CN112535693A

CN112535693A - Mixed lactobacillus for preventing and treating ulcerative colitis and application thereof

Info

Publication number: CN112535693A
Application number: CN202011458234.2A
Authority: CN
Inventors: 李柏良; 史佳鹭; 刘飞; 赵莉; 王松; 霍贵成
Original assignee: Northeast Agricultural University
Current assignee: Heilongjiang Youbet Dairy Co ltd
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-03-23
Anticipated expiration: 2040-12-10
Also published as: CN112535693B

Abstract

The invention discloses mixed lactobacillus for preventing and treating ulcerative colitis, which comprises lactobacillus acidophilus KLDS1.0901, lactobacillus helveticus KLDS1.8701 and lactobacillus plantarum KLDS 1.0318. The volume ratio of the lactobacillus acidophilus KLDS1.0901 to the lactobacillus helveticus KLDS1.8701 to the lactobacillus plantarum KLDS1.0318 is 1:1: 1; the MOI value of the mixed lactobacillus is 0.1-10. The invention also discloses an application of the mixed lactobacillus for preventing and treating ulcerative colitis. The mixed lactobacillus provided by the invention can restore the balance of intestinal flora of a patient and inhibit inflammatory reaction in the treatment of UC, and has less side effect than the traditional medicine treatment.

Description

Mixed lactobacillus for preventing and treating ulcerative colitis and application thereof

Technical Field

The invention relates to a mixed lactobacillus for preventing and treating ulcerative colitis and application thereof, belonging to the technical field of medicines.

Background

Ulcerative Colitis (UC) is a chronic nonspecific Inflammatory bowel disease that mainly involves the rectum, colon mucosa and submucosa, and is clinically mainly manifested by abdominal pain, diarrhea, bloody stool, etc., which seriously affects the quality of life of patients, even evolves into cancer, and is an Inflammatory Bowel Disease (IBDs) together with Crohn's Disease (CD). Epidemiological statistics show that the incidence rate of UC in the United states, Western Europe and other countries is high, the number of patients suffering from ulcerative colitis in China also increases year by year in recent years, and the patients tend to be younger. The etiology and pathogenesis of the disease are unknown, and may be related to genetics, immunity, nutrition, bacteria, viruses and other environmental factors.

The traditional drug treatment measures aiming at the UC at present are as follows:

(1) aminosalicylic acid medicine

Aminosalicylic acid can reduce the expression of inflammatory signaling factors, but is effective only in patients with mild or moderate UC, and the most widely used ones are Salazosulfapyridine (SASP) and mesalamine. SASP is prepared by connecting salicylic acid and sulfapyridine as carrier through azo bond, and decomposing into 5-aminosalicylic acid (5-aminosalicylic acid, 5-ASA) and sulfadiazine under hydrolysis of bacterial enzyme in colon, wherein sulfadiazine has certain side effect on human body. Mesalazine is a novel 5-ASA drug, removes toxic sulfadiazine in SASP, but has higher drug dependence.

(2) Glucocorticoids

Glucocorticoids have anti-inflammatory and immunomodulatory properties, acting primarily on immune and epithelial cells, but the drug has side effects and is drug dependent. The glucocorticoid which is mainly applied clinically comprises prednisone, hydrocortisone, sodium succinate hydrocortisone and a novel hormone budesonide, is suitable for patients with severe ulcerative colitis or patients who have ineffective 5-ASA treatment, has quick response and is not suitable for long-term maintenance of mild and moderate patients.

(3) Immunosuppressant

The immunosuppressant can inhibit the production of proinflammatory cytokines by limiting the number of T cells, so as to prevent immunoreactive inflammation, but can increase the infection risk of patients and has certain adverse reaction. Clinically, the immunosuppressants including azathioprine, methotrexate and cyclosporine are mainly applied to patients with steroid hormone dependence or remission stage.

(4) Antibody formulations

The antibody-based drugs can inhibit the expression of inflammatory signaling factors, but the therapeutic effect is reduced with the time, the price is high, the domestic research time is short, and the specific effect needs to be further evaluated. The clinical application of the monoclonal antibody is most widely Infliximab (IFX) and Adalimumab (ADA), and the Infliximab and the Adalimumab are respectively a human-mouse chimera and a fully humanized monoclonal antibody which are specifically combined with TNF-a, and can be specifically combined with the TNF-a to inhibit inflammatory reaction.

Because the traditional drug therapy has the limitations of poor effect, great side effect and strong dependence, research on a novel method for treating ulcerative colitis becomes a hotspot, the ulcerative colitis is a chronic disease with repeated attack and is divided into an active period and a remission period, and although clinical symptoms and pathology can be improved to the greatest extent after the induction remission therapy, the clinical symptoms and pathology can still be repeated, so that the probiotic bacteria capable of inducing remission, maintaining remission for a long time and treating UC are urgently needed to prevent and treat the ulcerative colitis.

Disclosure of Invention

The invention aims to solve the technical problem of providing a mixed lactobacillus for preventing and treating ulcerative colitis and application thereof.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

a mixed lactobacillus for preventing and treating ulcerative colitis comprises Lactobacillus acidophilus KLDS1.0901, Lactobacillus helveticus KLDS1.8701 and Lactobacillus plantarum KLDS 1.0318.

The volume ratio of the lactobacillus acidophilus KLDS1.0901 to the lactobacillus helveticus KLDS1.8701 to the lactobacillus plantarum KLDS1.0318 is 1:1: 1; the MOI value of the mixed lactobacillus is 0.1-10.

The mixed lactobacillus is applied to the preparation of the medicine for preventing ulcerative colitis.

The mixed lactobacillus is applied to the preparation of medicines for improving the length of colon, reducing spleen index, reducing MPO enzyme activity, inhibiting COX-2 transcription, inhibiting the secretion of PGE2 and reducing the level of proinflammatory cytokines; the proinflammatory cytokines include TNF-alpha, IL-1 beta and IL-6.

The mixed lactobacillus is applied to the preparation of the medicine for treating ulcerative colitis.

The mixed lactobacillus is applied to the preparation of the medicine for improving the permeability of the colon mucous membrane.

The mixed lactobacillus is applied to preparing medicines for improving the intestinal barrier function and improving the expression of colon tight junction protein.

The mixed lactobacillus is applied to the preparation of medicines for improving the expression of mucins MUC1 and MUC2 in colon tissues.

The mixed lactobacillus is applied to the preparation of the medicine for increasing the content of short-chain fatty acid SCFAs in intestinal contents; the short chain fatty acids SCFAs include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid.

The mixed lactobacillus is applied to the preparation of the medicine for adjusting the balance of the microbiota on the intestinal mucosa; the microbiota balance includes increasing firmicutes, decreasing bacteroidetes relative abundance, and increasing pilospirillum and ruminal clostridium9 relative abundance in the gut.

