CN112481175A - Lactobacillus rhamnosus capable of preventing and relieving ulcerative colitis and application thereof - Google Patents
Lactobacillus rhamnosus capable of preventing and relieving ulcerative colitis and application thereof Download PDFInfo
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- CN112481175A CN112481175A CN202011596053.6A CN202011596053A CN112481175A CN 112481175 A CN112481175 A CN 112481175A CN 202011596053 A CN202011596053 A CN 202011596053A CN 112481175 A CN112481175 A CN 112481175A
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Abstract
The invention discloses lactobacillus rhamnosus capable of preventing and relieving ulcerative colitis and application thereof, and belongs to the technical field of functional microorganisms. The lactobacillus rhamnosus CCFM1128 can tolerate the gastrointestinal environment of a human body, remarkably improves the weight loss during the diseased period of ulcerative colitis, improves the stool property and the hematochezia condition, reduces the injury of colonic mucosa, reduces the MPO activity, reduces the generation of proinflammatory factors TNF-alpha, IL-1 beta, IL-6 and IFN-gamma in colon, reduces the content of the proinflammatory factors IL-6 in serum, up-regulates the gene transcription level of colon tight junction related proteins Claudin-3, ZO-1, ZO-2 and Occludin, up-regulates the transcription level of antimicrobial peptide CRAMP in colon, improves the content of short-chain fatty acids in stool, improves the diversity of intestinal flora, and improves the abundance of the intestinal short-chain fatty acid producing bacteria Coprococcus in intestinal tract.
Description
Technical Field
The invention discloses lactobacillus rhamnosus capable of preventing and relieving ulcerative colitis and application thereof, and particularly relates to application of lactobacillus rhamnosus CCFM1128 in preparation of a functional microbial inoculum, food and/or a medicament, belonging to the technical field of functional microorganisms.
Background
Inflammatory Bowel Disease (IBD) is a common autoimmune Disease of the digestive system in western countries, with the incidence of Disease as high as 5-12 patients per hundred thousand population. With the increase of the living pressure of Chinese people and the change of the dietary regularity, the incidence of inflammatory bowel diseases in China is increasing day by day and is improved by more than 15 times compared with 20 years ago, so that more and more research institutions and scholars deeply explore the pathogenesis and treatment method of IBD. Ulcerative Colitis (UC) is a form of IBD, and the main clinical manifestations are chronic or subacute diarrhea, mucopurulent bloody stool and abdominal pain. UC is a continuous inflammation of the mucosal and submucosal layers of the colon, and the disease usually begins to spread from the rectum to the entire colon. The tight connection of intestinal tracts at the lesion is broken, and the expression quantity of proteins important in intestinal barriers such as transmembrane proteins Claudins, Ocplus and intracellular proteins ZO families (including ZO-1, ZO-2 and ZO-3) is reduced. The disease is seen at any age, but is most seen in 20-30 years old. In recent decades, with the economic development, UC has become a global disease, and the incidence rate of UC in china has also shown a rapid increase trend. UC has long course and easy recurrence, which not only affects the life quality of patients, but also increases the risk of intestinal cancer of patients. At present, the pathogenesis of UC is not clear, and researches show that UC is mainly related to immune factors, genetic factors and environmental factors. In recent years, more and more researches show that the incidence of UC is directly related to the abnormality of intestinal flora, so that the improvement of the intestinal flora structure of patients also becomes an important means for treating UC.
