Use of lactobacillus rhamnosus ZJUIDS07 in preparation of a drug for relieving primary dysmenorrhea

Abstract

This invention belongs to the field of microbial technology, specifically relating to a strain of *Lactobacillus rhamnosus* ZJUIDS07 with the effect of relieving primary dysmenorrhea and its application. This invention discloses a strain of *Lactobacillus rhamnosus* (ZJUIDS07). Lactobacillus rhamnosus The application of ZJUIDS07 in the preparation of drugs with the effect of relieving primary dysmenorrhea, Lactobacillus rhamnosus ( Lactobacillus rhamnosus The accession number for ZJUIDS07 is: CGMCCNO.23996. *Lactobacillus rhamnosus* ( Lactobacillus rhamnosus ZJUIDS07 regulates PGE2 and PGF 2a This level of fluid can help relieve menstrual cramps.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a strain of Lactobacillus rhamnosus ZJUIDS07 that has the effect of relieving primary dysmenorrhea and its application in the pharmaceutical field. Background Technology

[0002] Dysmenorrhea is a common chronic gynecological disease among young women, referring to lower abdominal pain and distension that occurs before, during, or after menstruation, often accompanied by symptoms such as lower back pain, dizziness, vomiting, and diarrhea. It is mainly divided into primary dysmenorrhea (PD) and secondary dysmenorrhea (SD). Primary dysmenorrhea is a spasmodic pain that occurs in women with normal pelvic structures, usually lasting 1-3 days. Secondary dysmenorrhea is often accompanied by organic lesions, generally caused by endometriosis, pelvic inflammatory disease, cervicitis, etc., and is mostly treated with surgery or medication. The incidence of dysmenorrhea is about 65% in women of reproductive age, with primary dysmenorrhea accounting for about 90% of the total incidence. Studies show that recurrent dysmenorrhea increases the incidence of chronic pain-related diseases such as irritable bowel syndrome, painful bladder syndrome, adenomyosis, and uterine fibroids later in life, posing a potential threat to the physical and mental health of women of reproductive age.

[0003] Currently, the accepted pathogenesis of primary dysmenorrhea is the prostaglandin (PGs) theory. Under normal circumstances, the shedding of the uterine lining forms menstrual blood clots that accumulate in the uterine cavity. However, due to the limited capacity of the uterine cavity and the narrow cervix, menstrual blood cannot be expelled spontaneously. At this time, the body produces prostaglandins to stimulate uterine contractions, thereby ensuring normal menstrual flow. When prostaglandin levels are too high, they can stimulate uterine blood vessels and nerves, causing pain. Clinically, nonsteroidal anti-inflammatory drugs (NSAIDs) that inhibit PG synthesis, such as ibuprofen and diclofenac sodium, are commonly used for treatment. Contraceptive drugs that reduce PG synthesis by ingesting exogenous estrogen to influence endogenous hormone secretion, altering ovarian endocrine patterns and the cyclical changes of the endometrium, are also used. Both methods have drawbacks such as numerous side effects, treatment intolerance, and cyclical medication, and cannot cure or prevent dysmenorrhea. In addition, traditional Chinese medicine has advantages in treating dysmenorrhea, including holistic regulation, fewer side effects, and personalized treatment, but it also has disadvantages such as slow onset of action, long treatment cycles, and unstable efficacy. Therefore, finding effective treatments for primary dysmenorrhea is of great significance to women's physical and mental health.

[0004] In recent years, the use of lactic acid bacteria to regulate gut microbiota for the treatment or relief of Parkinson's disease (PD) has garnered widespread attention. Currently, strains proven effective in alleviating PD or uterine and pelvic inflammatory disease include *Lactobacillus rhamnosus*, *Lactobacillus plantarum*, *Lactobacillus reuteri*, *Pediococcus lactis*, and *Weisseria gonorrhoeae*. *Lactobacillus rhamnosus*, as a type of lactobacillus, can colonize the human gut and exert probiotic effects when added to food or pharmaceuticals, such as enhancing immunity, maintaining gut microecological balance, inhibiting the growth of intestinal pathogens, improving lactose digestion, and providing antioxidant benefits. Due to its natural, safe, and highly effective probiotic properties, it shows promising application prospects in animal husbandry, food processing, and clinical treatment.