The invention has the following beneficial effects:

1. the research of the invention finds that human intestinal flora plays an important role in UC pathogenesis and possibly determines the severity of intestinal inflammation. Intestinal flora imbalance and mucosal barrier function disappear, so that intestinal pathogenic bacteria invade submucosa, microbial antigens are activated to cause host immune imbalance, and inflammatory cells are activated to cause organism inflammatory reaction, so that intervention on the intestinal flora becomes a potential treatment measure.

2. The mixed lactobacillus provided by the invention can restore the balance of intestinal flora of a patient and inhibit inflammatory reaction in the treatment of UC, and has less side effect than the traditional medicine treatment.

3. The invention adopts mixed lactobacillus to intervene on an LPS-induced RAW264.7 cell inflammation model, and evaluates the anti-inflammatory property of the mixed lactobacillus on a molecular level; the mixed lactobacillus intervenes in mouse ulcerative colitis induced by DSS, and is detected to have prevention and treatment effects on UC mice.

4. The invention researches the inhibition effect of mixed lactobacillus interfering LPS to induce RAW264.7 cells to generate inflammatory reaction based on NF-kB signal channel; DSS is used for inducing C57BL/6J male mice to generate an ulcerative colitis model, and pathological conditions of the mice are judged through DAI values, colon lengths and HE staining; measuring the IL-1 beta, IL-6 and TNF-a content and mRNA expression level in colon tissues, and evaluating the inhibition effect of the mixed lactobacillus on the inflammation of the mice; performing PCR analysis on the expression of Claudin-1, occludin and ZO-1 of the tight junction protein in colon tissues, and evaluating the repair effect of a target strain on intestinal barrier; analyzing the intestinal microbial structure in the mouse excrement by a 16SrDNA high-throughput sequencing technology, and evaluating the influence of a target strain and DSS on intestinal flora; changes in the levels of SCFAs in the mouse feces were detected by the gas chromatography-mass spectrometry method. The conclusion is that the mixed lactobacillus has the effect of preventing and treating UC, the effect is equivalent to that of salzosulfapyridine (SASP), and the limitations of poor treatment effect, large side effect and strong dependence of the traditional medicaments are avoided.

Drawings

FIG. 1 is a graph showing the effect of different MOI values of Lactobacillus on the viability of RAW264.7 cells in accordance with the present invention;

FIG. 2 is a graph showing the effect of Lactobacillus on the concentration of PGE2 in RAW264.7 cells induced by LPS and the expression level of COX-2 in the present invention;

FIG. 3 is a graph showing the effect of Lactobacillus in the present invention on the concentration and expression level of pro-inflammatory cytokines in RAW264.7 cells induced by LPS;

FIG. 4 is a graph showing the effect of Lactobacillus on the expression of TLR4 protein in RAW264.7 cells;

FIG. 5 is a graph showing the effect of Lactobacillus on NF-. kappa.B signaling pathway in RAW264.7 cells;

fig. 6 shows the weight and DAI score changes of mice in the present invention, wherein a is the weight change of mice, B is the DAI score, note: the lower case difference indicates significant difference (P < 0.05);

FIG. 7 is a graph showing the change in the length of the colon of a mouse in the present invention, wherein A is the colon of a mouse, B is the length of the colon, and Note: the lower case difference indicates significant difference (P < 0.05);

FIG. 8 is a graph of spleen indices for mice of the present invention, note: the lower case difference indicates significant difference (P < 0.05);

FIG. 9 is a graph showing HE staining of a colon tissue section in accordance with the present invention;

FIG. 10 is a graph of tissue damage scores for the present invention, noting: the lower case difference indicates significant difference (P < 0.05);

FIG. 11 is a chart showing the staining of AB-PAS on a colon tissue section according to the present invention;

FIG. 12 is a graph of the number of goblet cells in the present invention;

FIG. 13 is a graph of the myeloperoxidase activity of colon tissue of mice in the present invention;

FIG. 14 is a graph showing the concentration of PGE2 in colon tissue and the expression level of COX-2 in mouse according to the present invention;

FIG. 15 is a graph showing cytokine concentration and mRNA expression level in colon of mouse in accordance with the present invention;

FIG. 16 is a graph showing the concentration of D-lactic acid in serum of a mouse according to the present invention;

FIG. 17 is a graph showing the gene expression level of the tight junction protein in the present invention;

FIG. 18 is a graph showing the expression levels of mucin genes in the present invention, wherein A is the expression level of MUC1 gene and B is the expression level of MUC2 gene, note that: the lower case difference indicates significant difference (P < 0.05);

FIG. 19 is a TIC map of SCFAs standards in accordance with the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings.

1 materials and methods

1.1 Experimental materials

1.1.1 test strains: lactobacillus acidophilus (Lactobacillus acidophilus) KLDS1.0901, Lactobacillus helveticus (Lactobacillus helveticus) KLDS1.8701 and Lactobacillus plantarum (Lactobacillus plantarum) KLDS1.0318 were isolated from traditionally fermented dairy products in Sinkiang and preserved by the Collection of Industrial microorganism cultures in the Key laboratory of the department of Dairy science education of northeast university of agriculture.

1.1.2 Experimental cells: a mouse mononuclear macrophage RAW264.7 cell strain is a main cell for regulating and controlling inflammatory reaction and is widely applied to a macrophage inflammation model induced by lipopolysaccharide. The cell line is a gift from Zhao Xinhuai professor in the important laboratory of the department of dairy science and education.

1.1.3 Experimental animals: male C57BL/6J mice (7 weeks old).

1.1.4 Medium

1.1.4.1 bacterial culture medium

MRS liquid medium: after adding the components of the culture medium according to the proportion of (5.0 g of peptone, 5.0g of beef extract, 10.0g of tryptone, 5.0g of sodium acetate, 2.0g of dipotassium hydrogen phosphate, 5.0g of yeast powder, 0.25g of manganese sulfate, 0.58g of magnesium sulfate, 2.0g of diammonium hydrogen citrate, 801.0 g of tween-801.0 g and 20.0g of glucose), fixing the volume to 1L by using distilled water, fully and uniformly stirring, adjusting the pH to 6.2-6.4, sterilizing for 15min at the temperature of 121 ℃, and then storing in a refrigerator at the temperature of 4 ℃ for later use. MRS solid medium: add 16% agar per liter MRS broth.

Cell culture medium: complete high-sugar DMEM medium. High-glucose DMEM was supplemented with 10% heat-inactivated fetal bovine serum and 1% diabody (penicillin concentration 100U/mL and streptomycin 100. mu.g/mL), sterile filtered through a 0.22 μm filter, and stored in a refrigerator at 4 ℃ until needed.

