hemoglobin (HGB) content, with the post-fermentation group showing better results, especially the active ingredient group.

[0107] In summary, in mice with primary dysmenorrhea caused by cold stagnation and blood stasis, the fermented compound raw material group can significantly inhibit the writhing response and can achieve the effects of replenishing blood and improving dysmenorrhea by reducing the levels of prostaglandin PGF2 and angiotensin ADH in uterine tissue and increasing the content of hemoglobin (HGB).

[0108] Example 8

[0109] Human trials to verify (efficacy in removing dampness)

[0110] The experiment was conducted on women diagnosed with excessive dampness who voluntarily underwent intervention. The participants exhibited the following symptoms: ① Heaviness in the body: weakness in the limbs; ② Oily hair; ③ Digestive abnormalities: loss of appetite, abdominal distension, sticky or loose stools that easily stick to the toilet; ④ Thick, greasy tongue coating (white or yellow), with teeth marks on the edges; ⑥ Increased vaginal discharge: copious, thick vaginal discharge, possibly with an unpleasant odor; ⑦ Edema and skin problems: facial or lower limb edema, oily skin, recurring eczema, and acne; ⑧ Other symptoms: sticky mouth, bitter taste, morning fatigue, etc.; ⑨ During menstruation, significant abdominal pain and cold sensations, relieved by applying heat (cold dampness).

[0111] Exclusion criteria: Individuals found to be in a disease state through comprehensive physical examination; healthy individuals; pregnant or planning pregnancy women, and breastfeeding women; individuals suspected or confirmed to have a history of alcohol or drug abuse; patients currently participating in other drug clinical trials; and those unwilling to cooperate. A total of 40 female patients, aged 20-55 years (the youngest being 20 years old), were clinically verified and randomly divided into three groups: Experimental Group 1, taking 2g of the fermented raw material prepared in Example 5 of this invention before bedtime each night; Experimental Group 2, taking 2g of the probiotic preparation (100 million CFU / g) prepared in Example 4 of this invention before bedtime each night; Comparative Example 1, taking 2g of the raw material prepared in Comparative Example 1 of this invention before bedtime each night; and Comparative Example 2, taking 2g of the raw material prepared in Comparative Example 2 of this invention before bedtime each night.

[0112] A course of treatment lasts two weeks. Table 11 shows the results of each group after one course of treatment.

[0113] Table 11

[0114]

[0115] As can be seen from Table 11, the fermented product group 1 (Astragalus and Angelica probiotic fermented product) of the present invention has a good dampness-removing effect and its application effect in human trials is significant; the experimental group 2 (probiotic preparation) is worse than comparative example 1 and comparative example 2 in relieving digestive abnormalities; overall, the effects of the two comparative examples are not as good as those of experimental group 1 (fermented product group).

Claims

1. Streptococcus salivarius subsp. thermophilus HX-ST36, characterized by: Its accession number is CGMCCNo.32955.

2. The fermentation product of *Streptococcus thermophilus* subsp. as described in claim 1, characterized in that: The fermentation products include .

3. The fermentation product of *Streptococcus thermophilus* subsp. as described in claim 2, characterized in that: It is obtained by fermenting Angelica sinensis and Astragalus membranaceus with Streptococcus salivarius as described in claim 1.

4. The method for preparing the fermentation product of *Streptococcus thermophilus* as described in claim 3, characterized in that: The process includes the following steps: Astragalus powder and Angelica powder are mixed in a weight ratio of 5:1 to obtain a compound raw material. Then, the compound raw material is mixed with water to obtain 3-4 wt% fermentation raw material. Then, 3 wt% of HX-ST36 bacterial solution is inoculated into the fermentation raw material. After inoculation, fermentation is carried out at 37℃ for 24 hours. After that, the fermentation liquid is concentrated by rotary evaporation to obtain the fermentation concentrate, which is then vacuum freeze-dried to obtain the final product.

5. A probiotic preparation, characterized in that: It includes the fermentation product and freeze-drying protectant as described in claim 3.

6. The probiotic preparation as described in claim 5, characterized in that: The freeze-drying protectant comprises the following components by weight percentage: trehalose 5-20%, skim milk powder 15-25%, maltodextrin 1-5%, glycerol 2-5%, inulin 0.1-1%, glutamine 0.5-1%, and water as the balance.

7. The probiotic preparation as described in claim 5, characterized in that: The method for preparing the probiotic preparation includes the steps of streaking Streptococcus thermophilus subsp. as described in claim 1, picking single bacteria into liquid MRS medium, culturing at 37°C, and after two rounds of activation, centrifuging to obtain probiotic sludge.

8. The probiotic preparation as described in claim 7, characterized in that: The MRS culture medium comprises the following components by weight percentage: 1.0-1.5% soybean peptone, 0.5-1.5% yeast extract, 2.0-3.0% sucrose, 0.05-0.15% Tween, 0.1-0.2% dipotassium hydrogen phosphate heptahydrate, 0.4-0.6% sodium acetate trihydrate, 0.02-0.06% magnesium sulfate heptahydrate, 0.02-0.03% manganese sulfate monohydrate, 0.1-0.3% astragalus powder, 0.1-0.3% angelica powder, and water as the balance.

9. The application of the probiotic preparation as described in claim 5, characterized in that: Used in the preparation of drugs that improve iron bioavailability; or It is used in the preparation of drugs to improve dysmenorrhea.

Images