Lactobacillus kefiranofaciens JK-24 with characteristic of treating or relieving colitis and application of lactobacillus kefiranofaciens JK-24

Abstract

The invention discloses lactobacillus kefiranofaciens JK-24 with the characteristic of treating or relieving colitis and application of the lactobacillus kefiranofaciens JK-24, the lactobacillus kefiranofaciens JK-24 is preserved in the China Center for Type Culture Collection, the preservation time is July 3, 2024, the preservation number is CCTCC NO: M 20241456, the strain has remarkable bacteriostatic and antioxidant activity, and the lactobacillus kefiranofaciens JK-24 can be used for treating or relieving colitis. The traditional Chinese medicine composition can be used for remarkably treating or relieving abnormal increase of expression quantities of proinflammatory factors TNF-alpha, IL-12, IL-10 and IL-6 caused by DSS. The lactobacillus kefiranofaciens JK-24 can effectively improve the DSS-induced colitis symptom, can be used as a microbial medicine or product with a good industrial prospect, and has a potential clinical application value.

Description

Technical Field

[0001] The invention belongs to the field of microorganisms, and in particular relates to a kumiss-like lactobacillus JK-24 with the property of alleviating colitis and an application thereof. Background Art

[0002] Colitis is a common intestinal inflammatory disease characterized by chronic inflammation of the colonic mucosa. At present, methods for treating colitis include drug therapy, surgical treatment, and probiotic therapy. Among them, probiotic therapy has received widespread attention due to its high safety and small side effects. Lactic acid bacteria are Gram-positive bacteria that are widely present in nature. They have excellent probiotic properties, regulate intestinal flora, enhance intestinal barrier function and anti-inflammatory effects, and can relieve colitis symptoms. Existing lactic acid bacteria include Lactobacillus acidophilus, Lactobacillus lactis, Lactobacillus bulgaricus, Bifidobacterium, Kühfe-like Lactobacillus, etc. There are public reports in the prior art that Bifidobacterium, Lactobacillus plantarum 1201, and Bacillus licheniformis BL63516 have a regulatory effect on DSS-induced colitis in mice. Kühfe-like Lactobacillus is isolated from traditional fermented kühfe, but there is no report that Kühfe-like Lactobacillus has a regulatory effect on DSS-induced colitis in mice. Summary of the invention

[0003] Purpose of the invention: In order to solve the above technical problems, the present invention provides a strain of Kühm-like Lactobacillus JK-24, which has significant therapeutic effects on the treatment and prevention of inflammatory bowel disease and the relief of oxidative stress.

[0004] Another object of the present invention is to provide a use of Kfushin-like Lactobacillus JK-24 in treating or alleviating DSS-induced colitis.

[0005] Another object of the present invention is to provide an anti-inflammatory bacterial agent produced by the kumiss-like Lactobacillus JK-24 antibacterial agent and its application.

[0006] Technical solution: In order to achieve the above-mentioned purpose, the kumiss-like Lactobacillus JK-24 described in the present invention has been deposited in the China Center for Type Culture Collection on July 3, 2024, and the deposit number is CCTCC NO: M20241456.

[0007] The invention discloses an application of the kefir-like lactobacillus JK-24 in the preparation of a medicine or reagent for treating or alleviating colitis.

[0008] The invention discloses an application of the kefir-like lactobacillus JK-24 in the preparation of a medicine or reagent for treating or alleviating DSS-induced colitis.

[0009] Furthermore, the kefir-like Lactobacillus JK-24 treats or alleviates DSS-induced colitis, including changing the length of the colon and the structure of the colon tissue, while reducing the levels of inflammatory factors in serum and colon tissue and increasing the levels of antioxidant stress factors.

[0010] The anti-inflammatory bacterial agent produced by the kefir-like Lactobacillus JK-24 of the present invention is used for treating or relieving colitis.

[0011] Furthermore, the anti-inflammatory bacterial agent for alleviating colitis is used for treating and alleviating DSS-induced colitis.

[0012] Furthermore, the bacterial agent contains the kefir-like Lactobacillus JK-24 and / or the kefir-like Lactobacillus JK-24 culture.

[0013] Furthermore, the culture is a bacterial suspension obtained by culturing the kumiss-like Lactobacillus JK-24 and then adding physiological saline to resuspend the bacterial cells.

