Medical stone probiotic fermentation product and its application

Abstract

The present invention belongs to the technical field of probiotics, and specifically relates to a medical stone probiotic fermentation product and its application in intestinal flora regulation. The medical stone composite culture medium provided by the present invention can significantly promote the growth of Lactobacillus rhamnosus in acid, bile salt and artificial intestinal fluid. The composite culture medium is used to ferment Lactobacillus rhamnosus, and the fermentation product is used to treat diarrhea model rats, which can alleviate the diarrhea of ​​rats, increase the weight of rats and reduce the water content of feces. After treatment, the intestinal inflammation of rats recovered significantly, and it can be considered that the fermentation product has the effect of improving the condition, and the fermentation process can enhance the therapeutic effect.

Description

Technical field:

[0001] The invention belongs to the technical field of probiotics, and particularly relates to a medical stone probiotic fermentation product and application thereof in regulating intestinal flora. Background technology:

[0002] Medical stone was first recorded in the Compendium of Materia Medica. It is recorded in the Chinese Pharmacy Dictionary that medical stone is sweet, warm, non-toxic, and can nourish the spleen and stomach, remove cold and dampness. Modern research shows that medical stone has many characteristics such as good adsorption, dissolution, mineralization, and biological activity. The trace elements contained in medical stone can improve anti-fatigue, anti-hypoxia and immunity, and have the effects of fitness, longevity, anti-aging and disease resistance. It has a strong adsorption capacity for several elements harmful to the human body, such as cadmium, mercury, arsenic, and lead, and has a strong adsorption effect on chlorpromazine. At the same time, it also has a good adsorption capacity for Escherichia coli, Shigella dysenteriae, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, which can improve the gastrointestinal environment of the human body and enhance the peristalsis and digestion ability of the gastrointestinal tract of the human body. In addition, the use of medical stone as a new antibacterial agent carrier has also become a frontier field of current research.

[0003] Lactobacillus rhamnosus is an anaerobic, acid-resistant, non-spore-forming Gram-positive probiotic. After colonizing and multiplying in the human intestinal environment, Lactobacillus rhamnosus GG (abbreviated as Lactobacillus GG or LGG) attaches to the host's intestinal epithelial cells and can become a biological barrier of the intestinal mucosa. Moreover, LGG can regulate the structure and function of the microecological community in the host's intestine and bring it to a balanced state, thereby achieving the purpose of improving the function of the host's digestive system. Relevant studies have shown that the adhesion rate of LGG to adults is significantly higher than that to children, mainly because the intestinal environment of adults is more mature and suitable for LGG adhesion.

[0004] LGG can be used to prevent and treat diarrhea. Related studies have shown that after 29 infants took LGG preparations and ordinary cow's milk orally, LGG bacteria were detected in the feces of infants taking LGG. This shows that after taking it, the host uses LGG to regulate the intestinal flora, and the treatment effect of diarrhea in infants taking LGG preparations is better than that of taking ordinary cow's milk.

[0005] Based on this, the present invention discloses the effect of medical stone on the tolerance of Lactobacillus rhamnosus and its adsorption. Medical stone and Lactobacillus rhamnosus are fermented together to treat diarrhea model rats, thereby achieving the function of regulating intestinal flora and improving intestinal inflammation. Summary of the invention:

[0006] The invention relates to a medical stone and lactobacillus rhamnosus fermentation product and a preparation method thereof. The tolerance of lactobacillus rhamnosus in different environments is improved by medical stone fermentation, and the purpose of treating intestinal inflammation of diarrhea model animals and regulating intestinal flora is achieved by administering an effective amount of Chinese medical stone-lactobacillus rhamnosus fermentation product.

[0007] One of the technical solutions provided by the present invention is a composite culture medium, wherein the composite culture medium is prepared by preparing MRS culture medium with mineral water and then adding medical stone powder;

[0008] The MRS culture medium is composed of (g / L): 4.0-14.0 peptone, 3.0-12.0 beef powder, 1.0-5.0 yeast powder, 10.0-30.0 glucose, 1.0-3.0 dipotassium hydrogen phosphate, 1.0-5.0 diammonium hydrogen citrate, 2.0-8.0 sodium acetate, 0.1-1.0 magnesium sulfate, 0.01-0.5 manganese sulfate, 0.1-3.0 Tween 80, and the rest is solvent;

[0009] Preferably, the MRS medium is composed of (g / L): peptone 10.0, beef powder 8.0, yeast powder 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 80 1.0, and the rest is solvent;