High-glucose DMEM medium: that is, 10% heat-inactivated fetal bovine serum and 1% antibiotic (penicillin concentration 100U/mL, streptomycin 100. mu.g/mL) were added to a high-sugar DMEM medium, and the mixture was sterilized by filtration through a 0.22 μm filter and stored in a refrigerator at 4 ℃ until use.

1.2 Experimental methods

1.2.1 culture and preservation of the Strain

The experimental strain is inoculated into an MRS liquid culture medium in an inoculation amount of 2%, the culture is carried out at a constant temperature of 37 ℃, the continuous activation and passage are carried out for 2 times, and three-region streaking is carried out on the MRS solid culture medium. Culturing at 37 deg.C for 48h, selecting single colony, observing with gram stain, performing microscopic examination to determine whether it is single colony, subculturing for 2 times until activity is recovered, mixing culture solution of 3 rd generation with 50% glycerol at a ratio of 3:2, storing at-20 deg.C, and performing 16S rDNA identification.

And (3) counting strains: plate counting was used. Inoculating the test strain into MRS liquid culture medium at an inoculation amount of 2%, culturing at constant temperature of 37 ℃, continuously activating and passaging for 2 times, then coating on MRS solid culture medium, culturing at constant temperature of 37 ℃ for 48h, and then counting plates.

(1) Diluting: adding 0.9mL sterile PBS into an EP tube, shaking and uniformly mixing bacterial liquid, adding 0.1mL bacterial liquid into the EP tube, uniformly mixing, diluting by 10 times, sequentially carrying out gradient dilution, and diluting the original bacterial liquid to 10^-4、10^-5、10^-6、10^-7、10^-8CFU/mL。

(2) Coating: and (3) sucking 100uL of each gradient bacterial liquid, adding the gradient bacterial liquid into an MRS solid culture medium, placing a coating rod on the flame of an alcohol lamp for burning and sterilizing, after the temperature of the coating rod is reduced to a proper temperature, uniformly coating the bacterial liquid in the MRS solid culture medium, repeating each gradient for 3 times, and numbering.

(3) Counting: and (3) culturing the coated MRS solid culture medium at the constant temperature of 37 ℃ for 48h, and then selecting the colonies with the colony number of 30-300 for counting.

1.2.2 RAW246.7 cell culture and preservation

1.2.2.1 RAW246.7 cell activation

The RAW246.7 cell cryopreservation tube was taken out of the liquid nitrogen tank, and was rapidly placed in a 37 ℃ constant temperature water bath and repeatedly shaken to melt the cells. Quickly transferring to a super clean bench, adding 5mL of DMEM high-sugar culture medium into a 15mL sterile centrifuge tube, transferring the liquid in the frozen tube into the centrifuge tube, centrifuging (1000r/min, 5min), removing the supernatant, adding 1mL of culture medium into cell sediment, fully and uniformly blowing, transferring to a 50mL sterile cell culture bottle, adding 3mL of culture medium into the culture bottle, slightly shaking and uniformly mixing, adding 5% CO at 37 ℃, and stirring uniformly₂Culturing in an incubator. Replacing the fresh culture medium every 1 to 2 days, and carrying out passage when the cell growth state is good (the adherent growth of monolayer cells is 70 to 80 percent).

1.2.2.2 RAW246.7 cell passages

When the cell growth state is good (the adherent growth of the monolayer cells reaches 70% -80%), carrying out passage, discarding the original culture medium in a culture bottle, washing the monolayer cells for 2-3 times by 2mL sterile PBS, adding 1 mL0.25% pancreatin for digestion for 1-2min, then adding DMEM high-sugar culture medium to stop digestion, transferring to a 15mL sterile centrifuge tube, centrifuging (1000r/min, 5min), discarding the supernatant, adding 3mL culture medium into the cell sediment, fully and uniformly blowing, sucking 1mL cell suspension into 50mL culture bottles respectively, supplementing 3mL culture medium into each culture bottle, 37 ℃, and 5% CO₂Culturing in an incubator.

1.2.2.3 RAW246.7 cells cryopreserved

When the cells reach the passage standard, removing the original culture medium in a culture bottle, washing the monolayer cells for 2-3 times by using 2mL of sterile PBS, adding 1 mL0.25% pancreatin for digestion for 1-2min, then adding the culture medium to stop digestion, centrifuging for 5min at 1000r/min, removing the supernatant, adding 1mL of frozen stock solution (the frozen stock solution is prepared by 90% serum and 10% dimethyl sulfoxide (DMSO), keeping out of the shade during preparation, preparing in situ), fully and uniformly blowing, rapidly transferring to a sterile frozen stock tube, and sealing the mouth of the frozen stock tube by using a sealing film. Placing in a refrigerator at 4 deg.C for 30min, placing in a refrigerator at-20 deg.C for 2 hr, transferring to a refrigerator at-80 deg.C overnight, and freezing in liquid nitrogen for a long time.

1.2.3 study of in vitro anti-inflammatory mechanism of Lactobacillus

1.2.3.1 preparation of the bacterial suspension

Inoculating a test strain into an MRS liquid culture medium in an inoculation amount of 2%, culturing at a constant temperature of 37 ℃, continuously activating and passaging for 2 times, centrifuging for 10min at 8000g/min, discarding supernatant, washing thalli for 2-3 times by using sterile PBS, and resuspending the thalli with a high-sugar DMEM culture medium at a required concentration, wherein three strains of lactobacillus are mixed by using a mixed strain according to a volume ratio of 1:1: 1.

1.2.3.2 preparation of LPS solution

Dissolving 10mg of sterile LPS powder in 10mL of sterile PBS, mixing well to obtain 1mg/mL LPS stock solution, and standing at-20 deg.C. When in use, the solution is diluted to 1 mu g/mL LPS by adding high-sugar DMEM medium.

1.2.3.3 cell survival rates

The detection is carried out by using a CCK-8(Cell Counting Kit-8) method. The cells were grouped as: blank group (medium), control group (cells + medium), experimental group (medium + cells + mixed strain). Taking RAW264.7 cells in logarithmic growth phase, digesting, and uniformly blowing out at 2 × 10⁴The density of each well is inoculated on a 96-well plate, after the cells adhere to the wall, the culture solution is discarded, the cells are washed for 2 times by PBS, and each group has 6 multiple wells. Except for the blank group and the control group, mixed lactobacillus (MOI ═ 0.1, 1, 10, 100, 1000) was added to each well to treat the cells, the cells were gently shaken and put into an incubator to incubate for 24 hours, and then the cells were washed twice with PBS, and 10 μ L of CCK-8 solution was added to each well. After further incubation for 2h, absorbance was measured at 450nm using a microplate reader, and the relative survival rate of macrophages was calculated according to the following formula.