UC has unclear etiology and is difficult to cure. Aiming at the treatment of UC, the traditional medicines comprise mesalamine, sulfasalazine, hydrocortisone, prednisolone, azathioprine, methotrexate and the like, and the traditional medicines are mainly used for interfering in inflammatory bowel diseases by inhibiting signal pathways related to inflammation and regulating pathways for removing free radicals, oxides and the like, and are respectively suitable for treatment methods of patients with UC of different degrees. Patients with severe conditions are also treated directly by surgery to avoid organ necrosis or cancer. However, the surgical method only treats patients with UC from the viewpoint of relieving clinical symptoms to maintain the quality of life of patients and prevent other complications, and does not fundamentally solve the cause of UC. Because UC is a highly recurrent immune disease, long-term administration of chemically synthesized drugs can cause a large burden and a large number of side effects on the body of a patient. For the colon with serious pathological changes treated by surgical operation, there may be sequelae of intestinal obstruction, dysfunction, mucosa retraction at the ileostomy site, etc. after the operation. Therefore, in order to reduce the risk of complications of patients and improve the treatment efficiency, a safe and effective treatment and alleviation method applied to the long-term treatment process is urgently needed. In recent years, some special probiotic strains have been proposed to play a beneficial role in intervening in UC. Probiotics, as a class of microorganisms beneficial to human health, can exert beneficial health effects by changing intestinal microecology and other ways when a certain number of live probiotics are planted in host intestinal tracts, and have been applied to animal production as a substitute for antibiotic growth promoters. The lactobacillus is the most common probiotic bacteria, is safe and edible, has no side effect, and a large number of researches show that some special lactobacillus strains have good effects of resisting inflammation and regulating intestinal flora, but the effects have obvious individual difference in the lactobacillus. Therefore, some special strains with anti-inflammatory, intestinal barrier repair and antibacterial peptide production induction are screened in lactobacillus, and can be used for preventing or assisting in treating UC.
Disclosure of Invention
The invention provides Lactobacillus rhamnosus (Lactobacillus rhamnosus) CCFM1128 which is preserved in Guangdong province microorganism strain preservation center at 7-24 th of 2020 with the preservation number of GDMCC No. 61099.
The invention provides a microbial inoculum containing lactobacillus rhamnosus CCFM1128 disclosed in claim 1.
In one embodiment of the invention, the preparation method of the microbial inoculum comprises the following steps: culturing Lactobacillus rhamnosus CCFM1128 in MRS culture medium, culturing at 35-39 deg.C under anaerobic condition for 18-24 hr, washing with phosphate buffer solution with pH of 7.0-7.4 for 2-4 times, and resuspending with the protectant to make the bacteria concentration reach 1010CFU/mL; then, pre-culturing the suspension liquid at 37 ℃ for 50-70min under the anaerobic condition, then pre-freezing for 8-14h at-15 to-20 ℃, and carrying out vacuum freeze drying to obtain the fermentation inoculum.
In one embodiment of the invention, the lyoprotectant is 100g/L to 150g/L skimmed milk powder, and/or 100g/L to 150g/L maltodextrin, and/or 140g/L to 160g/L trehalose.
The invention provides application of the lactobacillus rhamnosus or the microbial inoculum in preparing products for preventing, relieving or improving ulcerative colitis symptoms.
In one embodiment of the invention, the product comprises a functional fermented food product, a pharmaceutical or a pharmaceutical composition.
In one embodiment of the present invention, the functional fermented food includes solid food, liquid food, semi-solid food.
In one embodiment of the invention, the fermented food comprises dairy products, bean products, fruit and vegetable products, and the dairy products comprise yoghourt, cream and cheese; the fruit and vegetable products comprise cucumber, carrot, beet, celery and cabbage products.
In one embodiment of the present invention, the medicament or pharmaceutical composition is applied to at least one of (a) to (e):
(a) reducing weight loss during the period of ulcerative colitis, improving stool character and hematochezia;
(b) regulating the transcription level of colon tight junction related protein and colon antibacterial peptide, and improving colon mucosa injury;
(c) reducing myeloperoxidase activity;
(d) reducing pro-inflammatory factors in colon and serum;
(e) improving short chain fatty acid content, improving intestinal flora, and improving intestinal flora diversity.
In one embodiment of the invention, the proinflammatory factors in the colon and serum comprise TNF- α, IL-1 β, IL-6, IFN- γ.
In one embodiment of the invention, the colon tight junction-related proteins include Claudin-3, ZO-1, ZO-2 and Occludin; the colonic antimicrobial peptides include CRAMP.
In one embodiment of the invention, the short chain fatty acids comprise acetic acid, butyric acid and/or propionic acid.
In one embodiment of the invention, improving the gut flora comprises increasing the abundance of short chain fatty acid producing bacteria Coprococcus in the gut
In one embodiment of the invention, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.