[0010] The preparation method of the mineralized water is as follows: powdering a medical stone sample, passing through a 40-325 mesh sieve, and obtaining medical stone powder; mixing the sieved medical stone powder with water at a mass volume ratio (g / mL) of 1-2:1-20, soaking at 35-39° C. and 100-150 rpm for 12-36 hours to obtain a medical stone mineralized water suspension, and filtering under reduced pressure and passing through a microfiltration membrane to obtain medical stone mineralized water;

[0011] Preferably, the medical stone is Chinese medical stone;

[0012] Preferably, the medical stone powder is 80-325 mesh;

[0013] Preferably, the microfiltration membrane is a 0.45 μm filter membrane;

[0014] Furthermore, the composite culture medium is prepared with 75% mineralized water, that is, mineralized water and purified water are mixed at a ratio of 75:25 to prepare 75% mineralized water;

[0015] Furthermore, the composite culture medium is supplemented with 2-10% (m / v) medical stone powder;

[0016] Furthermore, the medical stone powder is 40-325 mesh;

[0017] Preferably, 5% 160 mesh medical stone powder is added;

[0018] More preferably, the composite culture medium is composed of (g / L): peptone 10.0, beef powder 8.0, yeast powder 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 801.0, 160 mesh medical stone powder 50.0, and the rest is 75% mineral water;

[0019] Furthermore, the pH of the composite culture medium is 5.7±0.2.

[0020] The second technical solution provided by the present invention is the application of the composite culture medium described in the first technical solution;

[0021] Furthermore, the application is its application in the cultivation of Lactobacillus rhamnosus;

[0022] Furthermore, the application is an application in improving the tolerance of Lactobacillus rhamnosus;

[0023] The tolerance includes but is not limited to acidity, alkalinity, gastric juice, intestinal juice, and bile salt tolerance;

[0024] Furthermore, the application is to prepare the fermentation product of Lactobacillus rhamnosus obtained by culturing in the first technical solution, and the specific preparation method is as follows:

[0025] The culture medium is inoculated with Lactobacillus rhamnosus and cultured at 35-39°C and 100-150rpm for 12-16h.

[0026] Furthermore, according to 1×10 7 -5×10 10 CFU / mL accession of Lactobacillus rhamnosus;

[0027] The fermented product may be in liquid form, or may be freeze-dried and stored and used in solid form;

[0028] The third technical solution provided by the present invention is the use of the above fermentation product in the preparation of drugs for treating diarrhea, especially in the preparation of drugs for regulating intestinal flora.

[0029] Beneficial effects:

[0030] 1. The medical stone composite culture medium provided by the present invention can significantly promote the survival of Lactobacillus rhamnosus in acidic, bile salt and artificial intestinal fluid environments, and exhibits excellent acid resistance, bile salt resistance and artificial intestinal fluid resistance properties. The composite culture medium has a protective effect on Lactobacillus rhamnosus in an acidic environment of 2≤pH≤5, or at a bile salt concentration of 0.1-0.15%, and can significantly improve its survival ability in artificial intestinal fluid.

[0031] 2. The composite culture medium prepared by the present invention is used to ferment Lactobacillus rhamnosus, and the fermentation product is used to treat diarrhea model rats, which can alleviate the diarrhea of ​​rats, increase the weight of rats and reduce the water content of feces. After treatment, the intestinal inflammation of rats recovered significantly. It can be considered that the fermentation product has the effect of improving the condition, and the fermentation process can enhance the therapeutic effect. The fermentation product can reduce the expression of inflammatory factors in rat colon, so that the inflammatory symptoms of the colon are improved. After treatment, the number of Escherichia coli and Lactobacillus in the rat colon increased slightly, and the number of Bifidobacterium decreased significantly. The fermentation product has a certain regulatory effect on the key strains in the intestinal flora in the rat intestine. Description of the drawings:

[0032] Figure 1 Growth changes of Lactobacillus rhamnosus in composite medium fermentation system at different pH

[0033] Among them, Blank is the MRS culture medium system and Composite is the composite culture medium system.

[0034] Figure 2 The growth changes of Lactobacillus rhamnosus in the composite medium fermentation system in different bile salt concentrations

[0035] Among them, Blank is the MRS culture medium system and Composite is the composite culture medium system.