[0014] Preferably, the culture is a bacterial suspension obtained by culturing the kumiss-like Lactobacillus JK-24 in a culture medium for a period of time, centrifuging and adding physiological saline to resuspend the bacterial cells.

[0015] Furthermore, the content of kefir-like Lactobacillus JK-24 in the anti-inflammatory bacterial agent is not less than 1×10 9 CFU / mL.

[0016] The invention discloses an application of the kefir-like lactobacillus JK-24 in the preparation of medicines, functional foods, special medical formula foods or health products for relieving colitis.

[0017] Furthermore, the effect of the kefir-like Lactobacillus JK-24 on alleviating DSS-induced colon problems in mice includes but is not limited to being able to effectively improve changes in DAI in mice.

[0018] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0019] The kefir-like lactobacillus JK-24 isolated and screened by the present invention has significant probiotic properties, including good acid resistance, bile salt resistance, antibacterial activity and antioxidant capacity, and can significantly treat or alleviate the abnormal increase in the expression of proinflammatory factors TNF-α, IL-12, IL-10 and IL-6 caused by DSS. In addition, JK-24 is sensitive to multiple drugs, shows high safety and is non-pathogenic. The present invention also provides a study on the alleviating effect of JK-24 on DSS-induced colitis in mice, indicating that JK-24 can effectively improve the symptoms of DSS-induced enteritis and has potential clinical application value. The bacterial agent prepared using the kefir-like lactobacillus JK-24 of the present invention can effectively treat and alleviate DSS-induced colitis in mice, providing a new microbial practice and therapy for the treatment of colitis, and can be used as a microbial drug or product with good industrial prospects, with potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the colony morphology of Kühm-like Lactobacillus JK-24;

[0021] Figure 2 This is the weight change curve of mice in the present invention. # indicates P < 0.05, ## indicates P < 0.01, * indicates P < 0.05, and ** indicates P < 0.01;

[0022] Figure 3 The images are of bloody stools in each group of mice in the examples of the present invention;

[0023] Figure 4 This is the change curve of DAI score of mice in the present invention. # indicates P < 0.05, ## indicates P < 0.01, * indicates P < 0.05, and ** indicates P < 0.01;

[0024] Figure 5 This is a comparison chart of the effects of kefir-like Lactobacillus JK-24 on the colon length of each group of mice in the examples of the present invention;

[0025] Figure 6 This is a bar graph showing the effect of Kühf-like Lactobacillus JK-24 on the colon length of each group of mice in the examples of the present invention;

[0026] Figure 7 The effect of JK-24 on the colon tissue structure of mice in the present invention, Note: Black arrows indicate the loss of intestinal crypts, intestinal mucosal epithelium and intestinal glands; white asterisks indicate inflammatory cell infiltration; yellow arrows indicate gland loss and irregular arrangement; green arrows indicate colon mucosal epithelial cell defects;

[0027] Figure 8It is the serum inflammatory factor in the test result of mouse serum index in the example of the present invention, # indicates P < 0.05, ## indicates P < 0.01, * indicates P < 0.05, and ** indicates P < 0.01;

[0028] Fig. 9 is the oxidative stress factor in the serum of the mouse serum index determination results in the examples of the present invention, # indicates P < 0.05, ## indicates P < 0.01, * indicates P < 0.05, and ** indicates P < 0.01;

[0029] Fig.10 It is the index of mouse colon inflammatory factor in the present invention. # indicates P<0.05, ## indicates P<0.01; compared with the DSS group, * indicates P<0.05, ** indicates P<0.01. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0031] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources. Experimental methods without specific conditions specified in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0032] A total of 36 male C57BL / 6 mice [8 weeks old, specific pathogen-free (SPF), body weight (20±2) g] were purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.; the animals were fed and watered freely and kept in an environment with a room temperature of 22±2℃ and a humidity of 45±10%. After 7 days of adaptive feeding, they were used in subsequent experiments. Kumiss-like Lactobacillus JK-24 was isolated from the traditional fermented kumiss in Kezhou, Xinjiang.

[0033] Example 1

[0034] Identification of Kühler-like Lactobacillus JK-24

[0035] 1. Colony characteristics

[0036] A lactobacillus strain was isolated from the traditional fermented mare's milk in Kezhou, Xinjiang. The colony morphology is round, white and translucent, and the edges are irregular. Figure 1 .

