Lactobacillus rhamnosus capable of preventing or treating inflammatory bowel disease and promoting digestion and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to lactobacillus rhamnosus capable of preventing or treating inflammatory bowel diseases and promoting digestion and application of the lactobacillus rhamnosus, and belongs to the technical field of biological medicine. The invention provides a strain of lactobacillus rhamnosus R7970, wherein the preservation number of the lactobacillus rhamnosus R7970 is CGMCC (China General Microbiological Culture Collection Center) 22244, and the preservation number of the lactobacillus rhamnosus R7970 is CGMCC 22244. The lactobacillus rhamnosus R7970 can be used for effectively preventing inflammatory enteritis (IBD) and has a prevention effect on intestinal inflammation, and meanwhile, the lactobacillus rhamnosus R7970 can be used for improving the nutrition digestion and absorption functions of intestinal tracts. Therefore, the lactobacillus rhamnosus R7970 can be applied to prevention of IBD, can be used for preparing probiotic preparations, common foods, health foods and biological pharmacy for improving intestinal health, is convenient for industrial production, and has great popularization value.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus rhamnosus capable of preventing or treating inflammatory bowel disease and promoting digestion, and a use thereof. Background Art

[0002] Inflammatory bowel disease (IBD) includes Crohn's disease (CD) and ulcerative colitis (UC). The incidence of UC is relatively high, often accompanied by abdominal pain, diarrhea, and mucus, pus and blood in the stool. At the same time, there are a variety of extraintestinal manifestations, which can affect the eyes, joints, liver, gallbladder, and skin. With continuous in-depth research, it is found that microecological disorders in the intestine play an important role in the occurrence and development of inflammatory bowel disease. It is a chronic recurrent gastrointestinal disease with more young patients and seriously affects their quality of life. Since the 21st century, IBD has spread from Western countries to emerging industrialized countries, and the incidence of IBD has been increasing all over the world. In Asia, the incidence of IBD varies from country to country or region, with an average annual incidence of 1.50 cases per 100,000 people.

[0003] In the past 20 years, the number of IBD cases in my country has increased rapidly, and the disease process is characterized by progressiveness and destructiveness. The treatment of IBD focuses on inducing remission and preventing disease relapse. Depending on the severity of the disease, it starts with aminosalicylates and antibiotics, and gradually develops into corticosteroids, immunomodulators, and biological agents. These therapies are successful and effective, but may be accompanied by serious side effects, which increase the risk of disease relapse and the medical burden of managing IBD. With the increasingly in-depth study of intestinal microorganisms in the pathogenesis of IBD, the therapeutic potential of using probiotics to restore the balance of intestinal flora and improve intestinal function has gradually been explored.

[0004] There is currently no specific treatment for IBD. Intestinal microbial malnutrition plays an important role in human pathology, and microorganisms are a key factor in maintaining the dynamic balance of the immune system. As an auxiliary treatment, probiotics can effectively restore intestinal barrier function, regulate immunity, and improve the intestinal microenvironment. Existing clinical trials have confirmed that probiotics are effective in the treatment of inflammatory bowel disease with a low incidence of adverse reactions. This also provides a new treatment idea for IBD patients who have not achieved good results under conventional treatment or have difficulty tolerating adverse reactions.

[0005] The intestine is an important place for food digestion and absorption. Its digestion and absorption function directly determines the efficiency of nutrient utilization, thus affecting the growth of the body. The intestinal mucosa also serves as an important barrier for the body to resist the invasion of microorganisms and harmful substances. Probiotics, as a type of microecological preparation, can form an intestinal barrier when taken in sufficient amounts, competitively inhibit harmful bacteria, enrich the number of microbial populations, improve the body's digestion rate of nutrients, and accelerate the growth and development of animals. Summary of the invention

[0006] Therefore, the technical problem to be solved by the present invention is to provide a Lactobacillus rhamnosus that can prevent or treat inflammatory bowel disease and promote digestion and its use.

[0007] To this end, the present invention provides the following technical solutions:

[0008] The embodiment disclosed in the present invention relates to a Lactobacillus rhamnosus R7970, which is deposited in the General Microbiological Center of China National Committee for the Preservation of Microorganisms with a deposit number of CGMCC No.22244.

[0009] The embodiment disclosed in the present invention relates to a microbial agent containing the bacteria, culture, fermentation product or at least one metabolite of the Lactobacillus rhamnosus R7970.

[0010] In some embodiments, the effective bacterial cell count of Lactobacillus rhamnosus R7970 is not less than 2×10 6 CFU / mL or 2×10 6 CFU / g.

[0011] In some embodiments, the metabolites include at least one of organic acids, amino acids, fatty acids, and tryptophan metabolites.

[0012] In some embodiments, the organic acid comprises at least one of tartaric acid, formic acid, lactic acid, acetic acid, citric acid, and succinic acid.

[0013] In some embodiments, the amino acid includes at least one of threonine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine, and arginine.

[0014] In some embodiments, the fatty acid includes at least one of butyric acid, hexanoic acid, octanoic acid, decanoic acid, undecanoic acid, dodecanonic acid, tridecanonic acid, tetradecanonic acid, cis-9-tetradecanoic acid, pentadecanonic acid, hexadecanoic acid, cis-9-hexadecanoic acid, heptadecanonic acid, octadecanoic acid, cis-9-octadecanoic acid, cis, cis-9,12-octadecadienoic acid, cis, cis, cis-9,12,15-octadecatrienoic acid, cis-5,8,11,14,17-eicosapentaenoic acid, tricosanoic acid, tetracosanoic acid, and cis-4,7,10,13,16,19-docosahexaenoic acid.

[0015] In some embodiments, the tryptophan metabolite includes at least one of o-aminophenol, o-aminobenzoic acid, 3-indoleethanol, L-tryptophan, indolepropionic acid, DL-indole-3-lactic acid, indole-3-carboxaldehyde, indole-3-acetamide, β-indole-3-acetic acid, 2-ketoadipic acid, N-formylkynurenine, L-kynurenine, picolinic acid, nicotinic acid, quinolinic acid, kynurenic acid, and xanthine acid.

