Lactobacillus rhamnosus QS001 and its use in preparing intestinal probiotic preparations

Abstract

The invention discloses a rhamnosus lactobacillus QS001 and a use thereof for preparing an intestinal probiotic preparation, wherein the rhamnosus lactobacillus QS001 is classified and named as rhamnosus lactobacillus Lacticaseibacillus rhamnosus , deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, with the deposit number CGMCC No.29275. The rhamnosus casei bacteria QS001 is used in the preparation of intestinal probiotic preparations. The active ingredients of the probiotic preparations include the bacteria of the above-mentioned rhamnosus casei bacteria QS001. The rhamnosus casei bacteria QS001 is also used in the preparation of immune disorder regulators, drugs for increasing the concentration of anti-inflammatory cytokines and / or reducing the concentration of pro-inflammatory cytokines in colon tissue, and has the advantage of alleviating or treating inflammatory bowel disease and regulating immune disorders.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial probiotics, and particularly relates to a Lactobacillus rhamnosus QS001 strain and an application thereof in preparing an intestinal probiotic preparation. Background Art

[0002] Inflammatory bowel disease (IBD) is a chronic and recurrent intestinal inflammation that manifests itself in two main clinical phenotypes: ulcerative colitis (UC) and Crohn's disease (CD). Ulcerative colitis primarily involves confluent inflammation of the colonic mucosa, while Crohn's disease typically has transmural, lamellar features. The etiology of IBD is still unclear, but genetics and other factors (such as microbiota and diet) can lead to immune disorders, including altered immune responses to the microbiota, disruption of intestinal barrier function, and separation of the intestinal epithelium from immune cells, ultimately leading to chronic inflammation. Microorganisms can affect immune cells by contacting with antigens or crossing the intestinal epithelial barrier, thereby inducing immune responses.

[0003] Treatments for IBD include anti-inflammatory, immunomodulatory, and immunosuppressive drugs, as well as biological therapies targeting inflammatory cytokines (such as tumor necrosis factor or blocking immune cell homing). However, these therapies have significant side effects and high treatment costs. Compared with traditional treatments, probiotic supplementation is beneficial for both adult IBD and pediatric IBD. It has been reported that probiotics can alleviate colitis symptoms by regulating intestinal microbiota, enhancing mucosal barrier function, inhibiting the expression of inflammatory cytokines, and regulating the imbalance of immune response of the immune system.

[0004] Lactobacillus rhamnosus is a Gram-positive bacterium that exists in many types of fermented foods. It can survive in various environments including the intestines and vagina and has a wide range of probiotic properties. Lactobacillus rhamnosus is one of the normal intestinal flora. It can promote the abundance of beneficial bacteria such as Bifidobacterium, regulate intestinal flora, and prevent or treat acute and chronic diarrhea. At the same time, Lactobacillus rhamnosus also has the effect of increasing the body's immunity, and it is also helpful for allergies. For example, increasing the body's immunity is also helpful for relieving eczema. However, Lactobacillus rhamnosus increases the body's immunity and relieves allergies and eczema. These are only auxiliary effects, not fundamental therapeutic effects. Therefore, the use of Lactobacillus rhamnosus to develop probiotic preparations that can assist in the treatment of inflammatory bowel disease (IBD) and regulate immune disorders has a positive significance for regulating human immunity and improving intestinal function. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus rhamnosus QS001 which has the effects of alleviating or treating inflammatory bowel disease and regulating immune disorders, and the use of the strain in preparing an intestinal probiotic preparation.

[0006] The technical solution adopted by the present invention to solve the above technical problems is: a strain of Lactobacillus rhamnosus QS001, which is classified and named as Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 11, 2023, with the deposit number CGMCC No.29275.

[0007] The present invention also provides use of the Lactobacillus rhamnosus QS001 in preparing an intestinal probiotic preparation.

[0008] Preferably, the active ingredient of the intestinal probiotic preparation includes the bacteria of the above-mentioned Lactobacillus rhamnosus QS001.

[0009] Preferably, the effective bacterial cell number of Lactobacillus rhamnosus QS001 in the intestinal probiotic preparation is not less than 1×10 8 CFU / g.

[0010] Preferably, the intestinal probiotic preparation includes at least one of powder, granules, pills, capsules, tablets, pastes, liquid preparations, gels and sprays.

[0011] Preferably, the intestinal probiotic preparation is composed of the following raw materials and their weight parts: 4-6 weight parts of Lactobacillus rhamnosus powder, 20-40 weight parts of fruit powder, 10-20 weight parts of galacto-oligosaccharide, 4-5 weight parts of lactitol, xylitol or sugar alcohol, and 25-35 weight parts of maltodextrin.