[0037] 2.16S rRNA Identification

[0038] The 16S rRNA gene of the strain JK-24 was subjected to a PCR experiment using a bacterial universal primer. After the PCR reaction amplification was completed, a 1479bp fragment was successfully obtained. The PCR product was cloned and sequenced to obtain the 16SrDNA gene sequence of the strain, which was also 1479bp in length (the sequence is shown in SEQ ID NO.1). A BLAST sequence comparison was performed on the NCBI website, and the results showed that the sequence had a similarity of more than 98% with multiple strains of Kühm-like Lactobacillus. Based on the above data, it can be determined that the strain is Kühm-like Lactobacillus, named Kühm-like Lactobacillus JK-24.

[0039] The strain Kfusobacterium kumiss JK-24 of the present invention has been deposited in the China Center for Type Culture Collection on July 3, 2024, with the deposit address being Wuhan University, China, and the deposit number being CCTCC NO: M 20241456.

[0040] Example 2.

[0041] Activation of Kruskal Lactobacillus JK-24 and preparation of bacterial suspension

[0042] 1. Bacteria activation

[0043] The kumiss Lactobacillus JK-24 preserved with glycerol was melted at room temperature, and the kumiss Lactobacillus JK-24 participating in the experiment was inoculated into the prepared MRS solid medium, resuscitated and purified in the MRS solid medium, cultured in a 37° incubator for 24 hours, cultured for three generations, and observed under a microscope for no foreign bacteria. The kumiss Lactobacillus JK-24 in the MRS solid medium was inoculated into the MRS liquid medium, cultured in a 37° incubator for three generations, and observed under a microscope for no foreign bacteria in the medium.

[0044] 2. Preparation of bacterial suspension

[0045] The pure strain colony obtained in step 1 of this example was inoculated into MRS liquid culture medium and cultured in an incubator at 37°C for 24 h. The liquid culture was then centrifuged at 6000 rpm for 10 min. The supernatant was removed, and the bottom bacteria were left and rinsed twice with saline. The bacteria were then resuspended with saline. The concentration of the bacterial suspension was finally adjusted to 1×10 9 CFU / mL, used for oral gavage of mice.

[0046] Example 3

[0047] Animal grouping and model construction

[0048] 1. Animal Grouping

[0049] Eighteen mice were randomly divided into three groups (n=6): Control group, DSS model group, and JK-24+DSS group.

[0050] 2. Model Construction

[0051] The control group and the DSS modeling group received normal saline (0.1 mL / 10 g mouse body weight) by gavage every day for 14 consecutive days, and they were free to eat during this period. The mice in the JK-24+DSS group received a suspension of Lactobacillus kumiss JK-24 (1×10 9 CFU / mL, 0.1mL / 10g mouse body weight), daily gavage for 14 consecutive days, during which they were free to eat. Next, the mice in the Control group drank water freely, and the mice in the other groups drank 3% DSS aqueous solution freely for 7 days, during which they were free to eat. The vitality, weight, feces and anal bleeding of the mice were recorded, and the bleeding of the mice was observed in time after gavage every day. After 3 weeks of intervention, the mice were killed after fasting for 24 hours, and the colon was collected, and the intestinal tube from the anus to the colon was taken for the detection of relevant indicators.

[0052] Example 4

[0053] Disease Activity Index (DAI) Scoring of Mice

[0054] During the construction of the mouse enteritis model in Example 3, the weight changes, stool characteristics and blood in the stool of the mice were recorded every day. The DAI scoring table (Table 1) was formulated with reference to the DAI scoring method of Yang Yang et al. for scoring, where DAI = (weight loss score + blood in the stool score + stool characteristics score) / 3.

[0055] Table 1 DAI score table

[0056]

[0057] 1. Weight changes

[0058] Depend on Figure 2 The weight change curve of mice showed that during the experiment, compared with the Control group, the weight change curve of the DSS group was significantly lower (P < 0.05) than that of the Control group on the 4th day; and the weight change curve was extremely significantly lower (P < 0.01) than that of the Control group on the 5th day and thereafter. Compared with the DSS group, the weight change curve of the JK-24+DSS group was significantly higher (P < 0.05) than that of the DSS group on the 4th and 5th days; and the weight change curve was extremely significantly higher (P < 0.01) than that of the DSS group on the 6th day and thereafter.