[0005] Invention CN116042470A, entitled "Fermentation of Traditional Chinese Medicine Using a Novel Lactaseibacillus Rhamnosus Strain and Its Application," discloses a strain of Lactaseibacillus rhamnosus LR-K0916, with accession number GDMCC No: 63006. It can be used to treat reproductive system diseases, including vaginitis, uterine fibroids, dysmenorrhea, breast nodules, breast cancer, and menopausal syndrome. However, no corresponding experimental data is provided.

[0006] Invention CN107158041A, entitled "A Probiotic Composition for Treating Gynecological Inflammation," discloses a composition for treating gynecological inflammation, comprising the following components: resistant dextrin, inulin, stachyose, *Lactobacillus rhamnosus* TR08, and *Lactobacillus reuteri* TR02. The accession number for *Lactobacillus rhamnosus* TR08 is CCTCCNO.M2016548.

[0007] Invention CN106309506A, entitled "A Probiotic Composition with Menstrual Regulating Function and Its Application," discloses that a probiotic composition composed of Lactobacillus rhamnosus, Lactobacillus plantarum, and Bifidobacterium longum has a menstrual regulating function, particularly in inhibiting primary dysmenorrhea. However, Lactobacillus rhamnosus, when used alone, has almost no menstrual regulating function.

[0008] The invention CN112294892A, entitled "Fermented Siwu Decoction Beverage Composition with Dysmenorrhea Relief Effect and Preparation Method", discloses a fermented Siwu Decoction beverage composition with dysmenorrhea relief effect. The raw materials include white peony root, angelica root, chuanxiong rhizome, and prepared rehmannia root. The Chinese herbal raw materials white peony root, angelica root, chuanxiong rhizome, and prepared rehmannia root are fermented by a compound bacteria composed of Lactobacillus plantarum and Rhamnosus bacillus.

[0009] Lactobacillus rhamnosus ZJUIDS07 (accession number CGMCCNO.23996) is only known to have hypoglycemic function in CN116064285A; products with hypoglycemic function contain live bacterial preparations of Lactobacillus rhamnosus ZJUIDS07. Summary of the Invention

[0010] The technical problem to be solved by this invention is to provide a new use for Lactobacillus rhamnosus ZJUIDS07 – to relieve primary dysmenorrhea.

[0011] To address the aforementioned technical problems, this invention provides the application of Lactobacillus rhamnosus ZJUIDS07 in the preparation of a drug with the effect of relieving primary dysmenorrhea. The accession number of Lactobacillus rhamnosus ZJUIDS07 is: CGMCCNO.23996.

[0012] As an improvement to the application of this invention: Lactobacillus rhamnosus ZJUIDS07 regulates PGE2 / PGF 2a This level of fluid can help relieve menstrual cramps.

[0013] Explanation: Regulation of PGE2 and PGF 2a The level of the uterus can regulate rhythmic uterine contractions and inhibit uterine smooth muscle spasms, thereby relieving menstrual cramps.

[0014] As a further improvement to the application of the present invention: Lactobacillus rhamnosus ZJUIDS07 inhibits the secretion of TNF-α and IL-6, thereby achieving the effect of relieving dysmenorrhea.

[0015] As a further improvement to the application of the present invention, a traditional Chinese medicine preparation with the effect of relieving primary dysmenorrhea can also be prepared.

[0016] As a further improvement to the application of the present invention: Lactobacillus rhamnosus ZJUIDS07 was prepared to have a viable count of 1.0 × 10⁻⁶. 11 ~1.0×10 12 CFU / g live bacteria preparation.