[0012] Preferably, the intestinal probiotic preparation is composed of the following raw materials and their weight parts: 4-6 weight parts of Lactobacillus rhamnosus powder, 20-40 weight parts of fruit powder, 10-20 weight parts of galacto-oligosaccharide, 4-5 weight parts of lactitol, xylitol or sugar alcohol, 20-30 weight parts of skimmed milk powder, 5-10 weight parts of microcrystalline cellulose, and 0.5-1 weight part of magnesium stearate.

[0013] The present invention also provides use of the Lactobacillus rhamnosus QS001 in preparing an immune disorder regulator.

[0014] The present invention also provides use of the Lactobacillus rhamnosus QS001 in preparing drugs for alleviating and / or treating inflammatory bowel disease.

[0015] The present invention also provides the use of the above-mentioned Lactobacillus rhamnosus QS001 in preparing a drug for increasing the concentration of anti-inflammatory cytokines and / or reducing the concentration of pro-inflammatory cytokines in colon tissue, wherein the anti-inflammatory cytokines are interleukin-2 and interleukin-10, and the pro-inflammatory cytokines are interleukin-5 and interleukin-1β.

[0016] Compared with the prior art, the advantages of the present invention are: the present invention provides a strain of Lactobacillus rhamnosus QS001 and the use thereof in preparing an intestinal probiotic preparation; the screened and preserved strain of Lactobacillus rhamnosus QS001 is separated from naturally fermented milk, can alleviate and / or treat inflammatory bowel disease (IBD), increase the concentration of anti-inflammatory cytokines, reduce the secretion of pro-inflammatory cytokines, reduce inflammatory response, reduce colon tissue damage, and has the function of regulating immune disorders; can be used for preparing probiotic preparations, ordinary foods, health foods and biopharmaceuticals for improving intestinal health, is convenient for industrial production, and has broad application prospects.

[0017] The above-mentioned Lactobacillus rhamnosus QS001 is classified and named as Lactobacillus rhamnosus ( Lacticaseibacillus rhamnosus ), the deposit number is CGMCC No.29275, the deposit date is December 11, 2023, the depositor is the General Microbiology Center of China Culture Collection Administration, and the deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the microscopic morphology of Lactobacillus rhamnosus QS001;

[0019] Figure 2 The morphological characteristics of Lactobacillus rhamnosus QS001;

[0020] Figure 3 Prediction results for Lactobacillus rhamnosus QS001 on the iprobiotics platform;

[0021] Figure 4 Design of a group for the treatment of inflammatory bowel disease with Lactobacillus rhamnosus QS001;

[0022] Figure 5 The results showed that Lactobacillus rhamnosus QS001 significantly reduced the weight loss symptoms caused by IBD;

[0023] Figure 6 Lactobacillus rhamnosus QS001 significantly reduced the DAI score results;

[0024] Figure 7 The results showed that Lactobacillus rhamnosus QS001 significantly reduced the macroscopic lesion scores of IBD mice;

[0025] Figure 8 The results showed that Lactobacillus rhamnosus QS001 significantly reduced the shortening of colon length in IBD mice;

[0026] Fig. 9 The results showed that Lactobacillus rhamnosus QS001 significantly reduced the changes in colon weight in IBD mice;

[0027] Fig.10 The results showed that Lactobacillus rhamnosus QS001 significantly reduced the changes in spleen weight in IBD mice;

[0028] Fig.11 The results showed that Lactobacillus rhamnosus QS001 significantly reduced the GSH content in the colon of IBD mice;

[0029] Fig.12 The results showed that Lactobacillus rhamnosus QS001 significantly reduced the NO content in the colon of IBD mice;

[0030] Fig.13 The results show that Lactobacillus rhamnosus QS001 significantly reduced the inflammatory response, where A is IL-1β, B is the content of IL-5, C is IL-2, and D is IL-10. The intervention group was the Lactobacillus rhamnosus QS001 group. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below with reference to the accompanying drawings. 1. Specific embodiments

[0033] Example 1: Isolation and screening of target strains.

[0034] Weigh 0.5 g of naturally fermented milk sample and dilute it with 4.5 mL of sterile PBS buffer, then take 0.1 mL and spread it on the modified MRS solid medium, and culture it anaerobically at 37℃ for 72 h. After selecting typical colonies for Gram staining microscopy, select Gram-positive strains, and then streak them on the modified MRS solid medium for 2-3 times to obtain pure colonies. The formula of the modified MRS solid medium is: bacteriological peptone 10 g / L, beef extract powder 8 g / L, yeast extract powder 4 g / L, glucose 20 g / L, Tween-80 1 mL, K2HPO4 2g / L, sodium citrate 5g / L, ammonium citrate trihydrate 2 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·5H2O 0.05g / L, purified water 1000 mL, agar 15 g / L.