1.2.3.4 measurement of PGE2 and TNF-. alpha.IL-6 and IL-1. beta

ELISA method was used. The cells were grouped as: blank control group, LPS only treated group, LPS + KLDS1.0901 group, LPS + KLDS1.8701 group, LPS + KLDS1.0318 group and LPS + mixed lactobacillus group. Taking RAW264.7 cells in logarithmic growth phase at 2X 10⁶Density of individual/well was seeded in 6-well plates with 3 heavy wells per group. After the cells are attached to the wall, different lactobacilli (MOI is 10) are added for pretreatment for 1h, LPS with the final concentration of 1 mu g/mL is added, after incubation for 12h, the cell culture solution is centrifuged at 1000r/min for 5min at room temperature, and then the cell culture solution is taken. Standard curves were generated and the cell culture media were assayed for PGE2 and TNF-. alpha.IL-6 and IL-1. beta. according to the procedures described in the ELISA kit (commercially available).

1.2.3.5 inflammatory factor mRNA expression assay

A real-time fluorescent quantitative Polymerase Chain Reaction (PCR) method is used.

(1) Total RNA extraction in cells

RAW246.7 cells were grouped and processed accordingly as described in section 1.2.3.4. RAW264.7 cell suspension at 2X 10⁶The density of each well was inoculated in 6-well plates per wellGroups of 3 duplicate wells were pretreated with different lactobacilli for 1h, and after LPS stimulation for 12h, the cells were washed twice with PBS. Total RNA from cells was extracted using a Tiangen kit (commercially available), and the total RNA concentration was measured using a nucleic acid analyzer with no enzyme water as a control, and the A260/A280 value was between 1.8 and 2.0 for subsequent experiments.

(2) Generation of cDNA

According to GoScript of Promega^TMThe Reverse Transcription Mix kit prepares a reaction system on ice, and carries out Reverse Transcription reaction after being mixed gently to synthesize cDNA.

(3) Nucleotide sequence of primer

The relevant primers were designed and synthesized by Biotechnology engineering (Shanghai) GmbH.

(4) Real-time fluorescent quantitative PCR

2. mu.g of cDNA as template, according to Promega

The qPCR Master Mix kit indicates to prepare reaction solution, and a real-time fluorescent quantitative PCR system (ABI 7500) is used for two-step PCR amplification. Reaction conditions are as follows: pre-denaturation at 95 ℃ for 10min, PCR at 95 ℃ for 15s, and PCR at 60 ℃ for 1min, and circulating for 40 times, wherein the volume of the reaction system is 10 mu L. According to the Ct value of the target gene and the Ct value of the reference gene beta-actin, the expression is as follows^-ΔΔCtThe method calculates the relative expression level line analysis of the target gene mRNA in the sample.

1.2.3.6 Western blotting analysis

The Nuclear transcription factor inhibitor (NF) -kB, I kB), phosphorylated Nuclear transcription factor kappa-B and phosphorylated I kB protein expression levels in the NF-kB signal pathway were determined.

1.2.4 study of mechanism of Lactobacillus in vivo prevention and treatment of colitis and ulcerative colitis

1.2.4.1 preparation of bacterial suspensions

Inoculating the experimental strain into MRS liquid culture medium at 2%, culturing at constant temperature of 37 deg.C, activating and subculturing for 2 times, centrifuging at 8000g/min for 10min, discarding supernatant, and adding sterile PBS washing thallus for 2-3 times, and re-suspending with sterile physiological saline to regulate lactobacillus concentration to 1 × 10⁹CFU/mL, wherein the mixed strain is prepared by mixing three strains of lactobacillus according to the volume ratio of 1:1: 1.

1.2.4.2 mouse grouping and Experimental design

After feeding 60 male C57BL/6J mice (7 weeks old) for 1 week, the 60 mice were randomly divided into 6 groups of 10 mice each according to the following Table 1. The experiment was carried out for five weeks, the first four weeks were the prevention period, the gavage was carried out according to the following table, and then 3% DSS aqueous solution was drunk for 7 days except for the blank group, during which the feeding temperature was 22 ± 2 ℃, the humidity was 55% ± 5%, feeding and drinking water were freely carried out, 12h light-dark cycle.

TABLE 1 mouse experimental groups

In table 1, LAB1 is lactobacillus acidophilus KLDS 1.0901; LAB2 is Lactobacillus helveticus KLDS 1.8701; LAB3 is Lactobacillus plantarum KLDS 1.0318; mix is mixed lactobacillus of lactobacillus acidophilus KLDS1.0901, lactobacillus helveticus KLDS1.8701 and lactobacillus plantarum KLDS1.0318 in a volume ratio of 1:1: 1.

1.2.4.3 disease Activity index

Colitis severity was assessed by Disease Activity Index (DAI) (phloUC document E-disc). The DAI includes three aspects: body weight change, fecal characteristics and hematochezia status, specific scoring criteria are shown in table 2. During the molding period, body weight was measured every day, and stool properties and stool blood in the stool were observed. The fecal occult blood condition is measured by adopting an o-tolidine method, and the specific steps are as follows: (1) collecting the stool specimen as soon as possible and coating the stool specimen on a white porcelain plate; (2) dripping 2 drops (about 0.1mL) of o-tolylamine liquid at different positions of the excrement; (3) 2 drops (about 0.1mL) of the oxidizing agent were added dropwise, immediately timed and the color change observed. (4) Blue appears within 2min, fecal occult blood test is positive, and non-color development within 2min is negative.

TABLE 2 disease Activity index scores

1.2.4.4 Standard treatment

After the experiment is finished, blood is taken from the orbit of each group of mice after anesthesia, then the mice are killed by dislocation of cervical vertebrae, the abdominal part of the mice is disinfected by alcohol and the abdominal cavity is opened under the sterile environment, the colon is cut off, colon contents are carefully collected, then the colon contents are cleaned by PBS, the length of the complete colon is measured, one part of the colon is placed in 4 percent paraformaldehyde for detecting the pathological change of the microscopic tissue, and the rest part of the colon is stored in a refrigerator at minus 80 ℃ for subsequent detection experiments of inflammatory factors and the like. After standing the whole blood for a period of time, centrifuging at 3000g for 20min, collecting serum, and storing in a refrigerator at-80 ℃ for subsequent experiments.