[0059] 2. Blood in stool

[0060] The blood in the stool of each group of mice was recorded every day. Figure 3 .

[0061] 3. DAI Rating

[0062] Depend on Figure 4 It can be seen that during the experiment, the DAI score of the Control group decreased slightly, while the DAI scores of the DSS group and the JK-24+DSS group continued to increase. Compared with the Control group, the DAI scores of the DSS group and the JK-24+DSS group were significantly higher (P<0.01) than those of the Control group on the 3rd day and thereafter, and continued to increase; compared with the DSS group, the scores of the JK-24+DSS group were significantly lower (P<0.01) than those of the DSS group on the 5th day and thereafter.

[0063] It can be seen from the above experimental results that the kefir-like Lactobacillus JK-24 provided by the present invention can greatly improve the DAI score of mice, can well alleviate DSS-induced colitis in mice, and provide a new option for the treatment of colitis.

[0064] Example 5

[0065] Pathological examination of mouse colon

[0066] The 0.5-1 cm distal colon of the mice in the different treatment groups in Example 3 was selected, formalin was added, and HE staining sections were observed.

[0067] 1. Effect of Kfusobacterium kumiss JK-24 on the colon length of mice

[0068] The colon length of each group of mice was measured, and a comparison chart of the colon length of mice was made ( Figure 5 ) and histograms ( Figure 6 ). As shown in the figure, the colon length of the Control group was the longest, which was 5.4±0.58cm; compared with the Control group, the colon length of the DSS group was shorter than that of the Control group, which was 4.5±0.58cm; compared with the DSS group, the colon length of the JK-24+DSS group was longer than that of the DSS group, which was 5.2±0.36cm.

[0069] 2. Effects of Kfusobacterium kumiss JK-24 on the histological structure of mouse colon

[0070] like Figure 7As shown, the colon mucosal epithelial cells of the mice in the Control group were intact, the crypts were intact and the morphological structure was normal, the glands and goblet cells were neatly arranged, and there was no inflammatory cell infiltration. Compared with the Control group, the colons of the mice in the DSS group had no complete mucosal tissue structure, and the intestinal crypts, intestinal mucosal epithelium and intestinal glands were largely missing (indicated by the black arrows), and the inflammatory cell infiltration was obvious (indicated by the white asterisks). Compared with the DSS group, the colon mucosal tissue structure of the mice in the JK-24+DSS group was basically intact, the glands and goblet cells were relatively neatly arranged in most areas, the inflammatory cell infiltration area was significantly reduced, and only a small number of areas showed gland loss and irregular arrangement (indicated by the yellow arrows), and some areas had mild mucosal epithelial cell defects (indicated by the green arrows).

[0071] It can be seen from the above experimental results that the kefir-like Lactobacillus JK-24 provided by the present invention can effectively alleviate DSS-induced colitis in mice, improve colon length and colon tissue structure, and has great clinical research value.

[0072] Example 6

[0073] Detection of inflammatory factors and oxidative stress factors in serum and colon tissue of mice after treatment in Example 3

[0074] 1. Detection of mouse serum indicators

[0075] After removing the eyeballs of mice, blood was collected and centrifuged at 4000rpm for 10 minutes to obtain serum. The collected serum was stored at -80℃ for later use. ELISA detection kits for inflammatory factors such as IL-6, IL-10, IL-12, and TNF-α were used, and the content of inflammatory factors in serum was determined strictly according to the instructions of the kit.

[0076] 1.1 Results of mouse serum index determination

[0077] Depend on Figure 8 It can be seen that compared with the Control group, the inflammatory factor IL-12 in the DSS group was significantly higher (P < 0.05) than the Control group, and the inflammatory factors TNF-α, IL-10 and IL-6 were extremely significantly higher (P < 0.01) than the Control group; the inflammatory factor TNF-α in the JK-24+DSS group was extremely significantly higher (P < 0.01) than the Control group. Compared with the DSS group, the inflammatory factors IL-12 and IL-6 in the JK-24+DSS group were significantly lower (P < 0.05) than the DSS group, and IL-10 and TNF-α were extremely significantly lower (P < 0.01) than the DSS group.

