Application of lactobacillus rhamnosus ZJUIDS07 in preparation of medicine with efficacy of relieving primary dysmenorrhea

Abstract

The invention belongs to the technical field of food microorganisms, and particularly relates to lactobacillus rhamnosus ZJUIDS07 with the effect of relieving primary dysmenorrhea and application of the lactobacillus rhamnosus ZJUIDS07. The invention discloses an application of lactobacillus rhamnosus ZJUIDS07 in preparation of a medicine with an effect of relieving primary dysmenorrhea, and the preservation number of the lactobacillus rhamnosus ZJUIDS07 is CGMCC (China General Microbiological Culture Collection Center) NO.23996. The lactobacillus rhamnosus ZJUIDS07 can be used for preparing a medicine with an effect of relieving primary dysmenorrhea. According to the lactobacillus rhamnosus ZJUIDS07, the level of PGE2PGF2a is regulated and controlled, so that the effect of relieving dysmenorrhea is achieved, and the lactobacillus rhamnosus ZJUIDS07 can be used for regulating and controlling the level of PGE2PGF2a.

Description

Technical Field

[0001] The invention belongs to the technical field of food microorganisms, and specifically relates to a strain of Lactobacillus rhamnosus ZJUIDS07 having the effect of relieving primary dysmenorrhea and its application in the fields of food, health products and medicines. Background Art

[0002] Dysmenorrhea is one of the common chronic gynecological diseases in young women. It refers to lower abdominal pain and distension before, during or after menstruation, often accompanied by backache, dizziness, vomiting, diarrhea and other symptoms. It is mainly divided into primary dysmenorrhea (PD) and secondary dysmenorrhea (SD). Primary dysmenorrhea is spasmodic pain that occurs when the female pelvic structure is normal, usually lasting 1 to 3 days. Secondary pain is often accompanied by organic lesions, generally caused by endometriosis, pelvic inflammatory disease, cervicitis, etc., and is mostly treated with surgery or drugs. The incidence of dysmenorrhea accounts for about 65% of women of childbearing age, of which primary dysmenorrhea accounts for about 90% of the total incidence. Studies have shown that recurrent dysmenorrhea will increase the incidence of chronic pain diseases such as irritable bowel syndrome, bladder pain syndrome, adenomyosis, uterine fibroids, etc. in women in the future, and is a potential hidden danger to the physical and mental health of women of childbearing age.

[0003] At present, the recognized pathogenesis of primary dysmenorrhea is the prostaglandins (PGs) theory. Under normal circumstances, the endometrium sheds to form menstrual blood clots that accumulate in the uterine cavity, but the uterine cavity capacity is limited and the cervix is ​​narrow, so menstruation cannot be discharged on its own. At this time, the body produces prostaglandins to stimulate uterine contraction, thereby ensuring the normal discharge of menstruation. When the level of prostaglandins is too high, it will stimulate uterine blood vessels and nerves, causing pain. In clinical practice, non-steroidal anti-inflammatory drugs such as ibuprofen and diclofenac sodium that inhibit PGs synthesis are commonly used for treatment; and contraceptive drugs that affect endogenous hormone secretion by taking exogenous estrogens, change the ovarian endocrine pattern and the cyclical changes of the endometrium to reduce PGs synthesis. Both have shortcomings such as many side effects, treatment intolerance, and cyclical medication, and cannot cure or prevent dysmenorrhea. In addition, traditional Chinese medicine has the advantages of overall conditioning, few side effects, and personalized treatment in the treatment of dysmenorrhea, but it also has disadvantages such as slow onset, long medication cycle, and unstable efficacy. Therefore, finding an effective method to treat primary dysmenorrhea is of great significance to both women's physical and mental health.

[0004] In recent years, new methods of using lactic acid bacteria to regulate intestinal flora to treat or relieve PD have begun to attract widespread attention. At present, the strains that have been proven to have certain effects on relieving PD or uterine and pelvic inflammation mainly include Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus reuteri, Pediococcus acidilactici, Weissella, etc. As a type of lactic acid bacteria, Lactobacillus rhamnosus can be added to food or medicine to colonize in the human intestine to play a probiotic role, such as improving immunity, maintaining intestinal microecological balance, inhibiting the growth of intestinal pathogens, improving lactose digestion and anti-oxidation, etc. Due to its natural, safe and efficient probiotic properties, it has good application prospects in aquaculture, food processing industry and clinical treatment.