1.2.4.5 Colon histopathology observations and Scoring

(1) Embedding: fresh colon tissue is fixed by 4% paraformaldehyde for more than 24h, washed by running water for 30min, tissue is trimmed, and then put into a dehydration box to be dehydrated by gradient alcohol in sequence (75% ethanol for 4h, 85% ethanol for 2h, 90% ethanol for 2h, 95% ethanol for 1h, absolute ethanol for I30 min, absolute ethanol for II 30min, alcohol benzene for 10min, xylene for I10min, xylene for II 10min, paraffin I1h melted at 65 ℃, paraffin II melted at 65 ℃ for 1h, paraffin III melted at 65 ℃ for 1h, the wax-soaked tissue is embedded in an embedding machine (2) sliced into 5 mu m slices by a paraffin slicer, the tissue is flattened by a warm water at 40 ℃, a glass slide is taken out, and the slices are baked in a 60 ℃ oven (3) dyed, (a) the paraffin slices are dewaxed to water, the slices are put into xylene for 15min, xylene for II for 15min, xylene for III min and absolute ethanol for 5min in sequence, 5min of absolute ethyl alcohol II, 5min of 95% ethyl alcohol and 5min of 85% ethyl alcohol, and washing with tap water. (b) Hematoxylin staining: and (3) dyeing the slices in hematoxylin dyeing solution for 1-2min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water. (c) Eosin staining: and dyeing in eosin dye liquor for 2-3 min. (d) Dewatering and sealing: sequentially dehydrating (anhydrous ethanol I5 min, anhydrous ethanol II 5min, and anhydrous ethanol III 5min) and sealing with neutral gum; (e) microscopic examination and image acquisition and analysis.

The histological score is detailed in table 3.

TABLE 3 histological Scoring

1.2.4.6 AB-PAS (Alisin blue staining) staining

(1) The embedding and section (2) methods are the same as in section 1.2.4.5. (3) Dyeing: (a) paraffin section dewaxing to water: sequentially placing the slices into xylene I for 15min, xylene II for 15min, xylene III for 15 min-absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, 95% ethyl alcohol for 5min and 85% ethyl alcohol for 5min, and washing with tap water; (b) alisin blue staining: the slices are dyed in an Alisin blue dye solution for 5min, and washed with tap water for 2 min; (c) periodic acid dyeing: slicing into periodic acid dye solution for 15min, washing with tap water, and washing with distilled water twice; (d) and (3) performing snow dyeing: placing the slices into a Xuefer dye solution to dye for 30min in a dark place, and flushing for 5min by running water; (e) hematoxylin staining: staining the slices in hematoxylin staining solution for 3-5min, washing with tap water, differentiating the differentiation solution, washing with tap water, returning blue to the blue solution, and washing with running water; (f) dewatering and sealing: slicing, sequentially dehydrating (anhydrous ethanol I5 min, anhydrous ethanol II 5min, and anhydrous ethanol III 5min), clearing (xylene I5 min, xylene II 5min), and sealing with neutral gum; (g) microscopic examination and image acquisition and analysis.

1.2.4.7 determination of D-lactic acid in serum

Measured by ELISA method. And (3) measuring the content of the D-lactic acid in the serum according to the operation instruction of the ELISA kit.

1.2.4.8 colonic tissue Myeloperoxidase (MPO) Activity assay

Accurately weighing colon tissues, taking the reagent solution II as a homogenizing medium, adding the homogenizing medium according to the weight-volume ratio of 1:19 to prepare 5% tissue homogenate, taking 0.9mL of the 5% tissue homogenate, adding 0.1mL of the reagent III, fully mixing, carrying out water bath at 37 ℃ for 15min, and taking out to be tested. Adding 0.2mL of reagent IV and 3mL of color developing agent into 0.2mL of sample (adding 3mL of double distilled water into the control group), mixing well with water at 37 deg.CBathing for 30min, adding 0.05mL reagent seven, bathing for 10min at 60 deg.C, taking out, and immediately measuring absorbance at 460 nm. The calculation formula is as follows:

1.2.4.9 colonic tissue inflammatory factor assay: and (3) measuring by using an ELISA kit. Accurately weighing colon tissues, adding normal saline according to the weight-to-volume ratio of 1:9 to prepare 10% tissue homogenate, centrifuging the tissue homogenate at 4 ℃ for 20min at 2500 Xg, taking out supernate and determining the contents of PGE2, TNF-alpha, IL-1 beta, IL-6 and IL-10 according to the operation instruction of an ELISA kit.

1.2.4.10 colonic tissue inflammatory factor and intestinal barrier mRNA expression assay: as in section 2.2.3.5.

1.2.4.11 determination of short chain fatty acids in feces

(1) Sample preparation

Accurately weighing 0.8000 +/-0.010 g of cecum content, placing into a fecal sample box, treating with a HALO-F100 fecal treatment instrument to prepare 10% suspension, taking 500 mu L of suspension (if the suspension is a liquid culture medium sample, directly sucking 500 mu L, after the operation is carried out), placing into a 1.5mL centrifuge tube, adding 100 mu L of crotonic acid metaphosphoric acid solution, freezing at-30 ℃ for 24h, thawing, centrifuging at 8000rpm and 4 ℃ for 3min to remove protein and other impurities, taking supernatant, filtering with a 0.22 mu m water system filter, and performing on-machine determination.

(2) Instrumentation method

A chromatographic column: chromatography column Agilent DB-FFAP (capillary column 30m × 0.25mm ID × 0.25 um); column temperature: heating to 180 deg.C at 75 deg.C/min for 1min, and heating to 220 deg.C at 50 deg.C/min for 1 min; a sample inlet: temperature: 250 ℃, sample introduction: 1.0 μ L, split ratio: (5: 1); carrier gas: high purity nitrogen; flow rate: 2.5mL/min for 6.5min, 2.8mL/min 2 rising to 2.8mL/min for 2 min; a detector: FID; temperature: 250 ℃; tail blowing: 20 mL/min; hydrogen gas: 30 mL/min; air: 300 mL/min.

2 results and analysis

2.1 in vitro experiments

2.1.1 investigating the Effect of Lactobacillus on the viability of RAW246.7 cells

As shown in FIG. 1, after mixed Lactobacillus with different MOI values acted on RAW246.7 for 24h, compared with the control group, the mixed Lactobacillus with MOI values of 0.1, 1 and 10 did not have significant change on the cell survival rate, and the MOI values of 100(p <0.05) and 1000(p <0.01) had significant effect on the cell survival rate. The competition effect of nutrient substances between live bacteria and cells of the lactobacillus is prompted, and when the ratio of the number of the lactobacillus to the number of the cells is too large, the cells can die from non-drug pathogenicity. Subsequent studies will select mixed lactobacilli with an MOI of 10 to evaluate their anti-inflammatory efficacy and molecular mechanisms.