[0078] 2. Detection of oxidative stress factors in mouse serum

[0079] After the mice were killed, whole blood was collected, and the mouse blood was centrifuged at 4000r / min for 10min to obtain the supernatant, which was stored at -80°C for later use. The levels of catalase (CAT), myeloperoxidase (MPO), superoxide dismutase (SOD) and malondialdehyde (MDA) were determined using kits related to the levels of catalase (CAT), myeloperoxidase (MPO), superoxide dismutase (SOD) and malondialdehyde (MDA) according to the kit instructions.

[0080] 2.1 Detection results of oxidative stress factors in mouse serum

[0081] Depend on Fig. 9 It can be seen that compared with the Control group, the SOD and CAT in the DSS group were significantly lower (P < 0.01) than the Control group, and the MPO and MDA were significantly higher (P < 0.01) than the Control group; the MDA in the JK-24 + DSS group was significantly higher (P < 0.05) than the Control group. Compared with the DSS group, the SOD and CAT in the JK-24 + DSS group were significantly higher (P < 0.01) than the DSS group, the MPO was significantly lower (P < 0.05) than the DSS group, and the MDA was significantly lower (P < 0.01) than the DSS group.

[0082] 3. Determination of inflammatory factors in mouse colon tissue

[0083] The colon tissue samples were stored in cryovials, quickly frozen in liquid nitrogen, and stored at -80°C. The inflammatory factor ELISA detection kits such as IL-10 and TNF-α were used to determine the content of inflammatory factors in colon tissues in strict accordance with the instructions of the kits.

[0084] 3.1 Results of determination of inflammatory factors in mouse colon tissue

[0085] Depend on Fig.10 It can be seen that compared with the Control group, the inflammatory factors IL-10, IL-6 and TNF-α in the DSS group were significantly higher than those in the Control group (P < 0.01), and IL-12 was significantly higher than that in the Control group (P < 0.05); the inflammatory factor IL-6 in the JK-24+DSS group was significantly higher than that in the Control group (P < 0.01), and TNF-α was significantly higher than that in the Control group (P < 0.05). Compared with the DSS group, the inflammatory factors IL-10, IL-6, and TNF-α in the JK-24+DSS group were significantly lower than those in the DSS group (P < 0.01), and the inflammatory factor IL-12 was significantly lower than that in the DSS group (P < 0.05).

[0086] The above results show that the kefir-like Lactobacillus JK-24 provided by the present invention can reduce the levels of inflammatory factors in serum and colon tissue and increase the levels of antioxidant stress factors, which helps kefir-like Lactobacillus JK-24 to play a key role in the prevention and / or treatment of inflammatory bowel disease and other fields.

Claims

1. A strain of Kühn's lactobacillus JK-24 has been deposited in the China Center for Type Culture Collection on July 3, 2024, with a deposit number of CCTCC NO: M 20241456.

2. Use of the kefir-like Lactobacillus JK-24 according to claim 1 in the preparation of a medicine or reagent for treating or alleviating colitis.

3. Use of the kefir-like Lactobacillus JK-24 according to claim 1 in the preparation of a drug or reagent for treating or alleviating DSS-induced colitis.

4. The use according to claim 3, characterized in that: The kefir-like lactobacillus JK-24 treats or alleviates DSS-induced colitis by changing the length of the colon and the structure of the colon tissue, while reducing the levels of inflammatory factors in serum and colon tissue and increasing the levels of anti-oxidative stress factors.

5. An anti-inflammatory bacterial agent for treating or alleviating colitis produced by using the kefir-like Lactobacillus JK-24 described in claim 1.

6. The anti-inflammatory bacterial agent for treating or alleviating colitis according to claim 5, characterized in that: The anti-inflammatory bacterial agent is used to treat and alleviate DSS-induced colitis.

7. The use according to claim 5, characterized in that: The bacterial agent contains the kefir-like Lactobacillus JK-24 and / or the kefir-like Lactobacillus JK-24 culture.

8. The use according to claim 5, characterized in that: The culture is a bacterial suspension obtained by culturing the kumiss-like Lactobacillus JK-24 and then adding physiological saline to resuspend the bacterial cells.

9. The use according to claim 5, characterized in that: The content of kumiss-like Lactobacillus JK-24 in the anti-inflammatory bacterial agent is not less than 1×10 9 CFU / mL.

10. Use of the kefir-like Lactobacillus JK-24 according to claim 1 in the preparation of medicines, functional foods, special medical formula foods or health products for treating or alleviating colitis.

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