[0005] The invention of CN116042470A, "Chinese medicine fermentation using a novel strain of Lactobacillus rhamnosus and its application", discloses a Lactobacillus rhamnosus strain LR-K0916, whose deposit number is GDMCC No: 63006. It can be used to treat reproductive system diseases, including vaginitis, uterine fibroids, dysmenorrhea, breast nodules, breast cancer, menopausal syndrome, etc. However, no corresponding experimental data are given.

[0006] The invention of CN107158041A, "A probiotic composition for treating gynecological inflammation", discloses a composition for treating gynecological inflammation, including components: resistant dextrin, inulin, stachyose, Lactobacillus rhamnosus TR08, and Lactobacillus reuteri TR02. The deposit number of Lactobacillus rhamnosus TR08 is: CCTCCNO.M2016548.

[0007] The invention of CN106309506A, "A probiotic composition with menstrual regulation function and its application", states that the probiotic composition composed of Lactobacillus rhamnosus, Lactobacillus plantarum and Bifidobacterium longum has the function of regulating menstruation, especially can inhibit primary dysmenorrhea. However, when Lactobacillus rhamnosus is used alone, it has almost no function of regulating menstruation.

[0008] The invention of CN112294892A "Fermented Siwu Decoction Drink Composition with Effect of Alleviating Dysmenorrhea and Preparation Method" discloses a fermented Siwu Decoction Drink Composition with Effect of Alleviating Dysmenorrhea, the raw materials of which include white peony root, angelica root, chuanxiong rhizome and rehmannia root; the Chinese medicinal raw materials white peony root, angelica root, chuanxiong rhizome and rehmannia root are fermented by a composite bacteria composed of Lactobacillus plantarum and Bacillus rhamnosus.

[0009] Lactobacillus rhamnosus ZJUIDS07 (preservation number: CGMCCNO.23996) is only informed in CN116064285A that it has the function of lowering blood sugar; the products with the function of lowering blood sugar contain the live bacterial preparation of Lactobacillus rhamnosus ZJUIDS07. Summary of the invention

[0010] The technical problem to be solved by the present invention is to provide a new use of Lactobacillus rhamnosus ZJUIDS07 - relieving primary dysmenorrhea.

[0011] In order to solve the above technical problems, the present invention provides an application of Lactobacillus rhamnosus ZJUIDS07 in preparing a drug having the effect of relieving primary dysmenorrhea. The preservation number of Lactobacillus rhamnosus ZJUIDS07 is: CGMCCNO.23996.

[0012] As an improvement of the application of the present invention: Lactobacillus rhamnosus ZJUIDS07 regulates PGE2\PGF 2a level, thereby achieving the effect of relieving dysmenorrhea.

[0013] Description: Regulate PGE2\PGF 2a level, can regulate the rhythmic contraction of the uterus, inhibit the spasm of uterine smooth muscle, and thus achieve the effect of relieving dysmenorrhea.

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

[0015] As a further improvement of the application of the present invention, foods (including fermented milk products, etc.), health products, feeds, and Chinese medicine preparations having the efficacy of relieving primary dysmenorrhea can also be prepared.

[0016] As a further improvement of the application of the present invention: Lactobacillus rhamnosus ZJUIDS07 is prepared into a viable cell count of 1.0×10 11 ~1.0×10 12 CFU / g of live bacterial preparation.

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

[0018] The present invention focuses on regulating PGE2\PGF 2a The level of pain factors can be reduced to achieve the purpose of alleviating pain. Moreover, strain ZJUIDS07 has the function of regulating inflammatory factors in the body, which can achieve the effect of preventing dysmenorrhea and adjusting the menstrual cycle. Compared with other Lactobacillus rhamnosus described in the background technology, the Lactobacillus rhamnosus ZJUIDS07 of the present invention has a better effect in relieving primary dysmenorrhea, has a stronger ability to relieve pain and inflammation, and has a significant preventive and therapeutic effect when used alone, which can achieve the purpose of preventing dysmenorrhea and treating dysmenorrhea.