The invention has the beneficial effects that: the lactobacillus rhamnosus CCFM1128 provided by the invention can tolerate the gastrointestinal environment of a human body, remarkably reduce the weight loss during the illness period of ulcerative colitis, improve the stool character and the hematochezia condition, improve the injury of colonic mucosa, reduce the MPO activity, reduce the generation of proinflammatory factors TNF-alpha, IL-1 beta, IL-6 and IFN-gamma in colon, reduce the content of the proinflammatory factor IL-6 in serum, up-regulate the gene transcription level of colon tight junction related proteins Claudin-3, ZO-1, ZO-2 and Occludin, up-regulate the transcription level of colon antimicrobial peptide CRAMP, improve the content of short chain fatty acids in stool, improve the diversity of intestinal flora and improve the abundance of the Coprococcus generated by the short chain fatty acids in the intestinal tract.
The lactobacillus rhamnosus CCFM1128 can be used for preparing dairy products, bean products and fruit and vegetable products for preventing and/or assisting in treating ulcerative colitis. Can also be used for preparing probiotic preparations and medicaments for preventing and/or assisting in treating the ulcerative colitis, is used for assisting in improving the symptoms of the ulcerative colitis, and has very wide application prospect.
Biological material preservation
The Lactobacillus rhamnosus CCFM1128 provided by the invention is classified and named as Lactobacillus rhamnous, is preserved in Guangdong province microorganism strain preservation center in 7-24.2020, and is addressed to No. 59 floor 5 of Michelia Tokyo No. 100 of Guangzhou city, and the preservation number is GDMCC No: 61099.
drawings
FIG. 1 is a colony morphology diagram of Lactobacillus rhamnosus CCFM 1128;
FIG. 2 is a graph of the effect of Lactobacillus rhamnosus CCFM1128 on UC mouse body weight;
FIG. 3 is a graph of the effect of Lactobacillus rhamnosus CCFM1128 on colonic mucosa injury in UC mice;
FIG. 4 is a graph of the effect of Lactobacillus rhamnosus CCFM1128 on UC mouse colon histopathology score and colon MPO activity;
FIG. 5 is a graph showing the effect of Lactobacillus rhamnosus CCFM1128 on the proinflammatory factor content in the colon and serum of UC mice;
FIG. 6 is a graph of the effect of Lactobacillus rhamnosus CCFM1128 on UC mouse colon tight junction-related proteins;
FIG. 7 is a graph showing the effect of Lactobacillus rhamnosus CCFM1128 on the transcription level of antimicrobial peptide CRAMP in the colon of UC mice;
FIG. 8 is a graph of the effect of Lactobacillus rhamnosus CCFM1128 on the short chain fatty acid content in UC mouse feces;
FIG. 9 is a graph showing the effect of Lactobacillus rhamnosus CCFM1128 on the diversity of UC mouse intestinal flora (shannon index);
FIG. 10 is a graph of the effect of Lactobacillus rhamnosus CCFM1128 on the abundance of the genus Coprococcus in the UC mouse gut.
Detailed Description
Lactobacillus rhamnosus CCFM1128 has the following biological properties:
(1) the characteristics of the thallus are as follows: gram-positive, non-sporulating, immotile bacteria.
(2) Colony characteristics: aerobic or anaerobic culture for 36 hr to form obvious colony of 0.5-2mm diameter, round front shape, raised side shape, uneven edge, milky white, opaque, moist and smooth surface, and no pigment, as shown in figure 1.
(3) Growth characteristics: under aerobic or anaerobic conditions at a constant temperature of 37 ℃, the medium was cultured in mrss medium for about 15 hours to the end of log.
(4) Has better tolerance to simulated gastrointestinal fluid.
The method for obtaining the strain comprises the following steps:
(1) 1g of fresh faeces from healthy persons were taken. Enriching the sample in a culture medium containing sorbitol GM17 at 35 ℃ for 12 h;
(2) performing gradient dilution on the enriched sample, then coating the enriched sample on a GM17 solid plate added with 0.02% of olcresol purple, and culturing for 24-48 h;
(3) selecting single bacterial colony with obvious color changing circle and according with the basic morphology of lactobacillus, carrying out plate streaking purification, and screening and separating out lactobacillus;
(4) and culturing the single colony in a liquid GM17 culture solution for 24h, performing gram staining, and selecting gram-positive bacteria for subsequent tests.
Lactobacillus rhamnosus FNMGEL 5-1: is described in Huangdan graduation paper, research on comparative genome and partial physiological and biochemical characteristics of Lactobacillus rhamnosus in human intestinal tract, 2019.
mMRS culture medium: MRS medium +0.05g/100mL cysteine hydrochloride.