[0036] Figure 3 Growth changes of Lactobacillus rhamnosus in the composite medium fermentation system in artificial intestinal fluid

[0037] Among them, Blank is the MRS culture medium system and Composite is the composite culture medium system.

[0038] Figure 4 The changes in the pathological state of rats

[0039] Among them, Figure A shows the weight change of rats; Figure B shows the water content of rat feces;

[0040] Blank is the blank group, Model is the model group, Normal Saline is the natural recovery group, Probiotics is the probiotic group, and Fermentation is the fermentation group.

[0041] Figure 5 This is the H&E pathological section of rat colon.

[0042] Figure 6 The expression of inflammatory factors in intestinal tissue of rats

[0043] Among them, Figure A shows the content of TLR4 in intestinal tissue; Figure B shows the content of NF-κB in intestinal tissue.

[0044] Figure 7 It is the relative change of the expression of characteristic genes of different intestinal bacteria in rats (the blank group is the reference and is set to 1). Specific implementation method:

[0045] In order to make the purpose, technical solution and advantages of this patent more clear, this patent is further described in detail in combination with specific embodiments. It should be understood that the specific embodiments described here are only used to explain this patent and are not used to limit the present invention.

[0046] Preparation of medical stone mineralized water:

[0047] The Chinese medical stone sample was powdered and passed through a 40-325 mesh sieve to obtain medical stone powder. 20-40g of the sieved medical stone powder was taken, added with 200-400mL of deionized water, and soaked at 35-39°C and 100-150rpm for 12-36h to obtain medical stone mineralized water suspension, and then filtered under reduced pressure and passed through a 0.45μm filter membrane to obtain medical stone mineralized water.

[0048] The experimental strain used in the embodiment of the present invention is Lactobacillus rhamnosus CCTCC HB 20082747, which is a strain that has been made public before the application date and can be obtained by the public from the China Center for Type Culture Collection (CCTCC).

[0049] Unless otherwise specified, the pH of the culture medium in the embodiments of the present invention was 5.7±0.2.

[0050] The present invention will be further explained below in conjunction with specific embodiments.

[0051] Example 1: Effect of composite culture medium on acid resistance of Lactobacillus rhamnosus

[0052] 1 mol / L hydrochloric acid and 0.4% sodium hydroxide solution were used to adjust the MRS liquid medium and the composite medium to different pH values, namely 2.0, 3.0, 4.0, 5.0, 6.0, and 6.4. 3×10 8 For Lactobacillus rhamnosus with a CFU / mL, three parallel cultures were made in each group and samples were taken after culturing at 37°C and 125 rpm for 14 h. The number of viable bacteria was calculated by the gradient dilution plate counting method.

[0053] The composition of MRS medium is (g / L): peptone 10.0, beef powder 8.0, yeast powder 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 80 1.0, and the rest is sterile water;

[0054] The composition of the composite medium is (g / L): peptone 10.0, beef powder 8.0, yeast powder 4.0, glucose 20.0, dipotassium hydrogen phosphate 2.0, diammonium hydrogen citrate 2.0, sodium acetate 5.0, magnesium sulfate 0.2, manganese sulfate 0.04, Tween 80 1.0, 160 mesh Chinese medical stone powder 50, and the rest is 75% mineral water;

[0055] Preparation of mineralized water: grind Chinese medical stone to powder and pass it through an 80-mesh sieve to obtain medical stone powder; take 30 g of the sieved medical stone powder, add 300 mL of deionized water, and soak at 37°C, 125 rpm for 24 h to obtain medical stone mineralized water suspension, filter under reduced pressure and pass through a 0.45 μm filter membrane to obtain medical stone mineralized water, and mix the mineralized water and sterile water in a ratio of 75:25 to obtain 75% mineralized water.

[0056] The results are as follows Figure 1 As shown in the figure, when pH is 2≤≤5, the growth of probiotics in the composite culture medium fermentation system is significantly better than that in the common fermentation system. This indicates that in a medium to strong acidic environment, the composite culture medium fermentation system can significantly improve the acid resistance of probiotics.

[0057] Example 2: Effect of composite culture medium on bile tolerance of probiotics

[0058] Pig bile salts were added to the MRS liquid medium and the composite medium (same as in Example 1) to make the concentrations of 0% (control), 0.05%, 0.1%, 0.15%, 0.2%, and 0.3%, respectively. 3×10 8 CFU / mL of Lactobacillus rhamnosus, with 3 parallels per group, and samples were taken after culturing at 37°C and 125rpm for 14h, and the number of viable bacteria was calculated by the gradient dilution plate counting method.