[0016] The embodiment disclosed in the present invention relates to a method for preparing the microbial agent, comprising: expanding and culturing the Lactobacillus rhamnosus R7970.

[0017] The embodiments disclosed in the present invention relate to a use of the Lactobacillus rhamnosus R7970 or the microbial agent in any of the following:

[0018] (1) Use in the preparation of products for preventing, alleviating and / or treating inflammatory bowel disease (IBD);

[0019] (2) Use in the preparation of products for inhibiting weight loss, diarrhea and bloody stools;

[0020] (3) Use in the preparation of products that reduce blood inflammatory responses and / or reduce the concentration of pro-inflammatory cytokines;

[0021] (4) Use in the preparation of products that improve the nutrient digestion and / or nutrient absorption function of the intestine;

[0022] (5) Use in the preparation of products that promote bone health;

[0023] (6) Use in the preparation of products for increasing bone weight, bone hardness and / or bone elastic modulus;

[0024] (7) Use in the preparation of products that maintain or increase the relative abundance of beneficial intestinal bacteria.

[0025] In some embodiments, the nutrient absorption comprises calcium absorption.

[0026] In some embodiments, the product includes a functional food, a medicine, a health product, a functional care product or a functional auxiliary material.

[0027] In some embodiments, the product includes at least one of powders, granules, pills, capsules, tablets, pastes, liquid preparations, gels, sprays, and solid beverages.

[0028] The technical solution of the present invention has the following advantages:

[0029] 1. The present invention provides a strain of Lactobacillus rhamnosus R7970, the deposit number of which is CGMCC No. 22244; experiments have shown that the Lactobacillus rhamnosus R7970 can effectively prevent DSS-induced IBD (inflammatory bowel disease) symptoms, and the experiment verified that the R7970 group of mice had lower DAI scores, longer colon length, and higher body weight than the DSS group, indicating that R7970 can reduce the severity of colitis in mice. At the same time, it was verified that the levels of proinflammatory cytokines IL-1β, IL-6, and TNF-α, as well as anti-inflammatory factors IL-10 and IL-13, the levels of the three proinflammatory cytokines in the R7970 group were significantly lower than those in the DSS group (IL-1β, IL-6, and TNF-α), indicating that R7970 can reduce the levels of proinflammatory factors. The levels of IL-13 and IL-10 in the DSS group were lower than those in the R7970 group and the LGG group, indicating that R7970 can increase the levels of anti-inflammatory factors, and the R7970 group is superior to the LGG group.

[0030] Therefore, the Lactobacillus rhamnosus R7970 can be used in the prevention and / or treatment of IBD, and can be used to prepare probiotic preparations, common foods, health foods and biopharmaceuticals for improving intestinal health, is convenient for industrial production, and has great promotion value.

[0031] In addition, the present invention also detects the content of metabolites of Lactobacillus rhamnosus R7970, including organic acids, amino acids, fatty acids and tryptophan metabolites. Metabolism results show that the content of citric acid and succinic acid in R7970 is significantly higher than that of strain LGG, and R7970 has stronger growth-promoting and digestive functions in the body than LGG.

[0032] At the same time, free amino acids can promote muscle growth, improve immune system function, promote brain function, promote metabolism and other functions. The results of amino acid determination showed that the content of 12 free amino acids, including threonine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and arginine, in the R7970 strain was higher than that in the LGG strain, and had higher prebiotic value.

[0033] At the same time, the content of three short-chain fatty acids, butyric acid, caproic acid, and caprylic acid, in R7970 are all higher than that in LGG, and the content of capric acid is similar between the two. Short-chain fatty acids can help keep the intestines healthy and sealed, thereby preventing leaky gut syndrome and various problems related to leaky gut, such as IBS symptoms. The fatty acid metabolism results show that R7970 is more effective than LGG in treating IBD.

[0034] At the same time, a total of 19 tryptophan metabolites were detected in R7970 and LGG, of which 13 substances were found in R7970 strains at higher levels than in LGG, and N-acetyl 5-hydroxytryptamine and indole acrylic acid were not detected in LGG. As an important neurotransmitter, 5-hydroxytryptamine activates 5-hydroxytryptamine receptors, thereby exerting a wide range of effects on host physiology and affecting intestinal motility, secretion, vasodilation and nutrient absorption. It is believed to play an important role in maintaining intestinal homeostasis. Indole acrylic acid, a tryptophan indole metabolite, promotes intestinal epithelial barrier function and exhibits anti-inflammatory effects in IBD patients with intestinal flora imbalance. In summary, the results of tryptophan metabolite determination show that R7970 has more advantages than LGG in regulating intestinal health.

[0035] Furthermore, the Lactobacillus rhamnosus R7970 is a beneficial intestinal bacterium and is included in the list of bacteria that can be used in food. Therefore, the Lactobacillus rhamnosus R7970 and products whose active ingredients are the Lactobacillus rhamnosus R7970 have the advantage of good safety.

[0036] In addition, the Lactobacillus rhamnosus R7970 can be obtained by only using a culture medium for microbial culture. Therefore, the Lactobacillus rhamnosus R7970 and the product whose active ingredient is the Lactobacillus rhamnosus R7970 have the advantage of low cost.

[0037] 2. The present invention provides a strain of Lactobacillus rhamnosus R7970. Experiments have shown that regular intake of R7970 by rats can promote weight gain of rats within a normal range, significantly increase the activity of digestive enzymes secreted in the stomach, small intestine and pancreas, maintain the relative abundance of some beneficial bacteria in the intestine, enhance calcium absorption, enhance bone resistance to external forces, increase bone hardness, and promote bone health. Lactobacillus rhamnosus R7970 can improve the nutrient digestion and absorption function of the intestine.

[0038] Biomaterial Deposit

[0039] A strain of Lactobacillus rhamnosus R7970, taxonomically named Lactobacillus rhamnosus, was deposited in the General Microbiology Center of China Culture Collection Administration on April 27, 2021, with the deposit number CGMCC No.22244, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0041] Figure 1 This is a graph of DAI scores of different groups in Experimental Example 1 of the present invention;

[0042] Figure 2 Figure 1 shows the colon length of mice in different groups in Experimental Example 1 of the present invention.