[0035] Example 2: Identification of target strains.

[0036] 1. Microscopic morphology and culture characteristics: This strain is a Gram-positive bacterium observed under a microscope. It has no flagella, does not move, does not form spores, and requires oxygen. The bacteria are rod-shaped and have various shapes, mostly baseball-shaped. The average length (L) is 1.3559±0.1459μm, and the average width (D) is 0.4959±0.46μm. Figure 1 On the modified MRS solid medium, the colonies are smooth with complete edges, creamy white and opaque. Figure 2 ; The optimum growth temperature is 36℃-38℃, and the optimum pH value is 6.0-7.0.

[0037] 2. 16S rDNA sequence analysis: The isolated strains were subjected to physiological and biochemical identification and 16S rRNA molecular identification according to the Bergey Manual of Bacterial Identification using bacterial API50CH (bioMérieux). The results are shown in Table 1.

[0038] Table 1 Carbohydrate utilization results of Lactobacillus rhamnosus QS001

[0039]

[0040] The DNA sample of the strain was extracted and the sample was identified by 16S rRNA molecule by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. The molecular sequence was compared with NCBI database blastn, and QS001 was confirmed to be rhamnosus casei, and named rhamnosus casei ( Lacticaseibacillus rhamnosus ) QS001. The strain has been deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, and its classification name is Lactobacillus rhamnosus Lacticaseibacillus rhamnosus The deposit number is CGMCC No.29275, the deposit date is December 11, 2023, and the deposit address is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China.

[0041] 3. Basic genome information: The whole genome of Lactobacillus rhamnosus QS001 was assembled and its basic genome information was statistically analyzed. It was found that the genome of Lactobacillus rhamnosus QS001 contained 1 circular chromosome and two circular plasmid chromosomes, as shown in Table 2.

[0042] Table 2 Basic information of the genome of Lactobacillus rhamnosus QS001

[0043]

[0044] As shown in Table 2 , the size of the circular chromosome is 3,145,806 bp with a GC content of 46.1%, and the sizes of the plasmid chromosomes are 74,594 bp and 64,555 bp, with GC contents of 43.9% and 42.0%, respectively.

[0045] 4. Analysis of tolerance genes: The key to maintaining the viability of lactic acid bacteria is to improve the stress resistance of lactic acid bacteria and withstand a series of environmental pressures such as extreme temperature, osmotic pressure, pH, oxygen, etc. The tolerance-related functional genes of QS001 were annotated, and 6 genes related to acid tolerance (dapA, argH, metE, argR, argS, murE); 9 genes related to heat tolerance (dnaK, hrcA, dnaJ, grpE, dnaA, dnaG, dnaB, dnaE, dnaN); 1 gene related to oxygen tolerance (oxyR); 1 gene related to cold tolerance (clpC); 2 genes related to bile salt tolerance (purD, gcvH), see Table 3.

[0046] Table 3 Results of tolerance-related genes

[0047]

[0048] 5. Prebiotic-related genes: Prebiotic-related genes in the QS001 genome were annotated, and functional genes related to the production of riboflavin, lactic acid, extracellular polysaccharides, bioactive peptide synthesis, and acid and bile resistance were obtained (see Table 4).

[0049] Table 4 Probiotic-related genes

[0050]

[0051] The above strains were used to predict probiotics using the iProbiotics platform (there are more than 2282 probiotic strains enriched genes in the iProbiotics platform). Using the iProbiotics platform Model1: Probiotic Predictor analysis, it was found that the probability that QS001 is a probiotic is 99.87%, which is basically consistent with the prediction result of Lactobacillus rhamnosus GG strain, see Figure 3 shown.

[0052] 6. Drug resistance gene annotation: The Perfect and Strict algorithms in RGI5.1.0 software were used as screening criteria to compare the rhamnosus Lactobacillus QS001 and rhamnosus Lactobacillus GG strains with the CARD (The Comprehensive Antibiotic Resistance Database) database to predict antibiotic resistance-related genes in the strain genome. The results showed that no potential drug resistance-related genes were detected in the rhamnosus Lactobacillus QS001 and GG genomes.

[0053] In addition, the results were compared with the ResFinder database, which includes 2,817 drug-resistant gene sequences for 15 major categories and 49 antibiotics. The default parameters of the database, i.e., sequence similarity > 90% and sequence coverage > 60%, were used as screening criteria, and the results were consistent with the CARD results.

[0054] 7. Virulence factor annotation: The above strains were compared with the VFDB database (Virulence Factors of Pathogenic Bacteria) and the VirulenceFinder database, with sequence similarity > 90% and sequence coverage > 60% as screening criteria. The results showed that no potential virulence-related genes were detected in the genomes of Lactobacillus rhamnosus QS001 and Lactobacillus rhamnosus GG strains.