[0019] That is, in view of the current lack of research on the therapeutic effect of Lactobacillus rhamnosus in primary dysmenorrhea, the present invention provides a strain of Lactobacillus rhamnosus ZJUIDS07 and its application for alleviating primary dysmenorrhea, and investigates the effect of Lactobacillus rhamnosus on alleviating primary dysmenorrhea through a primary dysmenorrhea writhing model induced by estradiol benzoate and oxytocin in mice.

[0020] The lactobacillus rhamnosus of the present invention can be used to prepare a functional red date lactic acid fermented beverage applicable to food, can be used to prepare a live bacterial preparation having the effect of relieving primary dysmenorrhea, can be used to prepare fermented milk having the function of relieving primary dysmenorrhea, can be used to prepare bacterial powder having the function of relieving primary dysmenorrhea, can be used to prepare a Chinese medicine mixture having the function of relieving primary dysmenorrhea. Therefore, the lactobacillus rhamnosus of the present invention can be widely used in the development of prebiotic functional products related to relieving primary dysmenorrhea.

[0021] When Lactobacillus rhamnosus ZJUIDS07 of the present invention is actually used, it can be used in combination with Tongjingbao in the form of oral administration, and the dosage can be multiplied by the conventional conversion coefficient (10-12 times) according to the dosage of mice in the following Example 1. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The specific embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings;

[0023] Figure 1 This is the colony morphology of Lactobacillus rhamnosus ZJUIDS07;

[0024] Figure 2 This is a comparison chart of the changes in uterine weight of mice in each experimental group;

[0025] Figure 3 The writhing reaction pain indexes of mice in each group of the experiment: writhing reaction latency, writhing pain score, and writhing inhibition rate comparison chart;

[0026] Figure 4 Effect of Lactobacillus rhamnosus ZJUIDS07 on the pain biochemical indexes PGE2 and PGF in the serum of mice in each group 2a The impact of

[0027] Figure 5 The effect of Lactobacillus rhamnosus ZJUIDS07 on inflammatory biochemical indicators TNF-a and IL-6 in the serum of each group of mice; DETAILED DESCRIPTION

[0028] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto:

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

[0030] 1.1 Experimental animals

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

[0032] 1.2 Preparation of drug solution for intragastric administration

[0033] 1.2.1 Preparation of bacterial solution for intragastric administration

[0034] Lactobacillus rhamnosus ZJUIDS07 was frozen at -80 °C, and the strain was activated in MRS liquid medium at 37 °C for 16 h. After three passages, the strain was centrifuged at 5000 r for 10 min at 4 °C, the supernatant was discarded, the bacteria were collected, washed twice with PBS buffer (0.1 mol / L, pH = 6.8), and the bacterial solution concentration was adjusted to 1 × 10 9 CFU / mL, this was used as the bacterial solution and was gavaged on the same day after preparation.

[0035] 1.2.2 Preparation of Tongjingbao solution for intragastric administration

[0036] Tongjingbao granules were purchased from Zhongjing Wanxi Pharmaceutical Co., Ltd. Each animal was given 4 g / kg and gavaged on the same day after preparation (after dissolving in water). In the animal experiment, the gavage volume for each mouse was 200 μL.

[0037] 1.3. Establishment of the primary dysmenorrhea writhing model in mice

[0038] In this experiment, mice were fed a routine adaptive diet for 3 days, and then 5 mg / kg of estradiol benzoate was subcutaneously injected for 15 consecutive days. On the 15th day, 1 hour after the injection of estradiol benzoate, 100 U / kg of oxytocin was intraperitoneally injected to induce the primary dysmenorrhea writhing model in mice. Animal welfare and experimental procedures were carried out in accordance with the guidelines of the Regulations on the Management of Experimental Animals of Zhejiang University.