[0043] Figure 3 This is a graph showing the weight changes of mice in different groups in Experimental Example 1 of the present invention;

[0044] Figure 4 This is a graph of IL-6 concentrations in different groups in Experimental Example 1 of the present invention;

[0045] Figure 5 This is a graph of IL-1β concentrations in different groups in Experimental Example 1 of the present invention;

[0046] Figure 6 This is a graph of TNF-α concentrations in different groups in Experimental Example 1 of the present invention;

[0047] Figure 7 This is a graph of IL-10 concentrations in different groups in Experimental Example 1 of the present invention;

[0048] Figure 8 This is a graph of IL-13 concentrations in different groups in Experimental Example 1 of the present invention;

[0049] Fig. 9 This is a graph showing changes in rat body weight in Experimental Example 2 of the present invention;

[0050] Fig.10 This is a graph of pepsin activity in Experimental Example 2 of the present invention;

[0051] Fig.11 This is a diagram of pancreatic amylase activity in Experimental Example 2 of the present invention;

[0052] Fig.12 This is a graph of pancreatic lipase activity in Experimental Example 2 of the present invention;

[0053] Fig.13 This is a graph of lactase activity in Experimental Example 2 of the present invention;

[0054] Fig.14 This is a graph of sucrase activity in Experimental Example 2 of the present invention;

[0055] Fig.15 This is the aminopeptidase activity diagram in Experimental Example 2 of the present invention;

[0056] Fig.16 This is a graph of gastrin content in Experimental Example 2 of the present invention;

[0057] Fig.17 This is a graph of serum calcium ion content in Experimental Example 2 of the present invention;

[0058] Fig.18 This is a relative abundance diagram of different bacterial communities in Experimental Example 2 of the present invention;

[0059] Fig.19 This is a graph of rat bone weight in Experimental Example 2 of the present invention;

[0060] Fig. 20 This is a graph of rat bone hardness in Experimental Example 2 of the present invention;

[0061] Fig.21 This is the elastic modulus diagram in Experimental Example 2 of the present invention. DETAILED DESCRIPTION

[0062] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0063] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0064] Materials and Methods

[0065] The experimental strain Lactobacillus rhamnosus R7970 has been deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number of CGMCC No. 22244. Lactobacillus rhamnosus LGG was purchased from Chr. Hansen.

[0066] Table 1 Main instruments and equipment

[0067] name model factory HPLC UV detector U3000 Thermo Fisher Scientific Gas Chromatography-Mass Spectrometry Trace1310ISQ Thermo Fisher Scientific High Speed ​​Amino Acid Analyzer L-8900 Hitachi LC-MS / MS QTRAP 6500+ SCIEX Centrifuge 5424R Eppendorf Electronic balance AS 60 / 220.R2 RADWAG Ball Mill MM400 Retsch Multi-tube vortex mixer MIX-200 Shanghai Jingxin Ultrasonic cleaning machine KQ5200E Kunshan Shumei

[0068] The preparation method of the bacterial suspension or bacterial powder of Lactobacillus rhamnosus (R7970 or LGG) involved in the following embodiments comprises the following steps:

[0069] Preparation method of bacterial suspension: streak the bacterial liquid on MRS solid culture medium (commercially available), and culture it anaerobically at 37°C for 24 hours to obtain a single colony; pick a single colony and inoculate it in MRS liquid culture medium (commercially available), and culture it anaerobically at 37°C for 24 hours to activate it, and obtain an activated liquid; inoculate the activated liquid into MRS liquid culture medium at an inoculum amount of 2% (v / v), and culture it anaerobically at 37°C for 24 hours to obtain a bacterial liquid; centrifuge the bacterial liquid at 4°C and 8000×g for 10 minutes to obtain bacterial cells and supernatant; wash the bacterial cells with PBS buffer three times and resuspend them in PBS buffer until the bacterial concentration is ≥1×10 8 CFU / mL, and obtain Lactobacillus rhamnosus suspension.

[0070] Preparation method of bacterial powder: adding lyophilization protective agent glycerol to a suspension of Lactobacillus rhamnosus to a concentration of 25% by volume to obtain a mixed solution; vacuum freeze-drying the mixed solution to obtain Lactobacillus rhamnosus bacterial powder (viable cell count ≥ 1×10 11 CFU / g).

[0071] Example 1 Metabolomics Analysis of Lactobacillus rhamnosus R7970

[0072] 1. Determination of organic acids

[0073] Organic acids were determined by high performance liquid chromatography, and the instrument used was a high performance liquid chromatography ultraviolet detector. About 2 ml of bacterial suspension was taken as a sample, and the volume was fixed to 10 mL with deionized water solvent, and the sample was filtered with a 0.45 μm filter membrane. Ultra-high performance liquid chromatography (UPLC) was used for data acquisition, and the chromatographic conditions were as follows: chromatographic column: syncronis C18, Dim 250 mm × 4.6 mm, 5 μm; injection volume: 20 μL column temperature: 30 ° C; flow rate: 0.5 mL / min; mobile phase: acetonitrile: water (0.05 mol / L potassium dihydrogen phosphate adjusted with phosphoric acid to pH = 2.68) = 0.5: 99.5 (volume ratio), and the absorption peak was detected under ultraviolet wavelength of 210 nm.

[0074] The results of organic acid determination are shown in Table 2. Six organic acids were detected in R7970 and LGG, namely tartaric acid, formic acid, lactic acid, acetic acid, citric acid, and succinic acid. The contents of the above substances in the R7970 strain were higher than those in the LGG strain. Among them, the contents of citric acid and succinic acid in R7970 were significantly higher than those in the LGG strain. Citric acid has the effect of promoting the growth of the body and improving digestive function, mainly by inhibiting the growth of harmful bacteria, increasing the contraction strength and frequency of the smooth muscle of the digestive tract, promoting the activity of digestive enzymes, improving the morphology of the intestinal mucosa, and increasing the digestion and absorption area of ​​nutrients. Succinic acid, also known as succinic acid, has multiple physiological functions such as antibacterial, sedative, anticonvulsant and hypothermic. Citric acid and succinic acid are intermediate metabolites of the tricarboxylic acid cycle. The tricarboxylic acid cycle is a common metabolic pathway for the oxidation of carbohydrates, fats and proteins in the body, and is a common pathway for the oxidation function of the three main nutrients in the body. It shows that R7970 has stronger growth-promoting and digestive functions and has physiological functions such as antibacterial than LGG in the body.