[0059] The results are as follows Figure 2 As shown, when the bile salt concentration is 0.1-0.15%, the growth of probiotics in the composite culture medium fermentation system is significantly better than that in the common fermentation system, indicating that the composite culture medium fermentation system can significantly improve the bile salt tolerance of probiotics at this concentration of bile salt.

[0060] Example 3: Effect of composite culture medium on the resistance of probiotics to artificial intestinal fluid

[0061] Preparation of artificial intestinal fluid: Weigh 0.68 g of potassium dihydrogen phosphate, add 50 mL of water to dissolve, adjust the pH to 6.8 with 0.4% sodium hydroxide solution, take 1 g of trypsin, add appropriate amount of water to dissolve, combine the two liquids, add water to make up to 100 mL, and filter with a 0.45 μm sterile filter membrane.

[0062] 6 mL of artificial intestinal fluid was taken into a 10 mL centrifuge tube, and the probiotic fermentation broth grown in the MRS environment and the probiotic fermentation broth grown in the composite medium (MRS medium and composite medium are the same as those in Example 1) (inoculated at a concentration of 3 × 10 8 Rhamnosus Lactobacillus (CFU / mL) was inoculated into 2 mL of artificial intestinal fluid after being cultured at 37°C and 125 rpm for 14 h. Three parallel cultures were made in each group and cultured in a shaking incubator at 37°C and 125 rpm. Samples were taken at 0, 1, 2, 4, and 16 h, and the number of viable bacteria was calculated by the gradient dilution plate counting method.

[0063] The results are as follows Figure 3 As shown, during the time that food stays in the human intestine, under the same gradient dilution, the number of probiotic plate colonies grown in the composite culture medium environment is significantly greater than that in the MRS environment, indicating that the composite fermentation system can significantly improve the survival ability of probiotics in artificial intestinal fluid.

[0064] Example 4: Effects of fermentation products on pathological conditions in rats

[0065] 1. Preparation of drug solution

[0066] Fermentation group: MRS medium was prepared with 75% mineral water prepared with 80-mesh Chinese medical stone, and 160-mesh Chinese medical stone powder was added to the medium at a ratio of 5 g / 100 mL to prepare the fermentation group medium (the preparation method was the same as the composite medium in Example 1);

[0067] After inoculation of Lactobacillus rhamnosus, the culture was carried out at 37°C and 125 rpm for 14 h. After fermentation, samples were taken for coating and counting. The fermentation liquid was diluted to 10 according to the counting results. 8 CFU / mL, as fermentation broth.

[0068] Probiotic group: Lactobacillus rhamnosus was inoculated with MRS medium (same composition as in Example 1) and cultured at 37°C, 125 rpm for 14 h. Samples were taken for coating and counting. The fermentation broth was diluted to 10 8 CFU / mL, as a probiotic solution.

[0069] 2. Observation of pathological morphology

[0070] Fifty male SD rats (200±20g) were placed under conditions of 25±2℃, 12h / 12h light-dark cycle, and 60% relative humidity, with free access to food and water for 3 days to acclimate to the environment. They were then divided into 5 groups (blank group, model group, natural recovery group, fermentation group, and probiotic group. Unless otherwise specified, each group continued to have free access to food and water until 24h before sacrifice), among which:

[0071] Blank group: gavage with normal saline (2mL / 200g) all the time;

[0072] The other groups were gavaged with triple antibiotics (2 mL / 200 g) for 5 consecutive days (the start of gavage was counted as DAY1) to induce diarrhea in rats. Thereafter:

[0073] Model group: fasting and water deprivation for 24 h, 20% urethane (2 mL / 200 g) was injected intraperitoneally to anesthetize the rats, and the colon was quickly dissected, one part was immersed in 10% formalin and stored at 4°C, and the other part was stored at -80°C;

[0074] Natural recovery group: gavage with normal saline (2mL / 200g) every day;

[0075] Probiotic group and fermentation group: The corresponding drug solution (2mL / 200g) was gavaged orally for 5 consecutive days and feces were collected every day. After that, the five groups of rats were anesthetized and the colons were dissected.

[0076] Fecal samples were collected and weighed, and then dried at 105°C using a rapid moisture meter to determine dry weight. Fecal moisture content = (wet weight - dry weight) / wet weight × 100%.