Example 1: the lactobacillus rhamnosus CCFM1128 has good tolerance to simulated gastrointestinal fluid
Inoculating the refrigerated lactobacillus rhamnosus CCFM1128 into a mMRS culture medium (MRS culture medium + 0.05% cysteine hydrochloride), carrying out anaerobic culture at 37 ℃ for 48h, and carrying out subculture for 2-3 times by using a mMRS culture solution to obtain a culture solution of the lactobacillus rhamnosus CCFM 1128.
Taking 1mL of culture solution of lactobacillus rhamnosus CCFM1128, mixing with 9.0mL of artificial simulated gastric juice (mMRS culture medium containing 1% pepsin and pH 2.5), carrying out anaerobic culture at 37 ℃, sampling at 0h, 0.5h, 1h, 2h and 3h respectively, carrying out pouring culture by using mMRS agar culture medium, carrying out plate colony counting, measuring the viable count and calculating the survival rate. The survival rate is the ratio of the logarithmic viable count at the sampling time to the logarithmic viable count at the 0h time in the culture solution, and is expressed by%.
Adding 1mL of culture solution of lactobacillus rhamnosus CCFM1128 into 9mL of artificial simulated intestinal fluid (mMRS culture medium containing 0.3% of bovine bile salt, 1% of trypsin and pH 8.0), anaerobically culturing at 37 ℃, sampling at 0h, 0.5h, 1h, 2h, 3h and 4h respectively, pouring and culturing by using mMRS agar culture medium for counting plate colonies, measuring the viable count and calculating the survival rate.
The results of the experiment are shown in tables 1 and 2. The result shows that the lactobacillus rhamnosus CCFM1128 has better tolerance to the artificial gastrointestinal fluids.
TABLE 1 tolerance of Lactobacillus rhamnosus CCFM1128 in simulated gastric juice
TABLE 2 tolerance of Lactobacillus rhamnosus CCFM1128 in artificially simulated intestinal fluids
Example 2: the lactobacillus rhamnosus CCFM1128 has no toxic or side effect on C57BL/6J mice
Resuspending Lactobacillus rhamnosus CCFM1128 in 3g/100mL sucrose solution to make 5.0 × 109CFU/mL of bacterial suspension. 6 healthy male C57BL/6J mice with the weight of 14-16g are taken, after the mice are adapted to the environment for one week, the bacterial suspension with the concentration is administered for intragastric administration once a day (0.2 mL of intragastric administration is performed every time), and the death and weight conditions are recorded after one week of observation.
The results of these tests are listed in table 3. These results show that the feed concentration was 5.0X 109The CFU/mL lactobacillus rhamnosus CCFM1128 does not cause obvious influence on mice, and the weight of the mice has no obvious change and no death phenomenon. The mice had no apparent pathological symptoms in appearance.
TABLE 3 weight change and mortality in mice
| Time (sky) | 1 | 2 | 3 | 4 | 5 | 6 | 7 |
| Body weight (g) | 19.22±0.10 | 19.40±0.21 | 19.65±0.28 | 19.82±0.33 | 20.08±0.25 | 20.30±0.36 | 20.54±0.31 |
| Death situation | - | - | - | - | - | - | - |
Note: -: mice did not die
Example 3: influence of Lactobacillus rhamnosus CCFM1128 on UC mouse disease symptoms
30 healthy male C57BL/6J mice weighing 14-16g were acclimated for 1 week, 6 mice per group were randomized into 5 groups: blank group, model group, drug group, lactobacillus rhamnosus CCFM1128 intervention group (CCFM1128), lactobacillus rhamnosus FNMGEL5-1 control group (FNMGEL 5-1). The dosage of the gavage bacteria suspension is 5.0 × 109CFU/mL, resuspended in 3% (w/v) sucrose solution. Dextran Sodium Sulfate (DSS) was added to the drinking water at a final concentration of 2.5g/100mL (i.e., 2.5% (w/v)) at week 5 to induce colitis in mice. The grouping and treatment methods of the experimental animals are shown in Table 4:
TABLE 4 animal Experimental design
In the fifth week, i.e., the molding period (DSS treatment period), the body weight of the mice was regularly weighed every day and the percentage change thereof was calculated. In addition, the fecal characteristics of the mice were observed daily and classified into three grades: firstly, the excrement of a normal mouse is formed and is granular; second, stool is viscous and loose, but is "loose" without adhering to the anus; thirdly, the feces are watery or unformed and adhere to the anus, i.e. "loose feces". Meanwhile, the condition of occult blood of the mouse feces is measured by using a pilamitraz hole method, and if the mouse feces contains macroscopic reddish brown or bright red blood, macroscopic hematochezia is judged. Finally, the Disease Activity Index (DAI) of the mice was calculated according to the body weight, stool characteristics and hematochezia of the mice, and the scores of specific scoring items are shown in table 5.