[0075] Table 2 Organic acid content of R7970 and LGG (mg / kg)

[0076] Serial number name R7970 LGG P-value 1 oxalic acid Not detected (<2) Not detected (<2) - 2 tartaric acid 560.37 502.97 0.0045 3 Formic acid 1522.19 1485.51 0.2852 4 Malic Acid Not detected (<2) Not detected (<2) - 5 lactic acid 14796.53 14782.89 0.5392 6 Acetic acid 788.91 244.01 0.0001 7 Maleic acid Not detected (<2) Not detected (<2) - 8 Citric Acid 638.97 165.02 0.0002 9 Succinic acid 481.74 185.9 0.0001 10 Propionic acid Not detected (<2) Not detected (<2) -

[0077] 2. Determination of amino acids

[0078] The amino acid determination was carried out using a high-speed amino acid analyzer. A certain mass of bacterial suspension sample was accurately weighed into a 15ml centrifuge tube, 0.02mol / L hydrochloric acid 5mL was added, vortexed for 5 minutes, ultrasonically extracted for 5 minutes, kept away from light for 2 hours, centrifuged at 4000g for 10 minutes, 1mL of supernatant was accurately taken, 1mL of 6%-8% sulfosalicylic acid was added, vortexed for 1 minute, kept away from light for 1 hour, centrifuged at 15000g for 15 minutes, and the supernatant was filtered with a 0.22μm filter membrane for determination. The chromatographic conditions were as follows: chromatographic column: 2622# pH ion exchange column; column temperature: 57℃; post-column derivatization, derivatization reaction temperature 135℃; flow rate: derivatization reagent 0.35mL / min; injection volume: 20uL.

[0079] Free amino acids can promote muscle growth, improve immune system function, promote brain function, promote metabolism and many other functions. The results of amino acid determination are shown in the following table. The content of 12 free amino acids such as threonine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and arginine in R7970 strain is higher than that in LGG strain, and has higher prebiotic value.

[0080] Table 3 Amino acid content of R7970 and LGG (mg / 100g)

[0081] Serial number name R7970 LGG P-value 1 ASP free aspartic acid 1.503 1.649 0.4382 2 THR Free Threonine 0.388 0.191 0.0028 3 SER Free Serine 0.143 0.378 0.0018 4 GLU Free Glutamate 11.648 7.682 0.0184 5 GLY Free Glycine 0.489 0.166 0.0033 6 ALA Free Alanine 2.289 1.261 0.0149 7 CYS Free Cystine 1.604 1.459 0.0324 8 VAL Free valine 1.997 1.311 0.1245 9 MET Free Methionine 1.558 0.847 0.0013 10 ILE Free isoleucine 0.398 0.132 0.0024 11 LEU Free Leucine 1.053 0.249 0.0053 12 TYR Free Tyrosine 0.772 0.405 0.0043 13 PHE Free Phenylalanine 0.705 0.329 0.0056 14 LYS Free Lysine 0.177 0.328 0.0001 15 HIS Free Histidine 0.375 0.380 0.0005 16 ARG Free Arginine 0.889 0.524 0.0014 17 PRO Free Proline 2.657 4.103 0.0045

[0082] 3. Determination of fatty acids

[0083] Fatty acids were determined by gas chromatography-mass spectrometry: Weigh about 2 mL of uniform sample bacterial suspension and transfer it to a 250 mL flat-bottom flask, accurately add 2.0 mL of internal standard solution, add about 100 mg of pyrogallic acid, add a few grains of zeolite, then add 2 mL of 95% ethanol and 4 mL of water, and mix well.

[0084] Add 5 mL of ammonia water and mix well. Place the flask in a 70℃~80℃ water bath for hydrolysis for 20 minutes. Shake the flask every 5 minutes to mix the particles adhering to the wall of the flask into the solution. After the hydrolysis is completed, take out the flask and cool it to room temperature. Add 10 mL of 95% ethanol to the hydrolyzed sample and mix well. Transfer the hydrolyzate in the flask to a separatory funnel, rinse the flask and stopper with 50 mL of ether and petroleum ether mixture, add the rinse solution to the separatory funnel and cover it. Shake for 5 minutes and let it stand for 10 minutes. Collect the ether layer extract into a 250 mL flask. Repeat the above steps to extract the hydrolyzate 3 times, and finally rinse the separatory funnel with ether and petroleum ether mixture and collect it in a 250 mL flask. Concentrate to dryness on a rotary evaporator, add 8 mL of 2% sodium hydroxide methanol solution to the fat extract, connect a reflux condenser, and reflux on a 80℃±1℃ water bath until the oil droplets disappear. Add 7mL of 15% boron trifluoride methanol solution from the top of the reflux condenser and continue to reflux in a water bath at 80℃±1℃ for 2min. Rinse the reflux condenser with a small amount of water. Stop heating, remove the flask from the water bath, and quickly cool to room temperature. Accurately add 10mL~30mL of n-heptane, shake for 2min, then add saturated sodium chloride aqueous solution, and let stand to separate. Pipette about 5mL of the upper n-heptane extraction solution into a 25mL test tube, add about 3g~5g of anhydrous sodium sulfate, shake for 1min, let stand for 5min, and pipette the upper solution into the injection bottle for determination. Chromatographic conditions: chromatographic column: TG-5MS (30m×0.25mm×0.25μm); temperature program: 80℃ for 1min, then 10℃ / min to 200℃, then 5℃ / min to 250℃, and finally 270℃ at 2℃ / min, and then 3min; injection port temperature: 290℃; carrier gas flow rate: 1.2ml / min; splitless injection, valve opening time: 1min. Mass spectrometry conditions: ion source temperature: 280℃; transfer line temperature: 280℃; solvent delay time: 5.00min; scanning range: 30-400amu; ion source: EI source 70eV.