2.1.2 Effect of Lactobacillus on LPS-induced concentration of PGE2 and COX-2 expression in RAW264.7

As shown in FIG. 2A, the concentration of PGE2 was significantly increased (P <0.05) in the RAW264.7 cell culture medium when stimulated by LPS addition, as compared to the NC group, and the content of PGE2 was decreased (P <0.05) in the LAB1, LAB2, LAB3 and mixed Lactobacillus treatment, as compared to the LPS group. COX-2 was the synthetase of PGE2, and the PCR results showed that after 1. mu.g/mL LPS stimulation (FIG. 2B), the relative expression level of COX-2mRNA in RAW264.7 cells was significantly increased, and after pretreatment with LAB1, LAB2, LAB3 and mixed lactobacillus, the relative expression level of COX-2mRNA was significantly decreased compared with LPS group (P < 0.05). The results show that the lactobacillus can down-regulate COX-2 transcription level and inhibit PGE2 secretion.

2.1.3 Effect of Lactobacillus on LPS-induced RAW264.7 cytokine concentration and expression level

As shown in FIG. 3, LPS at 1. mu.g/mL stimulated a significant increase in the concentration of proinflammatory cytokines (TNF-. alpha., IL-1. beta., and IL-6) in RAW264.7 cells (P < 0.05). In contrast, TNF-. alpha.IL-1. beta. and IL-6 concentrations were reduced to different extents in the LAB1, LAB2, LAB3 and mixed Lactobacillus treatments compared to the LPS group. Meanwhile, PCR results show that after 1 mu g/mL of LPS is added for stimulation, the transcription levels of TNF-alpha, IL-1 beta and IL-6 of RAW264.7 cells are obviously increased, and after LAB1, LAB2, LAB3 and mixed lactobacillus pretreatment, the relative expression amounts of TNF-alpha, IL-1 beta and IL-6mRNA are obviously reduced compared with that of LPS groups (P is less than 0.05). The results show that the lactobacillus can down-regulate the transcription levels of TNF-alpha, IL-1 beta and IL-6 and inhibit the secretion of the cytokines.

2.1.4 Effect of Lactobacillus on expression of TLR4 protein in RAW264.7 cells

LPS binds to the TLR4 receptor and can stimulate inflammation-related signaling pathways, such as the NF-kappa B pathway. Therefore, the protein expression level of TLR4 in RAW264.7 macrophage cells was determined by using the Western blot method. As shown in fig. 4, the Western blotting analysis results show that the expression level of TLR4 protein of RAW264.7 cells in LPS group is significantly increased compared to NC group (p < 0.05). Whereas the protein expression levels of TLR4 were significantly reduced (p <0.05) for LAB1, LAB2, LAB3 and mixed lactobacillus group RAW264.7 cells compared to LPS group, indicating that lactobacillus intervention could inhibit binding of LPS to TLR4 receptors.

2.1.5 Effect of Lactobacillus on the NF-. kappa.B Signaling pathway in RAW264.7 cells

In order to investigate whether the NF-kB signal pathway has correlation in the anti-inflammatory action of lactobacillus, key proteins in the NF-kB signal pathway are determined. As shown in FIG. 5, Western blotting analysis results show that after the RAW246.7 cells are treated by LPS, compared with a control group, the expression level of I kappa B protein is obviously reduced, the expression level of p-I kappa B protein is obviously increased, the expression of NF-kappa B p65 protein in a cell nucleus is obviously increased, and the expression of NF-kappa B p65 protein in cytoplasm is obviously lower. In the prognosis of LAB1, LAB2, LAB3 and mixed lactobacillus stems, I κ B protein expression levels were significantly increased, phosphorylation levels were significantly decreased, and NF- κ B p65 nuclear entry was decreased and p65 expression in the cytoplasm was increased, compared to LPS group. The results show that the lactobacillus can relieve the inflammation degree by inhibiting NF-kB signal channels.

2.2 in vivo experiments

2.2.1 mouse body weight and DAI score changes

During modeling, the weight of the mice is monitored every day, the stool characters are observed, the fecal occult blood and fecal blood conditions of the mice are measured, and the mice are scored according to DAI scoring standards.

The body weight change during the molding of each group of mice is shown in fig. 6A. The NC group mice grew steadily throughout the molding period, while the other groups started to lose weight from day 3 of molding, but the SASP, LAB1, LAB2, LAB3 and the mixed group treatment significantly relieved the weight loss tendency compared to the DSS group. When the DSS solution is drunk on the 3 rd day, the feces of the mice in the DSS group become green, the feces of the mice are loose by the 6 th day, and a small part of the mice have bloody stool and are adhered to the anus. The intervention group also exhibited similar symptoms to those of the DSS group after drinking the DSS solution, but all of the symptoms were reduced compared to the DSS group. As shown in fig. 6B, score DAI, note: the difference in lower case letters indicates significant difference (P <0.05), by day 7, the DAI score was significantly higher in DSS group mice than in NC group (P <0.05), but each intervention group could significantly lower DAI score, and the mixed lactobacillus group was closer to the drug group.

2.2.2 mouse Colon Length variation

After 21 days of experiment, mice were sacrificed by dislocation and the intact colon was removed for measurement. Colon length of each mouse is shown in fig. 7A, the colon length is longest in the NC group and shortest in the DSS group, and compared with the NC group, DSS induction significantly decreased colon length, and the colon length in the other groups was longer than that in the DSS group. And as shown in fig. 7B, the colon length of mice in DSS group was significantly decreased (P <0.05) compared to NC group, and LAB1, LAB2 and LAB3 were able to increase colon length, but were not significantly different from DSS group. However, gavage treatment with mixed lactobacilli significantly increased the colon length (P <0.05) in mice compared to the DSS group, with the same trend (P <0.05) as the SASP group. This indicates that mixed lactobacilli can more effectively increase colon length in mice.

2.2.3 mouse spleen weight changes

Spleen index change of mice in each group is shown in a figure 8, compared with mice in an NC group, spleen index of the mice is obviously improved after DSS solution drinking (P <0.05), spleen index of the mice is reduced after SASP, LAB1, LAB2, LAB3 and lactobacillus mixis are dried, and the spleen index is obviously different, wherein the lactobacillus mixis has better effect (P <0.05) and has similar effect with the drug group.