[0077] The rats in each group were in good condition and had similar diets. After the antibiotic modeling, the rats showed loose fur, arched backs, and loose stools, which indicated that the modeling was effective. During the drug administration, the weight of the rats in each drug administration group gradually increased. Figure 4 As shown in A, on the third day after administration, the weight growth rate of each drug administration group exceeded that of the natural recovery group, and the weight gain of the fermentation group was the most significant; Figure 4 As shown in B, the water content of feces of rats in the fermentation group decreased steadily only after drug administration, indicating that the drug solution in the fermentation group can improve the diarrhea of ​​rats and alleviate the symptoms of rats.

[0078] Example 5: Effects of fermentation products on intestinal inflammation in rats

[0079] After the experiment in Example 4, frozen sections of the colon at the same position were taken from the experimental mice, and H&E staining was performed, and pathological observation was performed based on the staining results.

[0080] like Figure 5 As shown in the figure, the colon mucosa structure of rats in the normal group was normal, the epithelial cells were intact, the goblet cells were neatly arranged, and the cell layers were clear. The colon of rats in the model group had obvious edema, epithelial cell loss and erosion, goblet cell loss, reduced glandular density, and a large number of inflammatory cells infiltrated in the mucosal lamina propria. The colon of rats in the natural recovery group still had obvious edema, goblet cell fragmentation, ulceration, and inflammatory cell infiltration; the colon of rats in the probiotic group had no edema, but goblet cell fragmentation and inflammatory cell infiltration were still observed; the colon of rats in the fermentation group had no edema, the epithelial cells were neatly arranged, the glands were normal, and although there were still a small number of inflammatory cells, the overall morphology was similar to that of normal rats.

[0081] Example 6: Effects of fermentation products on intestinal inflammatory factors in rats.

[0082] TLR4 and NF-κB are highly correlated with intestinal tissue inflammation. NF-κB is involved in the release of proinflammatory factors and the repair of intestinal mucosal damage; TLR4 can activate the NF-κB signaling pathway, causing the inflammatory signal to continue to amplify.

[0083] Take 8cm of colon tissue from the same position of the experimental mouse in Example 4, repeatedly rinse with ice-cold PBS buffer to remove blood and fecal residue, dry and weigh, add 9 times ice-cold PBS in proportion, grind into 10% tissue homogenate with a tissue homogenizer under ice bath, centrifuge at 2500-3000r / min for 15min, and the supernatant is tested. Operate according to the instructions of TLR4 and NF-κB kit (enzyme-linked immunosorbent assay), and prepare a standard curve according to the standard product provided by the kit. The content of TLR4 and NF-κB in intestinal tissue is calculated by comparing with the standard curve.

[0084] like Figure 6 As shown, according to the kit instructions, the standard curves of TLR4 and NF-κB were y=10.819x+0.2073 and y=7.8713x-0.6943, respectively. The standard curve R 2 All of them were greater than 0.99, and the standard curve was valid. The contents of TLR4 and NF-κB in each drug-treated group were lower than those in the natural recovery group, especially the fermentation group, which was close to the blank control group, indicating that the levels of TLR4 and NF-κB in the rat colon decreased after drug administration, and the inflammation was alleviated, proving the effectiveness of the drug. Compared with the drug-treated groups, only the results of the fermentation group and the model group showed significant differences, and the results of the other two groups were not significantly different from those of the model group, proving that the fermentation group had a better effect.

[0085] Example 7: Effects of fermentation products on intestinal flora of rats.

[0086] Total bacteria were used as internal reference genes, and four intestinal bacteria, Bacteroid, Enterococcus, Escherichia coli, and Lactobacillus, were used as indicators. The above four intestinal bacteria are key strains in the intestinal flora and are often used for the analysis of intestinal flora. Among them, Escherichia coli is a common conditional pathogen, and its high abundance may cause intestinal diseases; Enterococcus can form biofilms on intestinal epithelial cells to protect intestinal epithelial cells from harmful substances, but virulence genes may appear in long-term evolution, causing widespread infection; Lactobacillus is a common probiotic that can secrete a variety of enzymes, participate in the metabolism of nutrients and maintain the homeostasis of intestinal flora; Bacteroidetes is a common dominant bacteria in the intestinal flora, accounting for about 1 / 4 of the total intestinal flora. It can be transformed into a conditional pathogen under certain conditions, but it also participates in the normal metabolic function of the intestine.