[0017] According to patent CN116064285A, Lactobacillus rhamnosus ZJUIDS07 exhibits good acid and bile salt resistance, making it suitable for the gastrointestinal environment and possessing strong colonization ability. The culture medium of Lactobacillus rhamnosus ZJUIDS07 shows no antibiotic resistance, indicating that this strain is sensitive to antibiotics, does not carry drug-resistant genes, and has good safety. Lactobacillus rhamnosus ZJUIDS07 possesses antibacterial activity and can inhibit the proliferation of pathogenic bacteria in the intestine. Lactobacillus rhamnosus ZJUIDS07 also exhibits strong antioxidant capacity, significantly higher than other existing strains of the same type.

[0018] The focus of this invention is on adjusting PGE2\PGF 2a The level of pain-causing factors is adjusted to reduce pain. Furthermore, strain ZJUIDS07 has the function of regulating inflammatory factors in the body, which can prevent dysmenorrhea and regulate the menstrual cycle. Compared with other Lactobacillus rhamnosus strains described in the background art, the Lactobacillus rhamnosus ZJUIDS07 of this invention has a better effect on relieving primary dysmenorrhea, a stronger ability to reduce pain and inflammation, and also has a significant preventive and therapeutic effect when used alone, achieving the purpose of preventing and treating dysmenorrhea.

[0019] Specifically, in response to the current lack of research on the therapeutic effect of Lactobacillus rhamnosus in primary dysmenorrhea, this invention provides a strain of Lactobacillus rhamnosus ZJUIDS07 with the effect of relieving primary dysmenorrhea and its application. The effect of Lactobacillus rhamnosus on relieving primary dysmenorrhea was investigated using a mouse model of primary dysmenorrhea writhing induced by a combination of estradiol benzoate and oxytocin.

[0020] The *Lactobacillus rhamnosus* of this invention can be used to prepare live bacterial preparations with the effect of relieving primary dysmenorrhea, can be used to prepare bacterial powder with the function of relieving primary dysmenorrhea, and can be used to prepare traditional Chinese medicine compound preparations with the function of relieving primary dysmenorrhea. Therefore, the *Lactobacillus rhamnosus* of this invention can be widely used to develop probiotic functional products related to relieving primary dysmenorrhea.

[0021] In practical use, the Lactobacillus rhamnosus ZJUIDS07 of this invention can be used in conjunction with a menstrual pain reliever orally. The dosage can be calculated by multiplying the dosage for mice in Example 1 below by a conventional conversion factor (10-12 times). Attached Figure Description

[0022] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings;

[0023] Figure 1 This is a colony morphology diagram of Lactobacillus rhamnosus ZJUIDS07.

[0024] Figure 2 This is a comparison chart of uterine weight changes in mice from different experimental groups;

[0025] Figure 3 A comparison chart of writhing response pain indices for mice in each experimental group: writhing response latency, writhing pain score, and writhing inhibition rate.

[0026] Figure 4 The effects of Lactobacillus rhamnosus ZJUIDS07 on the serum pain biochemical indicators PGE2 and PGF in mice of each experimental group. 2a The impact;

[0027] Figure 5 The effect of Lactobacillus rhamnosus ZJUIDS07 on the inflammatory biochemical markers TNF-α and IL-6 in the serum of mice in each experimental group. Detailed Implementation

[0028] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0029] Example 1: Determination of the ability of Lactobacillus rhamnosus ZJUIDS07 (accession number: CGMCC NO.23996) to relieve primary dysmenorrhea.

[0030] 1.1 Laboratory Animals

[0031] Thirty-five female C57BL / 6J mice were purchased from Beijing Vital River Laboratory Animal Co., Ltd. and housed in an SPF environment at the Laboratory Animal Center of Zhejiang University.