[0085] The fatty acid content is shown in the following table. A total of 21 fatty acids were detected. Generally speaking, short-chain fatty acids have the functions of promoting sodium absorption, regulating gastrointestinal function, and repairing damaged intestinal mucosa. They are indirect nutrients produced by intestinal microorganisms. Short-chain fatty acids are usually produced by the fermentation of beneficial bacteria in the intestines from foods such as dietary fiber, indigestible sugars, carbohydrates, and resistant starch. They are the main energy source for cells in the inner wall of the colon, can maintain normal intestinal function, and have a positive effect on human health. The content of three short-chain fatty acids, butyric acid, caproic acid, and caprylic acid in R7970 is higher than that in LGG, and the content of capric acid is similar in the two. Short-chain fatty acids can help keep the intestines healthy and sealed, thereby preventing leaky gut syndrome and various problems related to leaky gut, such as IBS symptoms. The results of fatty acid metabolism show that R7970 is more effective than LGG in the treatment of IBD diseases.

[0086] Table 4 Fatty acid content of R7970 and LGG (mg / kg)

[0087]

[0088]

[0089] 4. Determination of tryptophan metabolites

[0090] Pipette 50 μL of bacterial suspension into a centrifuge tube, add 250 μL of methanol solution (containing 10 μL of internal standard working solution with a concentration of 250 ng / mL) into the centrifuge tube, vortex for 3 min to mix, and place in a -20 °C refrigerator for 30 min;

[0091] Centrifuge at 12000 r / min for 10 min at 4°C, and transfer 150 μL of supernatant to a new centrifuge tube;

[0092] After centrifugation again, 100 μL of the supernatant was transferred to an injection bottle and temporarily stored in a -20°C refrigerator for LC-MS / MS analysis. The chromatographic conditions were as follows: chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm×2.1 mm id); mobile phase: phase A, ultrapure water (containing 0.1% formic acid); phase B, acetonitrile (containing 0.1% formic acid); flow rate 0.35 mL / min; column temperature 40°C; injection volume 5 μL; mobile phase gradient: 0 min A:B is 90:10 (V / V), 1 min A:B is 90:10 (V / V), 8 min A:B is 5:95 (V / V), 9.5 min A:B is 5:95 (V / V), 9.6 minA:B is 90:10 (V / V), 12 min A:B is 90:10 (V / V). The mass spectrometry conditions mainly include: electrospray ionization (ESI) temperature 550°C, mass spectrometry voltage 5500V in positive ion mode, mass spectrometry voltage -4500V in negative ion mode, and curtain gas (CUR) 35psi. In Q-Trap 6500+, each ion pair is scanned and detected according to the optimized declustering potential (DP) and collision energy (CE).

[0093] Tryptophan, as a communication medium between intestinal flora and the host, generates many tryptophan metabolites under the action of a series of endogenous enzymes or microbial metabolism. Its metabolites regulate various physiological and pathological processes to varying degrees, including growth regulation, metabolism, emotions and immune responses. Tryptophan deficiency directly leads to a reduction in metabolites, which is closely related to intestinal imbalance and even induces IBD. The tryptophan metabolites of R7970 and LGG are shown in the table below. A total of 19 metabolites were detected, of which 13 substances in the R7970 strain were higher than those in LGG, and the contents of N-acetyl 5-hydroxytryptamine and indole acrylic acid were not detected in LGG. As an important neurotransmitter, 5-hydroxytryptamine activates 5-hydroxytryptamine receptors, thereby exerting a wide range of effects on host physiology, and affects intestinal motility, secretion, vasodilation and nutrient absorption. It is believed to play an important role in maintaining intestinal homeostasis. Indole acrylic acid, a tryptophan indole metabolite, promotes intestinal epithelial barrier function and exhibits anti-inflammatory effects in IBD patients with intestinal flora imbalance. In summary, the results of tryptophan metabolite determination showed that R7970 had more advantages than LGG in regulating intestinal health.

[0094] Table 4 Content of tryptophan metabolites in R7970 and LGG

[0095]

[0096]

[0097] Experimental Example 1 Effect of Lactobacillus rhamnosus R7970 on DSS-induced inflammatory bowel disease in mice

[0098] This study was approved by the Ethics Committee of Inner Mongolia Agricultural University on August 12, 2022 (NND2022099). Twenty-four male C57BL / 6J mice (weight 20-23 g, 6 weeks old) were obtained from Beijing Huafukang Biotechnology Co., Ltd. (HFK, Beijing, China) with free access to food and water and housed under specific pathogen-free conditions with a 12-h light-dark cycle, a temperature of 22 ± 2 °C, and a humidity of 45 ± 10%. All animal experiments were performed in accordance with the guidelines of the Laboratory Animal Care and Ethics Committee of Inner Mongolia Agricultural University.

[0099] The specific steps are as follows:

[0100] 1. Experimental design

[0101] After 1 week of adaptive training, the mice were randomly divided into four groups (6 mice in each group): healthy control group (NC group), DSS group, R7970 group and LGG group. From day -7 to day 0, the mice in the NC group and DSS group drank distilled water freely, and the mice in the R7970 group were intragastrically gavaged with Lactobacillus rhamnosus R7970 (Lactobacillus rhamnosus R7970 powder was resuspended in 0.9% saline, 4×10 9 CFU / 0.2mL / mouse / day), and the LGG group was intragastrically gavaged with Lactobacillus rhamnosus LGG (Lactobacillus rhamnosus LGG powder was resuspended in 0.9% saline, 4×10 9 CFU / 0.2mL / mouse / day). During the whole experiment, the NC group had free access to sterile water; the other three groups were given 2.5% dextran sulfate sodium salt (DSS) aqueous solution every day from day 0 to 5 (DSS drinking water was replaced every 3, 13, and 23 days), and sterile water was replaced every 6-10 days. This process was considered as one cycle, and a total of 3 cycles were performed. During the whole experiment, the weight, health status (diarrhea, bloody stools, activity) and death of the mice were observed and recorded every day. Fresh feces were collected and stored at -80°C, and the mice were killed after being injected with volatile isoflurane. The contents of the colon were removed, and the length and weight of the colon were measured. Blood was collected from the mice, and serum was obtained by centrifugation (3000×g rpm, 10 minutes, 4°C). The serum samples were stored at -80°C until use.