TABLE 5 animal Disease Activity Index (DAI) scoring System
TABLE 6 disease Activity index
During modeling, the mice in the UC model group have obvious hematochezia, loose feces and weight loss, as shown in figure 2, the weight of the mice in the model group is remarkably reduced from 5 days, the weight loss rate is close to 10% on the seventh day, in addition, the disease activity index of the mice in the model group is remarkably increased from 4 days of modeling, and is increased to 2.72 +/-0.14 (table 7) on 7 days, and the intake of the lactobacillus rhamnosus CCFM1128 can remarkably reduce the weight loss of the mice in the UC, improve the feces character and the hematochezia condition and reduce the DAI value, which indicates that the lactobacillus rhamnosus CCFM1128 screened by the invention has the function of relieving the symptoms of the mice in the UC.
Example 4: application of lactobacillus rhamnosus CCFM1128 in improvement of colonic mucosa injury of mice
The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 3.
After mice were sacrificed on day 36, mouse colon tissues were collected, colonic MPO activity was determined according to MPO kit instructions, colonic paraffin sections were made and HE stained, the experimental steps were: (1) fixing a section of distal colon 1cm away from anus 1cm for 48h with 4% paraformaldehyde; (2) washing the fixed colon tissue with running water for 8h, dehydrating, sequentially dehydrating the sample with 70%, 80% and 90% ethanol solutions for 30min, respectively, and placing the sample at 95% and 100% for 2 times, each for 20 min; (3) putting the colon sample into a mixed solution of xylene and alcohol at a ratio of 1:1 for 15min, and then putting xylene I and xylene II into the mixed solution for 15min respectively; (4) transferring the colon tissue to mixed liquid of xylene and paraffin for 15min, adding paraffin I and paraffin II, and waxing for 1h respectively, and keeping the temperature at 60 ℃; (5) embedding the colon in a re-melted wax block by using a leica paraffin embedding machine; (6) slicing the embedded tissue by a tissue slicer to a thickness of 5 μm; (7) drying after sticking, and placing in an oven at 62 ℃ for 1 h.
HE staining procedure was as follows: (1) dewaxing the paraffin sections by dimethylbenzene I and dimethylbenzene II for 5min respectively, then putting the paraffin sections into 100%, 95%, 90%, 80% and 70% alcohol solutions for 3-5min respectively, and then putting the paraffin sections into distilled water for 3 min; (2) staining with hematoxylin for 20 s; (3) washing away unbound hematoxylin with distilled water; (4) eosin is dyed for 2s, the eosin is sequentially added into 95% ethanol I, II and 70% ethanol, and is quickly taken out, and then the eosin is added into 80% ethanol for 50-55 s and is added into absolute ethanol for 2 min; (5) putting the slices into a mixed solution of xylene and alcohol with a ratio of 1:1 for 1min, and then putting the slices into xylene I and xylene II for 2-3 min respectively; (6) encapsulating with neutral gum.
The results of the experiment are shown in fig. 3 and 4. As can be seen from FIG. 3, a large amount of degeneration and necrosis of mucosal epithelial cells can be seen in the colon of the model mouse, the number of goblet cells is obviously reduced, pathological changes such as intestinal crypt injury, inflammatory cell infiltration and the like are observed, the colon tissue injury score is obviously higher than that of a blank control group (FIG. 4), in addition, the colon MPO value is obviously increased (FIG. 4), the transgastric rhamnosus CCFM1128 obviously reduces the MPO activity of the model mouse and is close to that of the blank control group, the colon tissue injury score is obviously reduced, and the colon structure is close to that of the blank control group. The result shows that the lactobacillus rhamnosus CCFM1128 can obviously improve the colonic mucosa injury of UC mice, and the effect is superior to that of the medicines mesalazine and lactobacillus rhamnosus FNMGEL 5-1.