[0032] 1.2 Preparation of the oral medication solution

[0033] 1.2.1 Preparation of the oral bacterial solution

[0034] Lactobacillus rhamnosus ZJUIDS07 was cryopreserved at -80℃. The strain was activated in MRS liquid medium at 37℃ for 16 h. After three subcultures, the cells were centrifuged at 5000 rpm for 10 min at 4℃, the supernatant was discarded, and the cells were collected. The cells were washed twice with PBS buffer (0.1 mol / L, pH 6.8), and the bacterial concentration was adjusted to 1 × 10⁻⁶ cells / mL with PBS. 9 The bacterial culture was prepared at CFU / mL and administered by gavage on the same day.

[0035] 1.2.2 Preparation of the Gavage Dysmenorrhea Relief Solution

[0036] The dysmenorrhea relief granules were purchased commercially from Zhongjing Wanxi Pharmaceutical Co., Ltd. The dosage was 4g / kg per animal. After preparation (dissolving in water), the granules were administered by gavage on the same day. In animal experiments, the gavage volume for each mouse was 200μL.

[0037] 1.3. Establishment of a mouse model of primary dysmenorrhea with writhing posture

[0038] In this experiment, mice were given a routine acclimatization diet for 3 days, followed by subcutaneous injections of estradiol benzoate at 5 mg / kg for 15 consecutive days. On day 15, oxytocin was injected intraperitoneally at 100 U / kg one hour after the estradiol injection to induce a primary dysmenorrhea writhing model in mice. Animal welfare and experimental procedures were conducted in accordance with the guidelines of the Zhejiang University Laboratory Animal Management Regulations.

[0039] 1.4 Experimental Design of the Relief Effect of Lactobacillus rhamnosus ZJUIDS07 on a Mouse Model of Primary Dysmenorrhea with Writhing

[0040] After 3 days of routine acclimatization, female C57BL / 6J mice were randomly divided into four groups of five mice each (n=5): a normal control group, a model group, a positive control group receiving a menstrual irregularity treatment (DUT) solution, and a ZJUIDS07 intervention group. Except for the normal control group, which received saline injections for 15 days, the model group, the positive control group receiving DUT, and the ZJUIDS07 intervention group received estradiol benzoate injections for 15 consecutive days. Except for the normal and model groups, starting from day 3 of estradiol benzoate injection, the DUT group received 200 μL of DUT solution by gavage, and the ZJUIDS07 intervention group received 200 μL of a mixture of DUT and bacterial solution (mixed at a 1:1 volume ratio) by gavage for 12 consecutive days.

[0041] 1.4.1 Detection of writhing indices in mice

[0042] On day 15, the normal group, model group, positive control group (Dongjingbao), and ZJUIDS07 intervention group were injected with oxytocin 1 hour after estradiol benzoate injection. The writhing behavior of mice in each group was observed within 30 minutes, and the writhing response score, latency, and inhibition rate were recorded. The writhing response is an indicator for assessing uterine spasmodic contractions (pain) and can directly reflect whether the primary dysmenorrhea model has been successfully established. According to the four-level behavioral scoring system proposed by Schmauss, the writhing response score and latency were recorded, and the inhibition rate was calculated. The writhing latency refers to the time required from the start of oxytocin injection to the first writhing response in the mouse, recorded in minutes. The writhing response was scored using the standards in Table 1: writhing score = number of times at level 0 × 0 points + number of times at level 1 × 1 point + number of times at level 2 × 2 points + number of times at level 3 × 3 points. The writhing inhibition rate was calculated as follows: [(mean writhing response score of the model group - mean writhing response score of the ZJUIDS07 intervention group) / mean writhing score of the model group] × 100%. During the experiment, all other feeding conditions, such as temperature, humidity, light, and feed, remained consistent.