[0039] 1.4 Experimental design of the alleviating effect of Lactobacillus rhamnosus ZJUIDS07 on primary dysmenorrhea writhing model mice

[0040] After 3 days of routine adaptive feeding, female C57BL / 6J mice were randomly divided into 4 groups, 5 mice in each group (n=5), namely normal group, model group, positive control Tongjingbao group and strain ZJUIDS07 intervention group. Among them, except for the normal group injected with normal saline for 15 days, the model group, positive control Tongjingbao group and strain ZJUIDS07 intervention group were injected with estradiol benzoate for 15 consecutive days. Except for the normal group and model group, from the third day of estradiol benzoate injection, the Tongjingbao group was gavaged with 200ul Tongjingbao liquid, and the strain ZJUIDS07 intervention group was gavaged with 200ul of Tongjingbao and bacterial liquid (mixed in a 1:1 volume ratio), and the gavage was continued for 12 days.

[0041] 1.4.1 Detection of writhing indices in mice

[0042] On the 15th day, oxytocin was injected into the normal group, model group, positive control Tongjingbao group, and strain ZJUIDS07 intervention group 1 hour after the injection of estradiol benzoate. The writhing state of mice in each group was observed within 30 minutes, and the writhing reaction score, writhing reaction latency, and writhing reaction inhibition rate of each group were recorded. The writhing reaction is an indicator for evaluating uterine spasm contraction (pain), which can intuitively reflect whether the primary dysmenorrhea model is successfully established. According to the four-level behavioral scoring standard proposed by Schmauss, the writhing reaction score and writhing latency of mice were recorded, and then the writhing inhibition rate was calculated. The writhing latency refers to the time required from the injection of oxytocin to the first writhing reaction of the mouse, which is recorded in minutes; the writhing reaction was scored according to the standards in Table 1, and the writhing score = 0 level times × 0 points + 1 level times × 1 point + 2 level times × 2 points + 3 level times × 3 points. The writhing inhibition rate = [(average writhing reaction score of the model group - average writhing reaction score of the strain ZJUIDS07 intervention group) / average writhing score of the model group] × 100%. During the experiment, other feeding conditions such as temperature, humidity, light, and feed were the same.

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

[0044] After observing the writhing reaction, anesthetize the mouse immediately. Quickly remove the fresh uterine tissue of the mouse on ice, and observe the uterine tissue morphology, color, texture, and the presence of edema with the naked eye. Rinse the uterine tissue with pre-cooled PBS to remove residual blood, and cut the tissue into pieces after weighing. Add the chopped tissue and the corresponding volume of PBS to a homogenizer at a ratio of 1:9, grind thoroughly, and prepare a 10% tissue homogenate. Centrifuge the homogenate at 8500r / min at 4°C for 10 minutes, and take the supernatant. Use ELISA to detect PGF in the supernatant of uterine tissue. 2a , PGE2 (pain index), IL-6, TNF-a (inflammatory index). ELISA kits were purchased from Nanjing Jiancheng or Beijing Pulilai Reagent Company, and all the above were operated strictly according to the instructions. When a color reaction occurred, the absorbance value was immediately measured using a multifunctional microplate reader, and then the corresponding content was calculated according to the standard concentration curve.

[0045] Table 1 Grading criteria for pain in writhing reaction

[0046]

[0047] 1.5 General

[0048] During the experiment, the mice in the normal group were healthy in all aspects, with shiny fur, rapid activity, and good mental state. The mice in the model group were significantly less active than those in the normal group, with dull fur, decreased appetite, and reduced response to external environmental stimuli. The conditions of the mice in the Tongjingbao group and the strain intervention group improved, with restored fur gloss, increased food intake, increased water intake, and slightly increased exercise.