[0055] 8. SNP analysis: Mauve colinearity analysis was performed using QS001 as the reference strain. QS001 is highly similar to GG, with only local fragment insertions. In addition, there are 65,700 SNPs between QS001 and GG, and 13 unique genes were found in the QS001 genome (epsL, rfaB, yiaC, dld, menB, menE, sauU, nfrA, xylA, nanK, cysM, topB, panE), which are mainly related to extracellular polysaccharides, lactic acid and amino acids. The above results show that there are significant differences in the genomes of Lactobacillus rhamnosus QS001 and Lactobacillus rhamnosus GG strain (LGG).

[0056] Example 3: Gastrointestinal fluid tolerance of Lactobacillus rhamnosus QS001.

[0057] Preparation of simulated gastric fluid: Pepsin was added to a pH 2.5 (adjusted with 1 mol / L HCl) sterilized PBS buffer to a concentration of 3.5 g / L, and the solution was sterilized by filtration with a 0.22 μm microporous filter membrane to prepare simulated gastric fluid.

[0058] After culturing two generations of Lactobacillus rhamnosus QS001, the bacteria were washed twice by centrifugation, and the cells were collected. An equal amount of simulated gastric fluid with pH 2.5 was added to the culture medium, and the cells were aerobically cultured at 37°C for 3 h. The number of viable bacteria was determined by the MRS agar medium pouring method at 0 h and 3 h.

[0059] Preparation of simulated intestinal fluid: Add 0.1% trypsin and 1.8% ox bile salt by mass of sterile PBS buffer solution at pH 8.0 (adjusted with 0.1 mol / L NaOH) and filter with a 0.22 μm microporous filter membrane to sterilize and prepare simulated intestinal fluid.

[0060] After the bacterial solution was treated with simulated gastric juice for 3 hours, the bacteria were collected by centrifugation and washing twice, and then the simulated intestinal fluid equal to the simulated gastric juice was added to continue aerobic culture at 37°C. The number of viable bacteria was measured by the MRS agar medium pouring method at 0 h, 4 h, and 8 h. The method for viable bacteria counting was the pouring plate counting method, and the specific operation steps were as follows: Detection according to GB 4789.35-2016 method. Survival rate = [N1 / N0] × 100%, where N0 represents the number of viable bacteria at 0 h; N1 represents the number of viable bacteria after simulated digestion for 3 h or 8 h.

[0061] After Lactobacillus rhamnosus QS001 was treated with simulated gastric fluid and simulated intestinal fluid, the results are shown in Table 5 below.

[0062] Table 5 Survival of Lactobacillus rhamnosus QS001 in simulated gastrointestinal digestive fluid

[0063]

[0064] From the data in Table 5, it can be seen that the Lactobacillus rhamnosus QS001 screened in Example 1 has good tolerance characteristics, and the survival rate can reach 61.42% after being treated with simulated intestinal fluid for 8 hours. According to the gastrointestinal fluid tolerance effect of this example, it can be seen that Lactobacillus rhamnosus QS001 has good tolerance to gastrointestinal fluid in the intestine and has probiotic properties.

[0065] Example 4: The effect of Lactobacillus rhamnosus QS001 on enhancing the body's immunity.

[0066] 1. Experimental design: Thirty SPF-grade 6-week-old male C57BL / 6J mice were selected and randomly divided into three groups after adaptive feeding for 7 days, with 10 mice in each group: a healthy control group, a DSS (dextran sulfate sodium) model group, and a Lactobacillus rhamnosus QS001 live bacteria group (live bacteria count 1×10 9 CFU / g). During the whole experiment, the healthy control group drank sterile water freely; the model group was given 2.5% (w / v) DSS aqueous solution for 0-5 days, which was replaced with 0.2 mL of sterile water for 6-10 days. This process was considered as one cycle, and a total of 3 cycles were performed (DSS drinking water was replaced once every 3, 13, and 23 days); the rhamnosus lactobacillus QS001 live bacteria group was given 2.5% (w / v) DSS aqueous solution for 0-5 days, which was replaced with 0.2 mL of rhamnosus lactobacillus QS001 bacterial solution for 6-10 days. This process was considered as one cycle, and a total of 3 cycles were performed (the bacterial solution was replaced once every 3, 13, and 23 days). The experimental period was 30 days. The specific implementation design plan is as follows Figure 4 shown.

[0067] 2. Weight monitoring: Monitor the initial weight (0 d) after modeling and the weight after 10 d, 20 d, and 30 d of intervention. Weigh each mouse and calculate the average weight. See the table for specific results. Figure 5 .Depend on Figure 5 It can be seen that mice with inflammatory bowel disease induced by DSS sodium dextran sulfate will experience weight loss. After continuous gavage of live Lactobacillus rhamnosus QS001 for 30 days, the Lactobacillus rhamnosus QS001 group can significantly increase the body weight of IBD mice compared with the model group ( P <0.05).