[0043] 1.4.2 Detection of pain factors and inflammatory factors in mouse uterine tissue

[0044] After observing the writhing response, mice were immediately anesthetized. Fresh uterine tissue was quickly harvested from mice on ice, and its morphology, color, texture, and presence of edema were observed visually. The uterine tissue was rinsed with pre-cooled PBS to remove residual blood, weighed, and then minced. The minced tissue was added to a homogenizer with an equivalent volume of PBS at a ratio of 1:9 and thoroughly homogenized to prepare a 10% tissue homogenate. The homogenate was centrifuged at 8500 rpm for 10 min at 4°C, and the supernatant was collected. PGF in the uterine tissue supernatant was detected using ELISA. 2a The levels of PGE2 (a pain marker), IL-6, and TNF-α (an inflammatory marker) were measured. ELISA kits were purchased from Nanjing Jiancheng or Beijing Pulilai Reagent Co., Ltd., and all procedures were strictly followed according to the instructions. Upon the appearance of a colorimetric reaction, the absorbance was immediately measured using a multi-functional microplate reader, and the corresponding levels were then calculated based on the standard concentration curve.

[0045] Table 1. Rating criteria for pain during writhing response

[0046]

[0047] 1.5 General Case

[0048] During the experiment, the normal group mice showed good health in all aspects, with glossy fur, rapid activity, and good mental state. The model group mice showed significantly reduced activity, dull fur, decreased appetite, and reduced response to external environmental stimuli after modeling compared to the normal group mice. The condition of the dysmenorrhea relief group and the bacterial strain intervention group mice improved, with restored fur luster, increased food and water intake, and slightly increased activity.

[0049] 1.6 Analysis of Experimental Results

[0050] Figure 2 The results showed that the uterine weight of the model group mice was significantly higher than that of the normal group, indicating successful modeling. This may be related to the endometrial thickening, uterine enlargement, and uterine retention caused by continuous injection of estradiol benzoate. The main effect of estrogen on the uterus is to promote the proliferation of myometrial and endometrial cells. Estrogen stimulates the mitosis of endometrial glandular epithelial cells and stromal cells, causing the endometrium to proliferate from the basal layer. Under the influence of estrogen, the endometrium undergoes cyclical proliferation. During the proliferative phase of the menstrual cycle, estrogen levels gradually increase, glandular epithelial cells enlarge, nuclear division increases, and intracellular glycogen and other substances increase. Myometrial cells also increase in number and volume under the stimulation of estrogen, resulting in an increase in the overall volume and mass of the uterus.

[0051] Note: Compared with the normal group # p<0.05; compared with the model group, * p<0.05.

[0052] Figure 3 The results of a showed that the latency of the writhing response in the model group, the dysmenorrhea relief group, and the strain intervention group was significantly shorter than that in the normal group, while the latency of the writhing response in the dysmenorrhea relief group and the strain intervention group was significantly longer than that in the model group, indicating that Lactobacillus rhamnosus can prolong the interval between uterine contractions. Figure 3 The results of b indicate that the writhing response scores of the dysmenorrhea relief group and the strain intervention group were significantly lower than those of the model group, suggesting that the pain was reduced after strain intervention. Figure 3 The results showed that, compared with the model group, the inhibition rates of writhing response in both the Tongjingbao group and the bacterial strain intervention group were significantly improved, with the inhibition rate of writhing response in the bacterial strain intervention group reaching 36.7%. The three indicators—writhing response latency, writhing response pain score, and writhing response inhibition rate—all indicate that *Lactobacillus rhamnosus* has a certain effect on relieving primary dysmenorrhea in mice.

[0053] Note: Compared with the normal group # p<0.05, ## p<0.01, ### p<0.001; compared with the model group, * p<0.05, ** p<0.01.

[0054] Figure 4 The results showed that, compared with the normal group, the PGE2 content in the uterine supernatant of mice in the model group was significantly reduced; compared with the model group, the PGE2 content in the dysmenorrhea relief group and the strain intervention group was significantly increased, indicating that Lactobacillus rhamnosus promotes the secretion of PGE2. Figure 4 The results of b indicate that, compared with the normal group, the PGF levels in the uterine supernatant of mice in the model group and the bacterial strain intervention group were significantly higher. 2a The levels of PGF in mice in both the dysmenorrhea relief group and the bacterial strain intervention group were significantly increased. 2a The content was significantly lower than that in the model group, indicating that this strain reduces the body's PGF. 2a The level.