[0049] 1.6 Experimental Results Analysis

[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 that the model was successfully established. This may be related to the continuous injection of estradiol benzoate, which leads to endometrial thickening, uterine enlargement, and uterine hydrops. The main effect of estrogen on the uterus is to promote the proliferation of myometrial cells and endometrial cells. Estrogen stimulates the mitosis of endometrial glandular epithelial cells and stromal cells, causing the endometrium to proliferate from the basal layer. The endometrium will undergo cyclical hyperplasia under the action of estrogen. During the proliferation period of the menstrual cycle, estrogen levels gradually increase, glandular epithelial cells enlarge, nuclear division increases, and glycogen and other substances in the cells increase. Myometrial cells will also increase in cell 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 writhing reaction latency of the model group, Tongjingbao group, and strain intervention group was significantly lower than that of the normal group, and the writhing reaction latency of the Tongjingbao group and strain intervention group was significantly longer than that of the model group, indicating that Lactobacillus rhamnosus can prolong the interval between uterine contractions. Figure 3 The results of b showed that the writhing reaction scores of the Tongjingbao group and the strain intervention group were significantly lower than those of the model group, indicating that the pain was alleviated after the strain intervention. Figure 3 The results of c showed that compared with the model group, the inhibition rates of writhing reaction in the Tongjingbao group and the strain intervention group were significantly improved, among which the inhibition rate of writhing reaction in the strain intervention group reached 36.7%. The three indicators of writhing reaction latency within 30 minutes, writhing reaction pain score, and writhing reaction 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 of a showed that compared with the normal group, the PGE2 content in the uterine supernatant of the model group mice was significantly reduced; compared with the model group, the PGE2 content in the Tongjingbao group and the strain intervention group was significantly increased, indicating that Lactobacillus rhamnosus can promote the secretion of PGE2. Figure 4 The results of b showed that compared with the normal group, the PGF 2a The contents of PGF in mice in the Tongjingbao group and the strain intervention group were significantly increased. 2a The content was significantly lower than that in the model group, indicating that the strain would reduce the body's PGF 2a level.

[0055] 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. 2a PGF and PGE2 are most closely related to PD and jointly regulate the occurrence of dysmenorrhea. 2a It mainly promotes the contraction of uterine smooth muscle. When it binds to the receptor, it stimulates the afferent nerve fibers, causing vasoconstriction, reducing uterine blood flow, causing smooth muscle ischemia, and thus causing pain. PGE2 antagonizes PGF 2a PGE2 can not only inhibit excessive contraction of uterine smooth muscle, maintain vasodilation, and ensure adequate blood flow to the uterus, but also has anti-inflammatory effects and helps relieve pain. 2a level, regulate the rhythmic contraction of the uterus, and inhibit the spasm of uterine smooth muscle to achieve the effect of relieving dysmenorrhea.

[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 TNF-a content in the uterine supernatant of mice in the Tongjingbao group, strain intervention group and model group was significantly increased; compared with the model group, the TNF-a content in the Tongjingbao group and strain intervention group was significantly decreased, indicating that Lactobacillus rhamnosus would inhibit 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 of mice in the strain intervention group and the Tongjingbao group was significantly reduced, indicating that the strain would inhibit the body's IL-6 secretion.

[0058] Studies have shown that dysmenorrhea is closely related to inflammatory factors. IL-6 and TNF-a also play a complex role in the generation and regulation of pain. They can activate a series of inflammatory cascade reactions and promote the local accumulation of inflammatory cells in the uterus. These inflammatory cells will further promote the production of PGE2 and PGF 2a The secretion and release of Lactobacillus rhamnosus ZJUIDS07 can regulate the body's immune balance, inhibit the synthesis of inflammatory factors, block the inflammatory factor storm, maintain immune balance, and relieve dysmenorrhea.

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

[0060] 1.7 Comparative experiment:

[0061] The experiment was carried out with Lactobacillus rhamnosus LR-K0916, Lactobacillus rhamnosus TR08, Lactobacillus rhamnosus NAQU Plateau LR-1, Lactobacillus plantarum ZJUIDS04, and Lactobacillus rhamnosus BLCC2-0077 according to the above Example 1. The results were as follows: there was no significant difference in the PGE2 content in the supernatant of the uterine tissue between the above strain intervention groups and the model group, and PGF 2a Compared with the model group, the decrease rate of the content in the control group was very limited, only about 10% to 12%, which was far less than the decrease rate of 36.7% of the present invention. Therefore, it was shown that the above strains could not effectively relieve primary dysmenorrhea, and the strain ZJUIDS07 had a better effect in relieving primary dysmenorrhea.