Example 5: application of lactobacillus rhamnosus CCFM1128 in reducing proinflammatory factor content in colon and serum
The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 3.
After mice were sacrificed on day 36, mouse blood and colon tissue were collected. Centrifuging mouse blood at 3000 Xg and 4 deg.C for 10min, collecting supernatant, and determining IL-6 content in serum according to IL-6 enzyme linked immunosorbent assay kit detection method. Adding precooled PBS buffer solution into colon tissues of a mouse according to the proportion of 1:9 for tissue grinding, 12000 Xg, centrifuging for 15min, taking supernatant, and respectively determining the contents of TNF-alpha, IL-1 beta, IL-6 and IFN-gamma in the colon according to the detection method of the TNF-alpha, IL-1 beta, IL-6 and IFN-gamma ELISA kit.
The experimental result is shown in figure 5, the contents of proinflammatory factors TNF-alpha, IL-1 beta, IL-6 and IFN-gamma in the colon of a model group mouse are obviously increased, the content of IL-6 in serum is obviously increased, the intragastric rhamnose lactobacillus CCFM1128 obviously reduces the proinflammatory factors TNF-alpha, IL-1 beta, IL-6 and IFN-gamma in the colon of the mouse, simultaneously obviously reduces the content of IL-6 in the serum, and the effect is superior to that of the drug mesalazine.
Example 6: application of lactobacillus rhamnosus CCFM1128 in improving transcription levels of colon tight junction related protein and antibacterial peptide
The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 3.
After the mice were sacrificed on day 36, mouse colon tissues were collected to determine the transcription levels of Claudin-3, ZO-1, ZO-2, Occludin and the antimicrobial peptide CRAMP in the colon. The measurement method is as follows: the primer sequences of Claudin-3, ZO-1, ZO-2, Occludin, CRAMP and GAPDH were synthesized, and the primer information is shown in Table 6. 1cm of colon tissue of the same part of a mouse is taken and quickly placed into liquid nitrogen, the liquid nitrogen is placed into a refrigerator at minus 80 ℃ for freezing, the frozen colon tissue is taken out and placed into a 1.5mL enzyme-free centrifuge tube added with 1mL of TRIzol and 3 zirconium beads, the homogenate is fully carried out by a tissue grinding and homogenizing machine, and the standing is carried out for 5min at room temperature. 0.2mL of chloroform was added, followed by vigorous shaking for 30s and standing for 10 min. Followed by centrifugation at 12000g for 15min at 4 ℃. Carefully suck the upper aqueous phaseAdding into a new enzyme-free 1.5mL centrifuge tube, adding isopropanol with the same volume, slightly reversing the upper part and the lower part, mixing, and standing at room temperature for 10 min. Followed by centrifugation at 12000g for 15min at 4 ℃. The supernatant was discarded, 1mL of pre-cooled 75% ethanol was added, and the pellet was washed gently. Centrifuging at 4 deg.C for 5min at 12000g, carefully sucking to remove supernatant, blow-drying the precipitate in a super clean bench, and adding 50 μ L of enzyme-free ultrapure water to dissolve RNA. Determination of the extracted RNA concentration, OD, Using a Microspectrophotometer260/OD280The quality is qualified between 1.9 and 2.0. And (3) synthesizing cDNA by taking the total RNA with qualified extraction quality as a template according to the steps of the reverse transcription kit instruction. Carrying out q RT-PCR detection on cDNA obtained by reverse transcription, wherein a PCR system comprises: 5 μ L SYBR Green Supermix, 3 μ L deionized water, 0.5 μ L forward primer (10 μmol/L), 0.5 μ L reverse primer (10 μmol/L) and 1 μ L cDNA template (100ng/μ L). qPCR run program settings: 94 ℃ for 2min, (94 ℃, 30 s; 61 ℃, 30 s; 72 ℃, 20s)39 cycles; after the target gene is detected by Real-time PCR, GAPDH is taken as an internal reference gene, and 2 is adopted-ΔΔCTThe method carries out relative gene expression analysis.