[0055] Previous studies have shown that the main pathogenesis of primary dysmenorrhea is related to the disorder of prostaglandin levels in the body, and excessive secretion of prostaglandins is considered to be the main mechanism of primary dysmenorrhea. Among them, PGF... 2a PGE2 and PD are most closely related, jointly regulating the occurrence of dysmenorrhea. PGF 2a Its main function is to induce uterine smooth muscle contraction. When it binds to receptors, it stimulates afferent nerve fibers, causing vasoconstriction, reducing uterine blood flow, and resulting in smooth muscle ischemia, thus producing pain. PGE2, on the other hand, antagonizes PGF. 2a The effects of PGE2: PGE2 not only inhibits excessive contraction of uterine smooth muscle, maintains vasodilation, and ensures adequate blood flow to the uterus, but also has anti-inflammatory effects, helping to relieve pain. Lactobacillus rhamnosus ZJUIDS07 regulates PGE2 / PGF... 2a It regulates the rhythmic contractions of the uterus and inhibits uterine smooth muscle spasms, thereby relieving menstrual cramps.

[0056] Note: Compared with the normal group ## p<0.01, ### p<0.001; compared with the model group, * p<0.05, ** p<0.01, *** p<0.001.

[0057] Figure 5 The results showed that, compared with the normal group, the levels of TNF-a in the uterine supernatant of mice in the Tongjingbao group, the bacterial strain intervention group, and the model group were significantly increased; compared with the model group, the levels of TNF-a in the Tongjingbao group and the bacterial strain intervention group were significantly decreased, indicating that Lactobacillus rhamnosus inhibits the secretion of TNF-a. Figure 5 The results of b showed that the IL-6 level in the model group was significantly higher than that in the normal group; compared with the model group, the IL-6 content in the strain intervention group and the dysmenorrhea relief group was significantly reduced, indicating that the strain can inhibit the secretion of IL-6 in the body.

[0058] Studies have shown that dysmenorrhea is closely related to inflammatory factors, with IL-6 and TNF-α playing complex roles in the generation and regulation of pain. They can activate a series of inflammatory cascades, prompting inflammatory cells to accumulate locally in the uterus. These inflammatory cells further promote the production of PGE2 and PGF. 2a Lactobacillus rhamnosus ZJUIDS07 can alleviate menstrual cramps by regulating the body's immune balance, inhibiting the synthesis of inflammatory factors, blocking cytokine storms, and maintaining immune balance.

[0059] Note: Compared with the normal group ### p<0.001; compared with the model group, *** p<0.001.

[0060] 1.7 Comparative Experiment:

[0061] Lactobacillus rhamnosus LR-K0916, Lactobacillus rhamnosus TR08, Lactobacillus rhamnosus NAQU Plateau LR-1, Lactobacillus plantarum ZJUIDS04, and Lactobacillus rhamnosus BLCC2-0077 were subjected to experiments as described in Example 1 above. The results showed that the PGE2 content in the uterine tissue supernatant of the above-mentioned strain intervention groups was not significantly different from that of the model group, and PGF... 2a Compared with the model group, the content of [specific substance] decreased by a very limited rate, only about 10% to 12%, which is far less than the 36.7% decrease rate of the present invention. Therefore, it is shown that the above strains cannot effectively relieve dysmenorrhea, while strain ZJUIDS07 has a better effect on relieving primary dysmenorrhea.

[0062] Example 2: Preparation of Lactobacillus rhamnosus ZJUIDS07 fermentation starter with the effect of relieving primary dysmenorrhea

[0063] The original strain of *Lactobacillus rhamnosus* ZJUIDS07 was inoculated into 11% skim milk (sterilized at 115℃ for 15 min) and cultured at 37℃ for 18-24 h until curdling. This was repeated for two generations to obtain the mother starter culture. The mother starter culture was then inoculated at a 5% inoculation rate into 11% skim milk (sterilized at 115℃ for 15 min) and cultured at 37℃ for 18-24 h until curdling. At this point, the viable bacterial count reached 10-1. 11 -10 12 CFU / mL yields Lactobacillus rhamnosus ZJUIDS07 fermentation primordial.