[0062] Example 2: Preparation of red date lactic acid fermented beverage for relieving dysmenorrhea using Lactobacillus rhamnosus ZJUIDS07

[0063] 1. Processing process of fermented beverages:

[0064] Raw material preparation → red date liquid preparation → blending → sterilization → inoculation and fermentation → blending → homogenization → filling and sterilization → cooling.

[0065] 2. Operation points

[0066] (1) Raw material preparation: Select fresh red dates that are highly mature, free of pests and diseases, and free of rot to ensure the quality and taste of the product. Soak the red dates in clean water for 10-15 minutes, then rinse them with running water to remove impurities and dirt on the surface, and then remove the red date pits with a pitting machine or manually.

[0067] (2) Preparation of red date liquid: Mix pitted red dates with water in a mass ratio of 1:2, and cook in a pot for 20-30 minutes until the red dates are soft. Pour the steamed red dates together with the cooking water into a pulper and beat into a fine red date pulp. Filter as needed to remove residues and crude fibers to obtain a finer red date pulp.

[0068] (3) Preparation: Add an appropriate amount of sucrose, glucose and other sugar substances to the red date pulp to adjust the sweetness and taste of the beverage. The general amount added is 5%-10% of the mass of the red date pulp. At the same time, an appropriate amount of citric acid, malic acid and other acidulants can be added to adjust the acidity. The amount added is 0.1%-0.3%. The red date pulp, sugar substances, acidulants, etc. are fully stirred and mixed to ensure that the various ingredients are evenly distributed.

[0069] (4) Sterilization: The prepared red date pulp is sterilized at 100℃-120℃ for 10-30 seconds to kill harmful microorganisms and enzymes, ensuring the smooth fermentation process and product quality and safety. The sterilized red date pulp is quickly cooled to 37℃-40℃ to create suitable temperature conditions for lactic acid bacteria inoculation and fermentation.

[0070] (5) Inoculation and fermentation: Inoculate the Lactobacillus rhamnosus ZJUIDS07 strain into the sterilized culture medium and culture it at 37°C for 24 hours for activation. Inoculate the activated bacteria into the cooled red date pulp at an inoculum amount of 2%-5%, stir evenly, and make the lactic acid bacteria evenly distributed in the red date pulp. Pour the inoculated red date pulp into a fermentation tank or fermentation container, seal it well, and ferment it at a constant temperature of 37°C-40°C for 12-24 hours. During this period, stir it regularly or let it stand for fermentation until the pH value of the red date pulp drops to about 4.0-4.5, and a distinct lactic acid fermentation flavor is produced.

[0071] (6) Mixing and homogenization: According to the taste and quality requirements of the product, appropriate amounts of sweeteners, acidulants, stabilizers, etc. can be added to the fermented red date beverage for mixing, such as adding 0.1%-0.3% of xanthan gum, sodium carboxymethyl cellulose and other stabilizers to improve the stability and taste of the beverage. The mixed beverage is then homogenized at 50℃-60℃ and 15-20MPa to make the beverage more delicate and uniform.

[0072] (7) Filling, sterilization and cooling: The homogenized fermented red date beverage is filled into pre-cleaned and sterilized glass bottles or plastic bottles while hot, and the filling temperature is controlled at 70℃-80℃. After filling, sterilization is carried out immediately, and pasteurization can be used at a temperature of 75℃-85℃ for 15-20 minutes, and then quickly cooled to room temperature.

[0073] Example 3: Preparation of a Lactobacillus rhamnosus ZJUIDS07 starter culture having the effect of relieving primary dysmenorrhea

[0074] The original strain of Lactobacillus rhamnosus ZJUIDS07 was inoculated into 11% skim milk (sterilized at 115℃ for 15min), cultured at 37℃ for 18-24h until curdled, and activated for two generations continuously as the mother starter. The mother starter was inoculated into 11% skim milk (sterilized at 115℃ for 15min) at a 5% inoculation rate, cultured at 37℃ for 18-24h until curdled, and the number of viable bacteria could reach 10 11 -10 12 CFU / mL, and the Lactobacillus rhamnosus ZJUIDS07 fermentation agent was obtained.