[0068] The above studies show that Lactobacillus rhamnosus QS001 can effectively reduce the weight loss symptoms caused by inflammatory bowel disease.

[0069] 3. Determination of DAI score: Mouse feces were collected, and the disease activity index (DAI) of all mice was determined by scoring the weight, hemolytic reaction or blood and feces consistency of DSS-induced mice. The specific scoring criteria are as follows: Weight change rate score: 0=0%-weight gain; 1=0%-1=0%<weight change rate ≤-5%; 2=-5%<weight change rate ≤-10%; 3=-10%<weight change rate ≤-15%; 4=weight change rate>-15%. Fecal characteristics score; 0=normal feces; 1=wet feces; 2=loose feces; 3=unformed feces. Fecal occult blood score: 4=0s<purple time ≤10s; 3=10s<purple time ≤30s; 2=30s<purple time ≤60s; 1=60s<purple time ≤120s; 0=purple time>120s. Fecal samples were collected, quickly frozen in liquid nitrogen and stored in a -80°C refrigerator. Metabolic analysis was performed at -14°C. The specific results are shown in Figure 6 .Depend on Figure 6 It can be seen that DAI comprehensively evaluates the weight change rate, fecal characteristics and fecal occult blood of mice, that is, DAI score = weight change rate score + fecal characteristics score + fecal occult blood score. After 20 days of intervention with Lactobacillus rhamnosus QS001, the DAI score of the intervention group was significantly lower than that of the model group, and the clinical symptoms of inflammatory bowel disease were significantly improved.

[0070] The above studies show that Lactobacillus rhamnosus QS001 can reduce DAI scores, reduce inflammatory responses, effectively treat immune disorders and improve inflammatory bowel disease.

[0071] 4. Colon macroscopic score determination: Mice were killed by CO2 asphyxiation, the abdomen was opened, and the macroscopic appearance of the colon was examined. The score range of colon mucosa was 0 to 10 points, as follows:

[0072] 0 = no macroscopic changes;

[0073] 1 = only hyperemia;

[0074] 2 = mild mucosal edema, small erosions;

[0075] 3 = moderate edema with linear ulceration in one area;

[0076] 4 = inflammation / ulceration at ≥2 sites;

[0077] 5 = ulcer / inflammation extending >1 cm along the length of the colon;

[0078] 6-10 = If the lesion covers > 2 cm along the length of the colon, the score is increased by 1 point for each additional cm.

[0079] The use of DSS-induced mucosal damage in the distal colon wall. Compared with the healthy control group, the colon mucosa showed obvious congestion, deep ulcers, edema, and bleeding. At the same time, the mice also had diarrhea, soft stools, rectal bleeding, and weight loss. In addition, due to tissue edema, the proportion of the distal colon weight to the animal body weight increased significantly. Figure 7 It indicates that the macroscopic lesions were significantly reduced after taking Lactobacillus rhamnosus QS001. It can be seen that Lactobacillus rhamnosus QS001 can alleviate the symptoms of colon tissue damage caused by inflammatory bowel disease.

[0080] 5. Determination of colon length and weight: DSS-induced colitis is usually located in the colon and is accompanied by pathological symptoms of shortened colon length. Colon length and weight can not only measure the severity of colitis in mice, but are also important indicators for evaluating the body's immune response. Remove the mouse colon, open it longitudinally, gently wash the feces with saline, and measure the colon length. Use a sterilized 200 μL pipette tip to remove the contents of the lumen (mucosa) before weighing the organ. Measure the colon length (anus-end of the cecum) of each mouse after 10 d, 20 d, and 30 d of intervention, and record the length of the colon in centimeters. See the specific results. Figure 8 At the end of the experiment, the colon length of the mice in the intervention group was significantly longer than that in the model group ( Figure 8 ), and the colon weight was significantly lower than that of the model group ( Fig. 9 ), indicating that rhamnosus lactobacillus QS001 effectively alleviated the inflammatory edema of colon tissue induced by DSS. It can be seen that rhamnosus lactobacillus QS001 can effectively prevent the shortening of colon length, reduce and alleviate the symptoms of colon tissue damage caused by inflammatory bowel disease, and regulate intestinal immune disorders.

[0081] 6. Spleen weight measurement: The spleen is an important immune organ, and its weight increase is related to the degree of inflammation. Fig.10 As shown, after supplementation with Lactobacillus rhamnosus QS001, the spleen weight of mice in the intervention group decreased, and there was a significant difference compared with the model group, which can significantly reduce the inflammatory response caused by inflammatory bowel disease and effectively enhance the body's immunity.