[0064] Example 3: Preparation of bacterial powder using Lactobacillus rhamnosus ZJUIDS07, which has the effect of relieving primary dysmenorrhea.

[0065] Lactobacillus rhamnosus ZJUIDS07, stored at -80℃, was inoculated into 25 mL of MRS liquid medium using an inoculation loop and cultured at 37℃ for 18 h. Then, a 10% inoculum was transferred to 250 mL of sterilized MRS liquid medium. The medium was then incubated in a bacterial incubator at 37℃ for 24 h. The resulting fermentation broth was centrifuged at 3000 rpm for 10 min, the supernatant was discarded, and the bacterial sludge was collected. The sludge was washed twice with PBS to obtain the Lactobacillus rhamnosus ZJUIDS07 bacterial pellet.

[0066] The obtained bacterial precipitate was added to physiological saline and mixed well to form a bacterial suspension. After adding the prepared cryoprotectant solution, the suspension was dispensed into lyophilization vials. The lyophilization vials containing the bacterial suspension were placed in a pre-freezing chamber at -80°C and frozen for 2.5 hours. After complete freezing, the pre-frozen sample was placed in a vacuum freeze dryer at -55°C and a vacuum degree of 0.5 mbar and freeze-dried for 30 hours to obtain *Lactobacillus rhamnosus* ZJUIDS07 bacterial powder.

[0067] Note: Protectant solution formula: Dissolve 4.1g trehalose, 2.8g monosodium glutamate, and 8g sucrose in 10mL of 40℃ distilled water, filter and sterilize using a 0.22um microporous membrane, and then add to 90mL of sterile water to obtain the protectant solution.

[0068] Example 4: Preparation of post-biotics using Lactobacillus rhamnosus ZJUIDS07, which has the effect of relieving primary dysmenorrhea.

[0069] A single colony of *Lactobacillus rhamnosus* ZJUIDS07 was inoculated into 50 mL of MRS liquid medium and incubated at 37°C for 18 h. It was then activated again by inoculating 5% of the colony into 250 mL of MRS liquid medium and incubated at 37°C for 24 h. Finally, the activated *Lactobacillus rhamnosus* ZJUIDS07 was inoculated at 5% in a 10 L fermenter for high-density anaerobic culture at 37°C and pH 6.8 for 18 h. Afterward, the colony was centrifuged at 8000 rpm and 4°C for 15 min, the supernatant was discarded, and the bacterial precipitate was collected. The bacterial cells were washed twice with sterile PBS buffer (pH 7.0). This yielded *Lactobacillus rhamnosus* ZJUIDS07 bacterial sludge.

[0070] The collected Lactobacillus rhamnosus ZJUIDS07 bacterial sludge, bacterial cells, or fermentation broth is sterilized at 121℃ under high pressure for 15 minutes. After sterilization, the inactivated bacterial sludge, bacterial cells, or fermentation broth is collected, vacuum-packed, or aseptically filled and used directly or added to other products.

[0071] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. Lactobacillus rhamnosus ( Lactobacillus rhamnosus The application of ZJUIDS07 in the preparation of drugs that relieve primary dysmenorrhea is characterized by: Lactobacillus rhamnosus ( Lactobacillus rhamnosus The collection number for ZJUIDS07 is: CGMCCNO.23996; Lactobacillus rhamnosus ( Lactobacillus rhamnosus ZJUIDS07 regulates PGE2 and PGF. 2a It can reduce and inhibit the secretion of TNF-α and IL-6, thereby relieving menstrual cramps.

2. The application according to claim 1, characterized in that: Lactobacillus rhamnosus ( Lactobacillus rhamnosus The viable bacterial count prepared by ZJUIDS07 was 1.0 × 10⁻⁶. 11 ~1.0×10 12 CFU / g live bacteria preparation.

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