The results of the experiment are shown in FIGS. 6 and 7. As can be seen from figure 6, four main tight junction proteins Claudin-3, ZO-1, ZO-2 and Occludin in the colons of the mice in the model group are all obviously reduced, and the transgastric rhamnosus CCFM1128 obviously improves the transcription levels of the four tight junction related proteins in the colons, and the effect of the drug is superior to that of mesalazine. As can be seen from FIG. 7, the transcriptional level of the antimicrobial peptide CRAMP in the colon is significantly increased after the intervention of Lactobacillus rhamnosus CCFM1128, and the relative mRNA transcriptional level is about 3 times that of the blank group.
Primers used in Table 7
Example 7: lactobacillus rhamnosus CCFM1128 can increase content of short-chain fatty acids in UC mouse feces
The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 3.
Fresh feces of mice collected at the end of the experiment were frozen at-80 ℃.The feces were first lyophilized, 50mg of feces were weighed, resuspended in 500. mu.L of saturated NaCl solution, and 20. mu.L of 10% H was added2SO4Acidifying; adding 800 μ L anhydrous ether, shaking, extracting fatty acid, and centrifuging 18000g for 15 min; taking the upper layer of ether phase, adding 0.25g of anhydrous Na2SO4Drying is carried out; standing for 30min, centrifuging at 18000g for 10min, collecting the upper diethyl ether phase, and determining short chain fatty acid content in lyophilized feces of mice by GC-MS. An Rtx-Wax column (column length 30m, inner diameter. 25 μm) was used; the carrier gas is He, and the flow rate is 2 mL/min; the sample introduction volume is 1 mu L, the temperature is increased to 140 ℃ according to the speed of 7.5 ℃/min, then the temperature is increased to 200 ℃ according to the speed of 60 ℃/min, the temperature is kept for 3min, and the ionization temperature is 20 ℃; the analysis adopts a full scanning mode, and the concentration of each short-chain fatty acid is calculated by obtaining a standard curve through an external standard method.
The results of the experiment are shown in FIG. 8. The experimental results show that compared with the blank group, the total amount of short-chain fatty acids in the feces of the model group mice is reduced, and particularly, the content of butyric acid is obviously reduced; the intragastric administration treatment of the lactobacillus rhamnosus CCFM1128 obviously improves the content of short-chain fatty acids in the mouse excrement, including acetic acid, propionic acid, butyric acid and the content of total short-chain fatty acids, and the effect is superior to that of the medicaments mesalazine and lactobacillus rhamnosus FNMGEL 5-1.
Example 8: lactobacillus rhamnosus CCFM1128 capable of improving UC mouse intestinal flora diversity
The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 3.
At the end of the test, fresh mouse excrement is collected and frozen at-80 ℃, DNA in the excrement is extracted by using a DNA rapid extraction kit according to the steps of the instruction method, the V3-V4 region of the 16S rDNA of the sample is subjected to PCR amplification, the amplified fragment is purified and recovered by using a gel recovery kit, high-throughput sequencing is carried out on an Illumina misseq pe300 platform, and the amplicon is analyzed by using a QIIME2 analysis platform.
PCR amplification primers:
F：5’-AYTGGGYDTAAAGNG-3’，SEQ ID NO.13；
R：5’-TACNVGGGTATCTAATCC-3’，SEQ ID NO.14。
the results of the experiment are shown in FIG. 9. The experimental results show that compared with the blank group, the shannon index of the mice in the model group is obviously reduced, which indicates that the diversity of intestinal flora of the DSS colitis mice is obviously reduced. After intervention of lactobacillus rhamnosus, the shannon index is obviously improved, which shows that the lactobacillus rhamnosus can improve the diversity of intestinal flora of colitis mice.
Example 9: lactobacillus rhamnosus CCFM1128 can improve the abundance of short-chain fatty acid bacteria Coprococcus in UC mouse intestinal tract
The grouping, modeling and handling methods of the C57BL/6J mice were the same as in example 3.
At the end of the test, fresh mouse excrement is collected and frozen at-80 ℃, DNA in the excrement is extracted by using a DNA rapid extraction kit according to the steps of the instruction method, the V3-V4 region of the 16S rDNA of the sample is subjected to PCR amplification, the amplified fragment is purified and recovered by using a gel recovery kit, high-throughput sequencing is carried out on an Illumina misseq pe300 platform, and the amplicon is analyzed by using a QIIME2 analysis platform (primers are shown as SEQ ID NO.13 and 14).