[0082] 7. Tissue SH group (GSH) content determination: The formation of thiobarbituric acid reactive substances (TBARS) was measured by spectrophotometry and the content of malondialdehyde in colon samples was calculated to determine lipid peroxidation. The GSH content in colon homogenate was determined by reducing Ellman's reagent with GSH in phosphate buffer at pH 8 to generate 5,5-dithio-2-nitrobenzoic acid (DTNB).

[0083] Fig.11 The results show that Lactobacillus rhamnosus QS001 can reduce the GSH (one of the key antioxidant components) consumption. After DSS induction, the concentration of GSH in colon tissue decreased significantly, while the consumption of Lactobacillus rhamnosus QS001 effectively controlled the decrease in GSH content. This suggests that Lactobacillus rhamnosus QS001 can alleviate the symptoms of inflammatory colitis.

[0084] 8. Determination of nitric oxide production: The colonic nitric oxide level was estimated by the acidic Griess reaction. An equal volume of colon homogenate and nitrate reduction test reagent (Griess reagent) were mixed, and its absorbance was measured at a wavelength of 450 nanometers. The concentration of nitric oxide was then determined by the plunger method, and the result was expressed in micrograms per milligram of wet tissue.

[0085] Depend on Fig.12 It can be seen that compared with the healthy control group, the NO content increased significantly after DSS induction ( P <0.05). However, the NO content in mice fed with Lactobacillus rhamnosus QS001 was significantly lower than that in the model group ( P <0.05). It can be seen that Lactobacillus rhamnosus QS001 can effectively alleviate intestinal inflammation and immune disorders caused by DSS.

[0086] 9. Immune factor determination: The colon tissue was homogenized in phosphate buffered saline using a homogenizer. The colon tissue homogenate was then centrifuged at 20,000 Íg for 20 minutes to obtain the supernatant. The ELISA kit was used to determine the content of anti-inflammatory cytokines IL-2 and IL-10, as well as the content of pro-inflammatory cytokines IL-1β and IL-5 in the colon samples, and the values ​​were expressed in pg / mg tissue.

[0087] The results are as follows Fig.13 As shown in the figure, after supplementation with Lactobacillus rhamnosus QS001, the serum IL-2 ( Fig.13 C) and IL-10 ( Fig.13 The content of D) in the model group was significantly higher than that in the model group, while IL-1β ( Fig.13 A) and IL-5 ( Fig.13The content of IL-10 in the QS001 group was about 20% lower than that in the control group, but nearly 25% higher than that in the model group ( Fig.13 D in the figure); the content of IL-1β was about 35% higher than that of the control group and nearly 20% lower than that of the model group ( Fig.13 A in the figure), Lactobacillus rhamnosus QS001 has the effect of increasing the concentration of anti-inflammatory cytokines and reducing the concentration of pro-inflammatory cytokines. The balance of pro-inflammatory cytokines and anti-inflammatory cytokines helps to control inflammation. Both have obvious immunomodulatory ability, that is, under the background of DSS, Lactobacillus rhamnosus QS001 can make the body's immune level close to the normal feeding group and significantly different from the model group. It can be seen that Lactobacillus rhamnosus QS001 can significantly alleviate the body's inflammatory response caused by inflammatory bowel disease and enhance the body's immunity.

[0088] 10. Statistical analysis: SPSS software was used to analyze the data. This study mainly involved the efficacy differences between the probiotic group and the model group. Considering the skewed distribution and nonparametric nature of our data, the Mann-Whitney test was used to compare the data differences between the probiotic group and the model group or between two different time points; the chi-square test was used to compare the data. Unless otherwise stated, all tests were P <0.05 statistical significance, and the data are expressed as mean ± standard error.

[0089] In summary, the Lactobacillus rhamnosus QS001 screened in Example 1 can effectively inhibit the increase in DAI score and weight loss caused by DSS-induced colitis in mice, increase colon length and reduce colon weight, actively alleviate the inflammatory edema caused by IBD, effectively alleviate the body's inflammatory response, and thus enhance the body's immunity.

[0090] Example 5: Verification of the ability of Lactobacillus rhamnosus QS001 to inhibit harmful bacteria.

[0091] The antibacterial effect of Lactobacillus rhamnosus QS001 screened in Example 1 was verified. The indicator bacteria specifically included: Escherichia coli (the designated culture medium was eosin methylene blue agar medium), Staphylococcus aureus (the designated culture medium was beef extract peptone medium), Salmonella enteritidis (the designated culture medium was three sugar iron medium), Candida albicans (the designated culture medium was YM medium), and Fusobacterium nucleatum polymorpha (the designated culture medium was Sabour agar medium). The above strains were all purchased from Testo Biotechnology Co., Ltd.