[0087] Weigh 0.20g of fecal samples from each group of experimental mice on DAY10 in Example 4 into a 2mL sterile EP tube, and extract total fecal DNA according to the steps in the DNA extraction kit manual. The concentration and purity of the extracted product were determined using a nucleic acid protein detector. A 10μL PCR reaction system was used, including 5μL of fluorescent dye SYBR green, 0.6μL of DNA template, 0.2μL of upstream and downstream primers, 1μL of ROX, and 3μL of double distilled water. Fluorescence quantitative PCR reaction conditions: 95℃ denaturation for 30s, 95℃5s, 60℃1min for a total of 40 cycles, and 72℃ extension for 10min. Negative controls were set for each experiment. In order to detect the specificity of the PCR product, the temperature was gradually increased from 60℃ to 95℃ (0.5℃ per cycle) during quantitative PCR, and the SYBRGreen signal was detected at the end of each cycle to draw a melting curve. Each sample was repeated 3 times.

[0088] Table 1: Sequences of primers for bacterial amplification

[0089]

[0090]

[0091] The expression multiple of characteristic genes in each group is the ratio of the CT value of qPCR of the gene in this group to that of the normal group (the dotted line is the normal blank group, which is regarded as 1). When the ratio approaches 1, the corresponding bacterial abundance of the gene in this group is close to the normal blank group. A value greater than or less than 1 indicates an increase or decrease in bacterial abundance, respectively.

[0092] like Figure 7As shown, compared with the normal blank group (the expression multiple of characteristic genes in each group is the ratio of the CT value of the gene qPCR of the group to the normal group, and when the ratio is close to 1, the corresponding bacterial abundance of the gene in the group is close to the normal group, and greater than or less than 1 indicates an increase or decrease in bacterial abundance, respectively), the number of Enterococcus faecalis and Escherichia coli in the model group rats increased significantly, and the number of Bacteroides and Lactobacillus decreased significantly, indicating that the rat modeling was successful in this experiment, and the corresponding intestinal bacteria had negative changes. Compared with the natural recovery group, the number of Bacteroides and Lactobacillus in each drug administration group, especially the fermentation group, increased slightly, and the content of Enterococcus faecalis and Escherichia coli decreased to a certain extent, indicating that the key strains in the intestinal flora in the rat intestine had certain regulatory changes after drug administration, showing a trend of restoring the normal flora structure.

[0093] The foregoing description and diagrams include illustrative embodiments of the present invention. The foregoing embodiments and methods may vary based on the abilities, experience, and preferences of those skilled in the art. The method steps listed in a certain order do not limit the order of the method steps of the present invention. The foregoing description and diagrams are merely explanations and illustrations of the present invention, and the present invention is not limited to such limitations, except for the limitations within the scope of the claims. Those skilled in the art may modify and vary the present invention without departing from the scope of the present invention.

Claims

1. A Lactobacillus rhamnosus fermentation product, characterized in that The preparation method is as follows: the composite culture medium is inoculated with Lactobacillus rhamnosus, and cultured at 35-39° C. and 100-150 rpm for 12-16 hours to obtain the obtained product; The composite culture medium is prepared by preparing MRS culture medium with mineral water and adding Chinese medical stone powder; The deposit number of the Lactobacillus rhamnosus is CCTCC HB 20082747; The composite culture medium is composed of (g / L): 10.0% peptone, 8.0% beef powder, 4.0% yeast powder, 20.0% glucose, 2.0% dipotassium hydrogen phosphate, 2.0% diammonium hydrogen citrate, 5.0% sodium acetate, 0.2% magnesium sulfate, 0.04% manganese sulfate, 1.0% Tween 80, 50.0% 160-mesh Chinese medical stone powder, and the rest is 75% mineral water; The preparation method of the mineralized water is as follows: a Chinese medical stone sample is pulverized and passed through a 40-325 mesh sieve to obtain medical stone powder; the sieved medical stone powder is mixed with water at a mass volume ratio (g / mL) of 1-2:1-20, and soaked at 35-39° C. and 100-150 rpm for 12-36 hours to obtain a medical stone mineralized water suspension, and the suspension is filtered under reduced pressure and then passed through a microfiltration membrane to obtain the medical stone mineralized water suspension.

2. The Lactobacillus rhamnosus fermentation product according to claim 1, characterized in that The fermented product is in liquid form, or can be freeze-dried and stored and used in solid form.

3. Use of the Lactobacillus rhamnosus fermentation product according to claim 1 in preparing a drug for treating diarrhea.

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