[0075] Example 4: Preparation of fermented milk using Lactobacillus rhamnosus ZJUIDS07 with the efficacy of relieving primary dysmenorrhea

[0076] First mix the sugar substances, stabilizers, etc. with a small amount of milk to dissolve, and then stir thoroughly with the remaining milk to mix evenly. Preheat the mixed milk to 50℃-60℃ to soften the fat globules and improve the homogenization effect. Use a high-pressure homogenizer to perform homogenization at a pressure of 14MPa-21MPa to break up and reduce the fat globules, evenly disperse them in the milk, improve the viscosity and stability of the fermented milk, and prevent the fat from floating. Then sterilize at 90℃-95℃ for 5 minutes, quickly cool the sterilized milk to 40℃-45℃, and inoculate the starter in Example 3 at a 5% inoculation rate. Pour the inoculated milk into a fermentation tank or fermentation container and incubate at 40℃.

[0077] Ferment at a constant temperature of -45℃ for 2h-4h, during which it can be stirred regularly or left to ferment until the pH value of the milk drops to about 4.0-4.5, producing a distinct lactic acid fermentation flavor, and the tissue state is in a coagulated state without excessive whey separation. After fermentation is completed, the fermented milk is quickly cooled to below 10℃ to terminate the fermentation and control the increase in acidity, while making the tissue state of the fermented milk more stable. Fill the cooled fermented milk into pre-cleaned and sterilized packaging containers, such as glass bottles, plastic bottles, etc. The filled fermented milk is placed in a cold storage at 0℃-6℃ and refrigerated for about 12h to promote the production of aromatic substances and increase the viscosity and taste of the product.

[0078] Example 5: Preparation of bacterial powder using Lactobacillus rhamnosus ZJUIDS07 with the efficacy of relieving primary dysmenorrhea

[0079] Use an inoculation loop to inoculate the Lactobacillus rhamnosus ZJUIDS07 stored at -80°C into 25mL MRS liquid culture medium and culture at 37°C for 18h. Then transfer 10% of the inoculation volume to the sterilized 250mL MRS liquid culture medium. Place it in a bacterial incubator and culture it at 37°C for 24h. Centrifuge the resulting fermentation liquid at 3000r for 10min, discard the supernatant to collect the bacterial sludge, and rinse the bacterial sludge twice with PBS. The bacterial precipitate of Lactobacillus rhamnosus ZJUIDS07 can be obtained.

[0080] Add physiological saline to the obtained bacterial precipitate and mix it into bacterial solution, add the prepared protective agent solution and then divide it into freeze-dried bottles. Put the freeze-dried bottle containing the bacterial solution into a pre-freezing chamber at -80°C and freeze it for 2.5 hours. After freezing completely, put the pre-frozen sample into a vacuum freeze dryer with a cold trap temperature of -55°C and a vacuum degree of 0.5mbar and freeze-dry it for 30 hours to obtain Lactobacillus rhamnosus ZJUIDS07 bacterial powder.

[0081] Note: Preservative solution formula: 4.1g trehalose, 2.8g sodium glutamate, and 8g sucrose are dissolved in 10mL 40℃ distilled water, filtered and sterilized with a 0.22um microporous filter membrane, and then added to 90mL sterile water to obtain the preservative solution.

[0082] Example 6: Preparation of postbiotics using Lactobacillus rhamnosus ZJUIDS07 with the efficacy of relieving primary dysmenorrhea

[0083] Pick a single colony of Lactobacillus rhamnosus ZJUIDS07 and inoculate it in 50mL MRS liquid culture medium, and place it in a 37°C incubator for 18h. Activate it again in 250mL MRS liquid culture medium at a 5% inoculation amount, and place it in a 37°C incubator for 24h. Finally, the activated Lactobacillus rhamnosus ZJUIDS07 is cultured in a 10L fermenter at a high-density anaerobic culture with a 5% inoculation amount, and cultured for 18h at 37°C and pH 6.8. After that, centrifuge it at 8000r / min and 4°C for 15min, discard the supernatant, collect the bacterial precipitate, and rinse the bacterial body twice with sterile PBS buffer (pH 7.0). You can get the Lactobacillus rhamnosus ZJUIDS07 bacterial mud.