The experimental results are shown in FIG. 10. The experimental result shows that compared with a normal control group, the abundance of the genus Coprococcus which produces the short-chain fatty acid in the mouse feces of the model group is remarkably reduced, the lactobacillus rhamnosus CCFM1128 after gastric perfusion can improve the flora change caused by DSS, and the abundance of the genus Coprococcus which produces the short-chain fatty acid in the UC mouse feces is remarkably improved.
Example 10: application of lactobacillus rhamnosus CCFM1128 in preparation of fermented food
Preparation of a microbial inoculum: culturing Lactobacillus rhamnosus CCFM1128 in MRS culture medium, culturing at 35-39 deg.C under anaerobic condition for 18-24 hr, washing with phosphate buffer solution with pH of 7.0-7.4 for 2-4 times, and resuspending with the protectant to make the bacteria concentration reach 1010CFU/mL; then, pre-culturing the suspension liquid for 50-70min under the anaerobic condition at the temperature of 37 ℃, pre-freezing for 8-14h at the temperature of-15 to-20 ℃, and carrying out vacuum freeze drying to obtain the zymophyte agent; the freeze-drying protective agent is 100g/L-150g/L skimmed milk powder, and/or 100g/L-150g/L maltodextrin, and/or 140g/L-160g/L trehalose.
Selecting fresh vegetables, cleaning, squeezing to obtain juice, performing high temperature instant sterilization, and performing sterilization at 140 deg.CAfter high-temperature heat sterilization is carried out for 2 seconds at the temperature of 37 ℃, the temperature is immediately reduced to 37 ℃, and then the lactobacillus rhamnosus CCFM1128 or the microbial inoculum leavening agent containing the lactobacillus rhamnosus CCFM1128 prepared by the invention is inoculated to ensure that the concentration of the lactobacillus rhamnosus CCFM1128 reaches 106More than CFU/mL, and refrigerating and storing at the temperature of 4 ℃ to obtain the fruit and vegetable beverage containing the live lactobacillus rhamnosus CCFM1128 bacteria.
The lactobacillus rhamnosus CCFM1128 can also be fermented to produce other fermented foods, wherein the fermented foods comprise yoghourt, cream, cheese, cucumber, carrot, beet, celery, cabbage products and the like.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
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Claims (10)
1. Lactobacillus rhamnosus (Lactobacillus rhamnosus) is preserved in Guangdong province microorganism strain collection center in 7-24 th of 2020 with the preservation number GDMCC No: 61099.
2. a microbial preparation comprising the Lactobacillus rhamnosus strain of claim 1.
3. Use of lactobacillus rhamnosus according to claim 1 or the bacterial agent according to claim 2 for the preparation of a product for preventing, alleviating or ameliorating the symptoms of ulcerative colitis.
4. Use according to claim 3, wherein the product comprises a functional fermented food product, a pharmaceutical or a pharmaceutical composition.
5. The use according to claim 4, wherein the functional fermented food comprises solid food, liquid food, semi-solid food.
6. The use according to claim 5, wherein the medicament or pharmaceutical composition is for use in at least one of (a) - (e):
(a) reducing weight loss during the period of ulcerative colitis, improving stool character and hematochezia;
(b) regulating the transcription level of colon tight junction related protein and colon antibacterial peptide, and improving colon mucosa injury;
(c) reducing myeloperoxidase activity;
(d) reducing pro-inflammatory factors in colon and serum;
(e) improving short chain fatty acid content, improving intestinal flora, and improving intestinal flora diversity.
7. The use according to claim 6, wherein the proinflammatory factors in the colon and serum comprise TNF- α, IL-1 β, IL-6, IFN- γ.
8. The use according to claim 7, wherein the colon tight junction-related proteins comprise Claudin-3, ZO-1, ZO-2 and Occludin; the colonic antimicrobial peptides include CRAMP.
9. Use according to claim 8, wherein the short chain fatty acids comprise acetic acid, butyric acid and/or propionic acid.
10. The use according to claim 9, wherein the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable excipient; the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.
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