[0102] 2. Disease Activity Index (DAI)

[0103] DAI is used to assess the severity of colitis and is the sum of weight loss, diarrhea, and blood in the stool.

[0104] Data results statistics:

[0105] Each index was evaluated on a 0-4 scale, and the final score was expressed as DAI = stool consistency score + weight change score + stool bleeding score. The specific scoring criteria are as follows: stool consistency scores range from 0 to 4, which are normal, slightly soft stool, loose stool, and watery stool, respectively; stool bleeding scores range from 0 to 4, which are normal, brown, red, and bloody stool, respectively; weight change scores are divided into 0 (<1%), 1 (2-5%), 2 (6-10%), 3 (11-15%), and 4 (≥16%) according to the percentage of weight loss.

[0106] DAI results are as follows Figure 1 As shown (the column results corresponding to each time point in the figure correspond to the NC group, DSS group, R7970 group and LGG group from left to right), on the 10th day, the DAI scores of mice in the DSS group, R7970 group and LGG group increased after the mice were gavaged with DSS aqueous solution. On the 20th and 30th days, the DAI scores of the R7970 group and LGG group decreased. In the column results at each time point, different letters between a, b, c, and d indicate statistical differences (P<0.05)

[0107] The length of mouse colon Figure 2 As shown, under the action of DSS, the colon lengths of mice in the DSS group, R7970 group, and LGG group were significantly shorter than that in the NC group. However, due to the previous intervention of R7970 and LGG, the colon lengths of the R7970 group and the LGG group were significantly longer than those of the DSS group, and there were statistical differences among the groups (P<0.05).

[0108] The weight changes of mice Figure 3 As shown in the figure (the column results corresponding to each time point in the figure correspond to the NC group, DSS group, R7970 group and LGG group from left to right), during the modeling period, the weight of mice in the NC group increased significantly (P < 0.05), while DSS could significantly inhibit the increase in the weight of mice (P < 0.05). On the 10th and 20th days, the weight of mice in the NC group was higher than that of the other three groups; on the 30th day, the weight of mice in the NC group was higher than that of the other three groups (P < 0.05), and the weight of mice in the R7970 and LGG groups was higher than that of the DSS group (P < 0.05). Different letters in a, b, c, and d in the figure indicate statistical differences (P < 0.05).

[0109] In summary, it was shown that R7970 and LGG could reduce the severity of colitis in mice. Meanwhile, indicators such as DAI score, colon length and body weight of mice showed that R7970 was better than LGG in reducing colitis.

[0110] 3. Quantitative analysis of inflammatory cytokines in serum

[0111] ProcartaPlexTM (EPX360-26092-901, Invitrogen, Thermofisher, Watham, MA, USA) was used to quantify the levels of multiple cytokines in serum, including interleukin (IL)-1β, IL-6, IL-10, IL-13, and tumor necrosis factor (TNF)-α. The experiments were performed in duplicate according to the manufacturer's instructions. Samples were incubated with appropriately diluted antibody-coupled beads for 18 h in a 96-well plate. The Bio-Plex 200 system detected the signals, and the Bio-Plex Manager software was used to analyze the data. Interleukin IL-6 ( Figure 4 )、(IL)-1β( Figure 5 )、IL-10( Figure 7 )、IL-13( Figure 8 ) and tumor necrosis factor (TNF)-α ( Figure 6 ). In the figure, different letters in a, b, c, and d indicate statistical differences (P<0.05).

[0112] The levels of proinflammatory cytokines IL-1β, IL-6 and TNF-α, as well as anti-inflammatory factors IL-10 and IL-13 in serum were measured. The levels of the three proinflammatory cytokines in the DSS group were significantly higher than those in the NC group, but the levels of the R7970 group and the LGG group were significantly lower than those in the DSS group (IL-1β, IL-6 and TNF-α), and the R7970 group was significantly lower than the LGG group, indicating that R7970 and LGG can reduce the levels of proinflammatory factors, and R7970 is more effective than LGG in reducing proinflammatory factors. The levels of IL-13 and IL-10 in the NC group were higher than those in the other three groups, while the levels of IL-13 and IL-10 in the DSS group were lower than those in the R7970 group and the LGG group, indicating that R7970 and LGG can increase the levels of anti-inflammatory factors, and the R7970 group is superior to the LGG group. In summary, the R7970 group can significantly reduce blood inflammatory response, and is significantly superior to the LGG group.

[0113] Experimental Example 2 Experiment on the Effect of Lactobacillus rhamnosus R7970 on Improving the Nutrition Digestion and Absorption Function of Intestinal Tract

[0114] The main test materials are as follows:

[0115] The model animals used were 6-week-old SD male rats. Among them, the weight range of SD rats was 210-230g. Experimental animals, maintenance feed, and rat bedding were purchased from Sbefor (Beijing) Biotechnology Co., Ltd. [SCXK (Beijing) 2019-0014]. During the experiment, all animals were kept in an SPF-level barrier environment with 12h / 12h day and night light and dark alternations, the ambient temperature was set at (22±2)℃, the ambient humidity was set at (50±10)%, and the model animals were free to eat and drink water. This experiment was approved by the Experimental Animal Welfare and Ethics Committee of Inner Mongolia Agricultural University (NND2023096).

[0116] Preparation of activated carbon solution: weigh 100g of gum arabic in a beaker, add 800mL of water, mix, boil until the solution is transparent, add 50g of activated carbon (activated carbon powder purchased from Sigma, Germany), boil three times to make activated carbon solution. After the solution is cooled, transfer, dilute to 1000mL with water, store at 4°C, and shake well before use.

[0117] Preparation of ordinary yogurt: The basic starter culture of yogurt (purchased from Beijing Ketuo Hengtong Biotechnology Co., Ltd., product number PYS-010) was inoculated into milk at an inoculum amount of 0.03‰ of the total mass, first fermented at 42°C to a pH of 4.6, and then cooled at 4°C for 5 min to break the emulsion, and ordinary yogurt (viable cell count ≥ 5×10 8 CFU / mL).