2.2.4 Colon histopathological changes in mice

Pathological changes of colon tissues of mice can be visually observed by H & E staining on colon tissue sections. As shown in fig. 9, epithelial cells of the mucosal layer of the mice in the NC group were intact, no necrosis and desquamation were observed, a large number of goblet cells were observed, and intestinal glands were abundant and aligned; the size of the submucosal gap is uniform, and no obvious pathological change is seen. The mucosal layer of mice in the DSS group is subjected to large-area necrosis, the crypt structure of the mucosal layer disappears, and inflammatory cells are subjected to diffuse infiltration; submucosa connective tissue proliferates, and interstitial spaces increase; the intestinal muscular layer is locally seen with muscle fiber gap enlargement, loose fiber arrangement and connective tissue hyperplasia among fibers. The intestinal mucosa of SASP, LAB1, LAB2, LAB3 and Mix groups has necrosis, crypt structure and goblet cell are damaged to different degrees, and inflammatory cell infiltration is accompanied, but the histological change of the mice is improved compared with DSS group mice. And as can be seen from the histological score chart 10, compared with the NC group, the histological score of the mice in the DSS group is remarkably improved, and the histological score of the mice can be remarkably reduced after the prognosis of LAB1, LAB2, LAB3 and mixed lactobacillus stem, wherein the mixed lactobacillus has the highest reduction degree (P <0.05) and has similar effect with SASP, which indicates that the mixed lactobacillus can more effectively reduce the pathological damage of colon tissues.

2.2.5 Lactobacillus effects on goblet cell distribution in Colon of mice

By AB-PAS staining of colon tissue sections, various goblet protein changes in the mouse colon can be visualized visually. As shown in FIG. 11, the neutral mucin-derived material was purple red and the acidic mucin-derived material was blue, and the number of neutrophil-secreting goblet cells and the number of acidic mucin-secreting goblet cells were counted based on the difference in staining of goblet cells. Compared with the NC group (FIG. 12), the DSS group had a significant decrease in the number of acid goblet cells and the number of neutral goblet cells, while LAB1, LAB2, LAB3 and mixed Lactobacillus acted on both goblet cells significantly, and the mixed Lactobacillus acted on the increase in the number of goblet cells better than that of single Lactobacillus.

2.2.6 mouse Colon tissue Myeloperoxidase (MPO) levels

MPO enzyme is a function and activation mark of central granulocyte, reflects the infiltration degree of neutrophil in colon tissue, and the higher the enzyme activity, the deeper the infiltration degree of inflammatory cells, which shows that the inflammatory reaction is more obvious. The change of the MPO enzyme activity of the colon tissues of each group of mice is shown in FIG. 13, and the MPO enzyme activity of the DSS group is obviously higher than that of the NC group, which indicates that the degree of inflammation of the model group is increased after the DSS is given. Compared with the DSS group, the MPO enzyme activity of each intervention group is reduced to a certain degree, especially the mixed lactobacillus can remarkably reduce the MPO enzyme activity (P is less than 0.05), and the mixed lactobacillus has similar effect with SASP, which shows that the intervention of the lactobacillus relieves inflammation to a certain degree and the mixed lactobacillus has the best relief effect.

2.2.7 mouse Colon tissue PGE2 concentration and COX-2 expression level

The concentration of PGE2 in colon tissue of each group of mice is shown in fig. 14A, and after DSS induction, the concentration of PGE2 was significantly higher (P <0.05) than in NC group. Compared with the DSS group, each intervention group can obviously reduce the rising of the level of PGE2 (P <0.05), and the mixed lactobacillus has the best effect. COX-2 is the synthetase of PGE2, and as shown in FIG. 14B, the PCR results showed that, compared with the NC group, COX-2 transcription was significantly upregulated (P <0.05) in the DSS group, whereas each intervention group was able to significantly inhibit this upregulation (P < 0.05). The mixed lactobacillus has the most obvious down-regulation tendency. The above results indicate that mixed lactobacilli are most effective in inhibiting COX-2 transcription and thus PGE2 secretion.

2.2.8 mouse Colon tissue cytokine concentration

Changes in cytokine levels play a major role in the pathogenesis of IBD. The ELISA method is adopted to measure the cytokine level in the colon of the mouse, and the result is shown in figure 15, compared with the NC group, the levels of proinflammatory cytokines TNF-alpha, IL-1 beta and IL-6 in the colon of the mouse in the DSS group are obviously increased (P is less than 0.05), while SASP, LAB1, LAB2, LAB3 and mixed lactobacillus all reduce the proinflammatory cytokine level and improve the anti-inflammatory factor level in different degrees. LAB1, LAB2, LAB3 were able to significantly reduce TNF-. alpha.levels (P >0.05), but were not significant for the reduction of IL-1. beta. and IL-6; the contents of TNF-alpha, IL-1 beta and IL-6 in the colon of the mice in the mixed lactobacillus group are all obviously reduced (P is more than 0.05), and the effect is similar to that of the drug group. Furthermore, the anti-inflammatory factor IL-10 levels in the DSS group were significantly reduced compared to the NC group (P <0.05), while the IL-10 levels in each intervention group were significantly higher (P < 0.05). Furthermore, there was no significant difference in IL-10 levels in the mixed lactobacilli compared to the control group (P > 0.05). The mRNA expression level of each cytokine varied similarly to the secretion level of the corresponding cytokine. Compared with the DSS group, the gene transcription level of proinflammatory cytokines TNF-alpha, IL-1 beta and IL-6 in the colon tissue of the mice treated by the DSS is obviously up-regulated; in the colon of mice in each intervention group, the level of the proinflammatory cytokines is reduced to a different extent than that in the DSS group. LAB1 and LAB2 were able to significantly reduce TNF-. alpha.and IL-6 levels (P >0.05) compared to the DSS group, but were not significant for IL-1. beta. lowering. Both LAB3 and the mixed Lactobacillus group significantly reduced the transcription levels of TNF- α, IL-1 β and IL-6 (P >0.05) and the mixed Lactobacillus group intervention group inhibited the reduction in transcription of these inflammatory cytokines more significantly, similar to the SASP group. In addition, DSS induction obviously reduces the transcription level of the anti-inflammatory factor IL-10, and each intervention group can obviously improve the transcription level (P >0.05), and the mixed lactobacillus has the most obvious improvement effect. Indicating that the mixed lactobacillus inhibits the expression of inflammatory cytokines in colon tissues of mice with DSS-induced colitis.

2.2.9 level of D-lactic acid in mouse serum

By detecting the concentration of the D-lactic acid in the serum, the intestinal mucosa repair can be effectively judged and the intestinal permeability change condition can be evaluated. As shown in fig. 16, the content of D-lactic acid was significantly higher in DSS group than in NC group (P < 0.05). The lactobacilli LAB1, LAB2, LAB3 and the Mix group were reduced compared to the DSS group and were significantly different, with the Mix group being the most reduced. Shows that the lactobacillus acidophilus KLDS1.0901, the lactobacillus helveticus KLDS1.8701, the lactobacillus plantarum KLDS1.0318 and the mixed lactobacillus can reduce the concentration of D-lactic acid in serum and improve the intestinal mucosa permeability of mice.