[0092] The indicator bacteria were inoculated from the cryopreserved tube into the specified culture medium and cultured at constant temperature for 18 h. The indicator bacteria were then diluted to 1×10 5CFU / mL of bacterial suspension, where the culture conditions for Escherichia coli, Staphylococcus aureus and Salmonella enteritidis are aerobic and 37°C, the culture conditions for Candida albicans are aerobic and 30°C, and the culture conditions for Fusobacterium nucleatum polymorphic subspecies are anaerobic and 37°C. The diluted indicator bacterial suspension is added to its designated culture medium at a volume fraction of 2%, and after being fully stirred, 20 mL is measured and poured into a sterilized plate, and the plate is gently shaken to evenly cover the culture medium. After solidification, the Oxford cup is placed at equal distances. The rhamnosus casei QS001 screened in Example 1 is dissolved in physiological saline to prepare a probiotic suspension with a concentration of 1 ml / L, and 150 μL of the probiotic suspension is slowly added to the Oxford cup with a pipette, and each treatment is repeated 3 times. Move the plate to a 4°C refrigerator for pre-diffusion for 4-10 hours, then take it out and place it in an incubator at a constant temperature (the temperature is the indicator bacteria culture temperature) and culture it upright until the inhibition zone is clear (about 12 hours). Use the inhibition zone method to determine the diameter of the inhibition zone. Measure each inhibition zone 3 times in different directions and record the average value.

[0093] Table 6 Diameters of inhibition zones of Lactobacillus rhamnosus QS001 against different microorganisms

[0094]

[0095] As shown in Table 6, the Lactobacillus rhamnosus QS001 screened in Example 1 can inhibit potentially harmful bacteria and has a broad-spectrum antibacterial ability; the diameters of the inhibition zones against Escherichia coli, Staphylococcus aureus, Salmonella enteritidis and Fusobacterium nucleatum polymorpha are all over 15 mm.

[0096] Experimental Example 6: Verification of the ability of Lactobacillus rhamnosus QS001 to promote the growth of beneficial bacteria.

[0097] The co-culture method was used to verify the growth-promoting effect of Lactobacillus rhamnosus QS001 on the common beneficial bacteria Bifidobacterium (using Bifidobacterium animalis lactis subsp. lactis, Bifidobacterium adolescentis and Bifidobacterium longum infantis subsp. infantis as examples). The experimental group was prepared by taking 5 mL of the probiotic suspension prepared in Example 5 with a concentration of 1 ml / L and adding it to 500 mL of Mupirocin lithium salt-modified MRS liquid culture medium (manufacturer: Beijing Luqiao Technology Co., Ltd.), and the control group was prepared by taking 500 mL of MRS liquid culture medium modified by Mupirocin lithium salt. Both the experimental group and the control group were inoculated with Bifidobacterium, so that the concentration of the Bifidobacterium strain after inoculation was 1×10 7 CFU / mL. After culturing at 37°C for 12 h under anaerobic conditions, the promoting effect of Lactobacillus rhamnosus QS001 on different bifidobacteria was evaluated by the number of viable bifidobacteria. The results are shown in Table 7 below.

[0098] Table 7 The promoting effect of Lactobacillus rhamnosus QS001 on different Bifidobacteria

[0099]

[0100] The promotion factor = the number of viable bacteria in the experimental group / the number of viable bacteria in the control group.

[0101] As shown in Table 7, the rhamnosus Lactobacillus QS001 screened in Example 1 can promote the growth of Bifidobacterium, and the promotion factors for Bifidobacterium animalis subsp. lactis, Bifidobacterium adolescentis, and Bifidobacterium longum subsp. infantis are 3.12 times, 2.21 times, and 1.35 times, respectively.

[0102] 2. Application Examples

[0103] Use of Lactobacillus rhamnosus QS001 in the preparation of intestinal probiotic preparations: The active ingredients of the intestinal probiotic preparations include the bacteria of Lactobacillus rhamnosus QS001 in the specific embodiment. The intestinal probiotic preparation can play a health role in any state of liquid, semi-solid and solid. Specifically, the intestinal probiotic preparation includes at least one of powder, granules, pills, capsules, tablets, pastes, liquid preparations, gels and sprays.

[0104] Example 1: The raw materials and weight proportions of the intestinal probiotic preparation are as follows: 5 weight parts of Lactobacillus rhamnosus QS001 powder, 32 weight parts of fruit powder, 18 weight parts of galacto-oligosaccharide, 4.5 weight parts of lactitol, and 30 weight parts of maltodextrin, wherein the number of viable probiotics in the intestinal probiotic preparation is greater than 1×10 10 CFU / g.