[0118] 1. Experimental Methods

[0119] 1.1 Preliminary study on improvement of gastrointestinal function in rats

[0120] After adaptive feeding (-1 week to 0 week), 48 SD rats were randomly divided into 4 groups, with 12 rats in each group. The R7970 group, LGG group, ordinary yogurt group and blank group (NC group) were all allowed to eat and drink freely during the experiment. The NC group was gavaged with 0.9% saline (0 week to 4 week, 2 mL / day), and the R7970 group and LGG group were gavaged with solutions containing active R7970 or LGG (Lactobacillus rhamnosus R7970 or LGG powder was resuspended in 0.9% saline, 0 week to 4 week, 2 mL / day, and the number of live bacteria in the R7970 group was higher than 1.0×10 9 CFU, the number of viable bacteria in the LGG group was higher than 1.0×10 9 CFU), the common group was gavaged with live bacterial yogurt (0-4 weeks, 2 mL / day, live bacterial count higher than 1.0×10 9CFU). Body weight and food intake of all SD rats were recorded at 0, 1, 2, 3, and 4 weeks, and fresh feces were collected. Blood supernatant, colon contents, stomach and small intestine contents were collected after being sacrificed at 4 weeks (28 days), and serum calcium ions, digestive enzyme activities (pepsin, lactase activity, sucrase activity, maltase activity, aminopeptidase, trypsin activity, pancreatic lipase activity, pancreatic amylase activity) and motilin content were detected.

[0121] 1.2 Rat femur biomechanics detection method

[0122] In the fourth week (28 days), 4 rats (a total of 16 rats) were selected from each group and fasted for 16 hours at 16:00. The normal group rats were gavaged with normal saline at 8:00 the next day, and the other groups were gavaged with 0.1 ml of loperamide hydrochloride (4 mg / kg) solution. 30 minutes later, 0.1 mL of activated carbon solution was given. The timing started from the gavage of activated carbon solution. After 30 minutes, the rats in each group were killed by dislocation of the cervical vertebrae. The abdominal cavity of the rats was opened, the mesentery was separated, and the intestinal tube from the upper end to the pylorus and the lower end to the ileocecum was taken and spread out. In the experiment, the biological force of the rat femur was directly measured using a texture analyzer. The parameters of the texture analyzer were set to a loading speed of 10 nm / min, and the probe continued to run for 2 mm after contacting the femur. After the texture analyzer was debugged normally, the wide side of the femur specimen was facing up, and the bending test could be started by clicking the run button on the operation interface. The elastic load, elastic disturbance, elastic modulus and other data could be read out through the bending curve.

[0123] 1.3 Metagenomic analysis of rat feces

[0124] The specific steps are as follows:

[0125] (1) Extraction of genomic DNA: The Tiangen magnetic bead kit was used to extract genomic DNA from feces.

[0126] (2) DNA sample testing: DNA purity and integrity were analyzed using 1% agarose gel electrophoresis (AGE); dsDNA Assay Kit in DNA was quantified using a 2.0 Flurometer (Life Technologies, CA, USA); an appropriate amount of sample was placed in a centrifuge tube and diluted with sterile water until the OD value was between 1.8 and 2.0.

[0127] (3) Library construction and sequencing: 1 μg of genomic DNA was collected from stool samples and sequenced using The library was constructed using UltraDNA Library Prep Kit for Illumina (NEB, USA). The fragments were randomly sheared into fragments of about 350 bp in length using a Covaris ultrasonic disruptor. The entire library preparation was completed through steps such as end repair, A-tailing, sequencing adapter addition, purification, and PCR amplification. After the library was constructed, Qubit 2.0 was used for preliminary quantification, and the library was diluted to 2 ng / ul. Then, the insert size of the library was detected using Agilent 2100. When the insert size met expectations, the effective concentration of the library was accurately quantified using the Q-PCR method (library effective concentration > 3 nM) to ensure the quality of the library. After the library inspection was qualified, different libraries were pooled according to the effective concentration and the target amount of data to be downloaded from the machine, and then Illumina PE150 sequencing was performed.

[0128] 2 Experimental results

[0129] 2.1. Weight

[0130] The changes of rat body weight from 0 to 28 hours are as follows Fig. 9 As shown in the figure, there was no significant difference in body weight changes among the groups from 0 to 14 days. After 21 days, the body weight of rats in the R7970 group was significantly higher than that of the other groups (P < 0.05), reaching 418g, indicating that feeding R7970 can significantly increase the body weight of rats.

[0131] 2.2 Digestive enzyme activity

[0132] (1) Digestive enzymes in the stomach

[0133] The activity of gastric pepsin in rats of different groups Fig.10 As shown in the figure, the order of pepsin activity in rats from high to low is R7970 group, NC group, common group and LGG group. The pepsin activity of rats fed with R7970 was significantly higher than that of the other groups. Different letters in a, b, c, and d in the figure indicate statistical differences (P<0.05).

[0134] (2) Pancreatic digestive enzymes

[0135] The activities of pancreatic amylase and pancreatic lipase in rat pancreas Fig.11 and Fig.12 As shown. Among them, the activities of pancreatic amylase and pancreatic lipase in rats fed with R7970 were significantly higher than those in the other groups, with values ​​of (580±14)U / g and (0.247±0.002)U / g, respectively, indicating that R7970 can increase the activities of pancreatic amylase and pancreatic lipase in the pancreas, and are significantly higher than those in the other groups. Different letters in a, b, c, and d in the figure indicate statistical differences (P<0.05).

[0136] (3) Small Intestinal Digestive Enzymes

[0137] Digestive enzymes in the small intestine Figures 13-15 As shown in the figure, the lactase activity from high to low is R7970 group, LGG group, NC group and common group, the sucrase activity from high to low is R7970 group, LGG group, common group and NC group, and the aminopeptidase activity is R7970 group, LGG group, common group and NC group, indicating that feeding R7970 is beneficial to the improvement of lactase, sucrase and aminopeptidase activities in the small intestine (P < 0.05). Different letters in a, b, c, and d in the figure indicate statistical differences (P < 0.05).