[0084] The collected Lactobacillus rhamnosus ZJUIDS07 bacterial mud, bacterial cells or fermentation liquid are sterilized at high temperature and high pressure at 121°C for 15 minutes, and then the inactivated bacterial mud, bacterial cells or fermentation liquid are collected and directly used or added to other products after vacuum packaging or aseptic filling.

[0085] Example 7: Preparation of probiotic milk powder using Lactobacillus rhamnosus ZJUIDS07 with the efficacy of relieving primary dysmenorrhea

[0086] 1. Preparation of Lactobacillus rhamnosus ZJUIDS07 powder

[0087] The freeze-dried bacterial powder of Lactobacillus rhamnosus ZJUIDS07 was prepared with reference to Example 5, and the number of viable bacteria in the bacterial powder was 1.0×10 11 ~1×

[0088] 10 12 CFU / g.

[0089] 2. Preparation of Formula Milk Powder

[0090] (1) Preliminary selection of raw materials: milk powder, fish meal, bone meal, grains, vegetable oil, additives: vitamins, trace elements, functional factors, and others;

[0091] (2) Automatic batching: put the obtained raw materials into the material bin according to the formula;

[0092] (3) Crushing: crush the weighed material through a crusher;

[0093] (4) Mixing: Add vegetable oil and trace elements to the crushed material and add it to the mixer to mix evenly;

[0094] (5) Extrusion: The mixed materials are made into granular materials through an extruder;

[0095] (6) Drying: The mixed materials are dried in a dryer with the temperature controlled at 65-70 degrees;

[0096] (7) Grading screening: The material flow is passed through a grading screen and the particle size is controlled at 2.5-5 mm.

[0097] 3. Preparation of probiotic formula milk powder

[0098] The bacterial powder prepared in 1 and the formula milk powder prepared in 2 were mixed evenly at a ratio of 1:100. The live bacteria in the final product were 8 CFU / g or above. After filling, the product is stored in the warehouse for sale.

[0099] Example 8: Preparation of fermented brown sugar using Lactobacillus rhamnosus ZJUIDS07 that has the effect of relieving primary dysmenorrhea 1. Prepare materials: brown sugar, deionized water, ZJUIDS07 bacterial powder

[0100] 2. Prepare brown sugar stock solution: Mix brown sugar and deionized water in a mass volume ratio of (40-240 g): (560-760 ml), control the water temperature at 30-60°C, and stir to dissolve to obtain brown sugar stock solution.

[0101] 3. Sterilization: Heat the brown sugar solution to 85-95℃ and sterilize for 10-30 minutes.

[0102] 4. Inoculation and fermentation: Add bacterial powder to the sterilized brown sugar solution at a rate of 0.01-0.1% of the total mass of the brown sugar solution, mix well, and ferment at 37°C ± 2°C for 24-72 hours.

[0103] 5. Terminate fermentation: Heat the brown sugar to 90-95°C, maintain for 10-20 minutes, and terminate fermentation.

[0104] Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by a person skilled in the art should be considered as the protection scope of the present invention.

Claims

1. Application of Lactobacillus rhamnosus ZJUIDS07 in the preparation of a drug for relieving primary dysmenorrhea, characterized in that: The accession number of Lactobacillus rhamnosus ZJUIDS07 is: CGMCCNO.23996.

2. The use according to claim 1, characterized in that: Lactobacillus rhamnosus ZJUIDS07 regulates PGE2\PGF 2a level, thereby achieving the effect of relieving dysmenorrhea.

3. The use according to claim 2, characterized in that: Lactobacillus rhamnosus ZJUIDS07 inhibits the secretion of TNF-a and IL-6, thereby achieving the effect of relieving dysmenorrhea.

4. The use according to any one of claims 1 to 3, characterized in that: The invention can also prepare food, health care products and feed having the effect of relieving primary dysmenorrhea.

5. The use according to any one of claims 1 to 4, characterized in that: Lactobacillus rhamnosus ZJUIDS07 was prepared to have a viable count of 1.0×10 11 ~1.0×10 12 CFU / g of live bacterial preparation.

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