2.2.10 mouse Colon tissue Claudin mRNA expression levels

The expression level of claudin mRNA in the colon of mice is shown in FIG. 17. Compared with the NC group, the DSS can obviously reduce the ZO-1, Occludin and Claudin-1 transcription levels (P <0.05), and has no obvious effect on the E-cadherin transcription level. Each intervention increased the level of claudin transcription to varying degrees. LAB1 and LAB2 were able to significantly increase ZO-1 and Occludin transcription levels (P >0.05), but were not significant for the Claudin-1 effect; both LAB3 and mixed lactobacillus can obviously improve the transcription levels of ZO-1, Occludin and Claudin-1 in colon of mice (P <0.05), wherein the mixed lactobacillus has the most obvious improvement effect. The results show that the mixed lactobacillus can most effectively improve the colon tight junction protein of the mice and improve the intestinal barrier function of the mice.

2.2.11 mouse Colon tissue mucin mRNA expression level

The expression level of mRNA of mucin (MUC1 and MUC2) in colon tissues of each group of mice is shown in FIG. 18, and the expression of the mucin MUC1 and MUC2 in the DSS group is remarkably reduced compared with that in the NC group (P < 0.05). Compared with the DSS group, the transcriptional levels of the colon tissues MUC1 and MUC2 of the mice of the SASP, LAB1, LAB2, LAB3 and Mix groups were significantly increased (P < 0.05). The mixed lactobacillus group MUC1 expression has no significant difference with the NC group (P <0.05), and MUC2 expression is higher than the NC group (P < 0.05). The results show that the mixed lactobacillus can improve the mucin expression of the colon tissue most obviously.

2.2.12 Structure and composition of intestinal flora

2.2.12.1 differences in gut microbial structure at the phylum level

At the phylum level, Bacteroides (Bacteroides), Firmicutes (Firmicutes), Proteobacteria (Proteobacteria) and Verrucomicrobia (Verrucomicrobia) are included. Compared with the NC group, the relative abundance of bacteroidetes and proteobacteria in intestinal tracts of the mice in the DSS group is increased, and the relative abundance of firmicutes and verrucomicrobia is reduced. Compared with the DSS group, the LAB1 group, the LAB2 group, the LAB3 group and the Mix group can reduce proteobacteria in intestinal tracts of mice, and the LAB1 group and the Mix group can improve the firmicutes and reduce the relative abundance of bacteroidetes. It can be seen that intervention with lactobacilli can improve the intestinal flora of mice at phylum level.

3.3.12.1 differences in gut microbial structure at genus level

In the intestinal microbial composition at the genus level, the abundance of Escherichia coli (Escherichia-Shigella), arbuscular (Alistipes), ri — sregniaceae RC9 (Rikenellaceae RC9), Bacteroides (Bacteroides) was increased in the intestine of mice in the DSS group compared to the NC group, while the abundance of these genera was decreased to various degrees in all of the LAB1 group, LAB2, LAB3, Mix group; however, the relative abundance of lachnospira (Lachnospiraceae NK4a136group), spirillum (Helicobacter), ruminal clostridium9 (ruministrodium 9), Akkermansia (Akkermansia) was reduced in the intestinal tract of mice in the DSS group compared to the NC group, whereas the relative abundance of Lachnospiraceae NK4a136group was increased in the intervention groups other than the LAB2 group and increased in all intervention groups compared to the DSS group. It is known that lactobacillus intervention can effectively reverse the changes in the microbial structure of colitis mice at the genus level.

2.2.13 short chain fatty acid content in the intestinal tract

As shown in FIG. 19, the TIC chart shows that each metabolite is well separated in color spectrum, and the peak shape is sharp and symmetrical, and mass spectrum quantification of each metabolite can be performed.

The results of the calibration curve for short fatty acids are shown in Table 4. And respectively calculating linearity and correlation coefficients of the components according to the proportion concentration ratio of the components in the total concentration by taking the concentration ratio of each component of the standard variety to the internal standard as the abscissa and the peak area ratio as the ordinate so as to investigate the linearity of the standard solution. The result shows that the linearity of each object to be detected in the linear range is good, and the correlation coefficient is larger than 0.99.

TABLE 4 Standard Curve Table of SCFAs

As shown in Table 5, compared with the NC group of mice, the contents of acetic acid, butyric acid and total acid in the caecum contents of the mice in the DSS group are obviously reduced (P is less than 0.05), and the contents of propionic acid, isobutyric acid, valeric acid and isovaleric acid are not obviously changed. The concentrations of acetic acid, butyric acid and total acid in the cecal contents of the mice after lactobacillus drying pretreatment were increased to different degrees compared with the DC group, wherein the acetic acid content in the mixed lactobacillus group was significantly different from that in the DSS group, but not significantly different from that in the NC group. The results show that the intervention of lactobacillus can increase the content of SCFAs in intestinal contents of mice, and the mixed lactobacillus has better effect than a single strain.

TABLE 5 SCFAs content table

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. Mixed lactobacillus for preventing and treating ulcerative colitis comprises lactobacillus acidophilus KLDS1.0901, lactobacillus helveticus KLDS1.8701 and lactobacillus plantarum KLDS 1.0318.

2. The lactobacillus mixtarus according to claim 1, wherein the lactobacillus acidophilus KLDS1.0901, lactobacillus helveticus KLDS1.8701 and lactobacillus plantarum KLDS1.0318 are in a volume ratio of 1:1: 1; the MOI value of the mixed lactobacillus is 0.1-10.

3. Use of a lactobacillus cocktail according to any of claims 1-2 in the preparation of a medicament for preventing ulcerative colitis.

4. The use according to claim 3, for the manufacture of a medicament for increasing colon length, decreasing spleen index, decreasing MPO enzyme activity, inhibiting COX-2 transcription, inhibiting secretion of PGE2, and decreasing levels of pro-inflammatory cytokines; the proinflammatory cytokines include TNF-alpha, IL-1 beta and IL-6.

5. Use of a lactobacillus cocktail according to any of claims 1-2 in the preparation of a medicament for treating ulcerative colitis.

6. Use according to claim 5, for the manufacture of a medicament for improving colonic mucosal permeability.

7. The use according to claim 5, for the manufacture of a medicament for improving gut barrier function and increasing expression of colonic claudin.

8. The use according to claim 7, for the preparation of a medicament for increasing the expression of mucins MUC1 and MUC2 in colon tissue.

9. Use according to claim 5, for the preparation of a medicament for increasing the content of short chain fatty acids, SCFAs, in the intestinal contents; the short chain fatty acids SCFAs include acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, and isovaleric acid.

10. Use according to claim 5, for the preparation of a medicament for regulating the microbiota balance on the intestinal mucosa; the microbiota balance includes increasing firmicutes, decreasing bacteroidetes relative abundance, and increasing pilospirillum and ruminal clostridium9 relative abundance in the gut.