[0138] 2.3 Gastrointestinal motility factors

[0139] Gastrointestinal motility factors Fig.16 As shown in the figure, the gastrin content of the R7970 group was 65±0.65pg / mL, the LGG group was 60±0.06pg / mL, the common group was 55±0.55pg / mL, and the NC group was 51±0.5pg / mL. The gastrin content of the R7970 group was significantly higher than that of the other three groups (P<0.05), indicating that feeding R7970 can increase the content of gastrointestinal motility hormone. Different letters in a, b, c, and d in the figure indicate statistical differences (P<0.05).

[0140] 2.4 Serum calcium

[0141] The serum calcium concentration of each group Fig.17 As shown in the figure, the calcium ion concentration in the R7970 group was significantly lower than that in the LGG group and the common group, indicating that feeding R7970 can promote the absorption of calcium ions in serum. Different letters in a, b, c, and d in the figure indicate statistical differences (P<0.05).

[0142] 2.5 Metagenomic flora

[0143] The relative abundance of different bacterial communities Fig.18 As shown in the data, the relative abundance of Escherichia coli and Enterococcus faecalis decreased over time, while the relative abundance of Akkermansia muciniphila (Akk bacteria) and Bifidobacterium increased. R7970 can increase the relative abundance of Akk bacteria and Bifidobacterium, and reduce the relative abundance of Escherichia coli and Enterococcus faecalis, indicating that it can effectively regulate the correlation between bacteria in the intestine, increase the complexity of the intestinal flora network, and have a positive effect on the stability of the intestinal flora.

[0144] 2.6 Biological tolerance of femur

[0145] The related indicators of femoral biological tolerance of experimental rats are as follows Figures 19 to 21As shown in the figure, the bone weight, bone hardness and elastic modulus of rats fed with R7970 were higher than those of the LGG group, the common group and the NC group (P < 0.05), indicating that R7970 helps to improve the biological tolerance of rat femurs and promote bone health. Different letters in a, b, c, and d in the figure indicate statistical differences (P < 0.05).

[0146] 3. Experimental conclusion

[0147] In summary, regular intake of R7970 can promote weight gain in rats within a normal range, significantly increase the activity of digestive enzymes secreted in the stomach, small intestine and pancreas, maintain the relative abundance of some beneficial bacteria in the intestine, promote the absorption of calcium ions in serum, enhance bone resistance to external forces, increase bone hardness, and promote bone health. Lactobacillus rhamnosus R7970 can improve the nutrient digestion and absorption function of the intestine.

[0148] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A Lactobacillus rhamnosus R7970, characterized in that The Lactobacillus rhamnosus is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number being CGMCC No.22244.

2. A microbial agent, characterized in that: A bacterial cell, culture, fermentation product or at least one metabolite thereof containing the Lactobacillus rhamnosus R7970 according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The effective bacterial count of Lactobacillus rhamnosus R7970 is no less than 2×10 6 CFU / mL or 2×10 6 CFU / g.

4. The microbial agent according to claim 2, characterized in that: The metabolites include at least one of organic acids, amino acids, fatty acids and tryptophan metabolites.

5. The microbial agent according to claim 4, characterized in that: The organic acid comprises at least one of tartaric acid, formic acid, lactic acid, acetic acid, citric acid and succinic acid; and / or, the amino acids include at least one of threonine, glutamic acid, glycine, alanine, cystine, valine, methionine, isoleucine, leucine, tyrosine, phenylalanine and arginine; And / or, the fatty acid includes at least one of butyric acid, caproic acid, caprylic acid, capric acid, undecanoic acid, dodecanonic acid, tridecanonic acid, tetradecanonic acid, cis-9-tetradecanoic acid, pentadecanoic acid, hexadecanoic acid, cis-9-hexadecanoic acid, heptadecanonic acid, octadecanoic acid, cis-9-octadecanoic acid, cis, cis-9,12-octadecadienoic acid, cis, cis, cis-9,12,15-octadecatrienoic acid, cis-5,8,11,14,17-eicosapentaenoic acid, tricosanoic acid, tetracosanoic acid, cis-4,7,10,13,16,19-docosahexaenoic acid; And / or, the tryptophan metabolite includes at least one of o-aminophenol, o-aminobenzoic acid, 3-indoleethanol, L-tryptophan, indolepropionic acid, DL-indole-3-lactic acid, indole-3-carboxaldehyde, indole-3-acetamide, β-indole-3-acetic acid, 2-ketoadipic acid, N-formylkynurenine, L-kynurenine, picolinic acid, niacin, quinolinic acid, kynurenic acid, and xanthine acid.

6. A method for preparing the microbial agent according to any one of claims 2 to 5, characterized in that: include: The Lactobacillus rhamnosus R7970 is cultured in an expanded manner.

7. Use of the Lactobacillus rhamnosus R7970 according to claim 1 or the microbial agent according to any one of claims 2 to 5 in any of the following: (1) Use in the preparation of products for preventing, alleviating and / or treating inflammatory bowel disease (IBD); (2) Use in the preparation of products for inhibiting weight loss, diarrhea and bloody stools; (3) Use in the preparation of products that reduce blood inflammatory responses, increase the concentration of anti-inflammatory cytokines and / or reduce the concentration of pro-inflammatory cytokines; (4) Use in the preparation of products that improve the nutrient digestion and / or nutrient absorption function of the intestine; (5) Use in the preparation of products that promote bone health; (6) Use in the preparation of products for increasing bone weight, bone hardness and / or bone elastic modulus; (7) Use in the preparation of products that maintain or increase the relative abundance of beneficial intestinal bacteria.

8. The use according to claim 7, characterized in that The nutrient absorption includes calcium absorption.

9. The use according to claim 7 or 8, characterized in that The products include functional foods, medicines, health products, functional care products or functional auxiliary materials.

10. The use according to any one of claims 7 to 9, characterized in that: The product comprises at least one of powders, granules, pills, capsules, tablets, pastes, liquid preparations, gels, sprays, and solid beverages.

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