[0105] Example 2: The raw materials and weight proportions of the intestinal probiotic preparation are as follows: 4 weight parts of Lactobacillus rhamnosus QS001 powder, 40 weight parts of fruit powder, 15 weight parts of galacto-oligosaccharide, 5 weight parts of lactitol, and 25 weight parts of maltodextrin, wherein the number of viable bacteria in the intestinal probiotic preparation is greater than 1×10 10 CFU / g.

[0106] Example 3: The raw materials and weight proportions of the intestinal probiotic preparation are as follows: 6 weight parts of Lactobacillus rhamnosus QS001 powder, 25 weight parts of fruit powder, 20 weight parts of galacto-oligosaccharide, 4 weight parts of lactitol, and 35 weight parts of maltodextrin, wherein the number of viable bacteria in the intestinal probiotic preparation is greater than 1×10 10 CFU / g.

[0107] Example 4: The raw materials and weight proportions of the intestinal probiotic preparation are as follows: 4.5 weight parts of Lactobacillus rhamnosus QS001 powder, 25 weight parts of skimmed milk powder, 25 weight parts of fruit powder, 12 weight parts of galacto-oligosaccharide, 4.5 weight parts of xylitol, 8 weight parts of microcrystalline cellulose, and 0.8 weight parts of magnesium stearate, wherein the number of viable bacteria in the intestinal probiotic preparation is greater than 1×109 CFU / g.

[0108] Example 5: The raw materials and weight proportions of the intestinal probiotic preparation are as follows: 4 weight parts of Lactobacillus rhamnosus QS001 powder, 30 weight parts of skimmed milk powder, 20 weight parts of fruit powder, 15 weight parts of galacto-oligosaccharide, 4 weight parts of xylitol, 10 weight parts of microcrystalline cellulose, and 0.5 weight parts of magnesium stearate, wherein the number of viable bacteria in the intestinal probiotic preparation is greater than 1×10 9 CFU / g.

[0109] Example 6: The raw materials and weight proportions of the intestinal probiotic preparation are as follows: 5 weight parts of Lactobacillus rhamnosus QS001 powder, 20 weight parts of skimmed milk powder, 30 weight parts of fruit powder, 10 weight parts of galacto-oligosaccharide, 5 weight parts of xylitol, 5 weight parts of microcrystalline cellulose, and 1 weight part of magnesium stearate, wherein the number of viable bacteria in the intestinal probiotic preparation is greater than 1×10 8 CFU / g.

[0110] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A strain of Lactobacillus rhamnosus QS001, characterized in that: The rhamnosus casei bacteria QS001 was classified and named as rhamnosus casei bacteria ( Lacticaseibacillus rhamnosus ), the deposit number is CGMCC No.29275.

2. Use of the Lactobacillus rhamnosus QS001 according to claim 1 in the preparation of intestinal probiotic preparations.

3. The use of Lactobacillus rhamnosus QS001 in the preparation of intestinal probiotic preparations according to claim 2, characterized in that: The active ingredient of the intestinal probiotic preparation includes the bacteria of Lactobacillus rhamnosus QS001 described in claim 1.

4. The use of Lactobacillus rhamnosus QS001 in the preparation of intestinal probiotic preparations according to claim 3, characterized in that: The effective bacterial count of Lactobacillus rhamnosus QS001 in the intestinal probiotic preparation is not less than 1×10 8 CFU / g.

5. The use of Lactobacillus rhamnosus QS001 in the preparation of intestinal probiotic preparations according to claim 3, characterized in that: The intestinal probiotic preparation comprises at least one of powder, granule, pill, capsule, tablet, paste, liquid preparation, gel and spray.

6. The use of Lactobacillus rhamnosus QS001 in the preparation of intestinal probiotic preparations according to claim 5, characterized in that The intestinal probiotic preparation is composed of the following raw materials and their weight parts: 4-6 weight parts of rhamnosus lactobacillus powder, 20-40 weight parts of fruit powder, 10-20 weight parts of galacto-oligosaccharide, 4-5 weight parts of sugar alcohol, and 25-35 weight parts of maltodextrin.

7. The use of Lactobacillus rhamnosus QS001 in the preparation of intestinal probiotic preparations according to claim 5, characterized in that The intestinal probiotic preparation is composed of the following raw materials and their weight parts: 4-6 weight parts of rhamnosus lactobacillus powder, 20-40 weight parts of fruit powder, 10-20 weight parts of galacto-oligosaccharide, 4-5 weight parts of sugar alcohol, 20-30 weight parts of skimmed milk powder, 5-10 weight parts of microcrystalline cellulose, and 0.5-1 weight part of magnesium stearate.

8. Use of the Lactobacillus rhamnosus QS001 according to claim 1 in the preparation of a drug for alleviating and / or treating inflammatory bowel disease.

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