Composition containing lactobacillus plantarum for promoting secretion of butyric acid metabolite and application of composition

Abstract

The invention relates to the field of lactobacillus plantarum, and particularly discloses a composition containing lactobacillus plantarum for promoting butyric acid metabolite secretion and application of the composition. The composition containing lactobacillus plantarum capable of promoting butyric acid metabolite secretion comprises lactobacillus plantarum UJS001 viable bacteria and / or dead thalli and metabolites thereof, and / or lactobacillus plantarum UJS002 viable bacteria and / or dead thalli and metabolites thereof, and / or lactobacillus plantarum UJS003 viable bacteria and / or dead thalli and metabolites thereof, and / or lactobacillus plantarum UJS003 viable bacteria and / or dead thalli and metabolites thereof. The preservation number of the lactobacillus plantarum UJS001 is CGMCC No: 28821, the preservation number of the lactobacillus plantarum UJS002 is CGMCC No: 28822, and the preservation number of the lactobacillus plantarum UJS003 is CGMCC No: 28823. The composition containing lactobacillus plantarum for promoting butyric acid metabolite secretion has the advantages of promoting enrichment of butyric acid producing flora in intestinal microorganisms, promoting secretion of butyric acid and derivatives thereof, improving the barrier effect of intestinal tracts and further preventing and / or treating colitis.

Description

Technical Field

[0001] The present application relates to the field of Lactobacillus plantarum, and more specifically, to a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites and its application. Background Art

[0002] Colitis is a common intestinal disease that usually manifests as inflammation of the colon mucosa, which may cause abdominal pain, diarrhea, blood in the stool and other symptoms. The causes of colitis are varied, including infection, autoimmune response, genetic factors, etc. At present, the treatment methods for colitis mainly include drug therapy, surgical treatment and lifestyle adjustment, etc. However, for some patients, traditional treatment methods may not be effective, so researchers have been looking for more effective treatments.

[0003] In recent years, with the in-depth study of intestinal microbiota, people have gradually realized the close relationship between intestinal microbiota and intestinal health. Intestinal microbiota is not only involved in food digestion and energy metabolism, but also affects the host's immune system and inflammatory response. Lactobacillus plantarum is a probiotic that is widely present in nature. It can have a positive impact on intestinal health by regulating the balance of intestinal flora, enhancing intestinal barrier function, and inhibiting the growth of harmful bacteria. Butyrate metabolite is one of the short-chain fatty acids produced by intestinal microbial fermentation, which has multiple biological functions such as anti-inflammatory and antioxidant. Butyric acid can reduce the inflammatory response of the intestinal mucosa and regulate the activity of intestinal immune cells, thus having a potential therapeutic effect on diseases such as colitis.

[0004] In summary, developing Lactobacillus plantarum that can promote the secretion of butyrate metabolites to provide a new therapeutic strategy for the treatment of colitis is an urgent problem to be solved. Summary of the invention

[0005] In order to obtain a Lactobacillus plantarum strain capable of promoting the secretion of butyrate metabolites for use in the prevention and treatment of colitis, the present application provides a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites and its application.

[0006] In a first aspect, the present application provides a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites, using the following technical solution:

[0007] A composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites, comprising live bacteria and / or dead bacteria of Lactobacillus plantarum UJS001 and metabolites thereof, and / or live bacteria and / or dead bacteria of Lactobacillus plantarum UJS002 and metabolites thereof, and / or live bacteria and / or dead bacteria of Lactobacillus plantarum UJS003 and metabolites thereof;

[0008] The Lactobacillus plantarum UJS001 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28821;

[0009] The Lactobacillus plantarum UJS002 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28822;

[0010] The plant lactobacillus UJS003 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28823.

[0011] Preferably, the 16s rDNA sequence of the Lactobacillus plantarum UJS001 is shown as SEQ ID NO.1.

[0012] Preferably, the 16s rDNA sequence of the Lactobacillus plantarum UJS002 is shown as SEQ ID NO.2.

[0013] Preferably, the 16s rDNA sequence of the Lactobacillus plantarum UJS003 is shown as SEQ ID NO.3.

[0014] Preferably, the Lactobacillus plantarum UJS001, the Lactobacillus plantarum UJS002, and the Lactobacillus plantarum UJS003 are all isolated and screened from sauerkraut samples in Zhenjiang City, Jiangsu Province.

[0015] Preferably, the composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites is a pharmaceutical composition, a health product composition, and / or a food composition.

[0016] Preferably, the composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites also includes pharmaceutical excipients, health care product excipients, and / or food excipients.

[0017] In a second aspect, the present application provides an application of a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites, using the following technical solution:

[0018] An application of a composition containing plant lactobacillus that promotes the secretion of butyric acid metabolites is used to promote the enrichment of butyric acid-producing bacteria in intestinal microorganisms.

[0019] Preferably, the butyrate-producing bacteria group includes one or more of Akkermansia, Butyricoccus, Ruminococcus, Clostridium, Butyricoccus, Alternaria, and Coprococcus.

[0020] In a third aspect, the present application provides a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites for use in preventing and / or treating colitis, using the following technical solution:

[0021] Use of a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites in preventing and / or treating colitis.

[0022] Preferably, the composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites improves the intestinal barrier function of mice by promoting the secretion of butyrate and its derivatives by intestinal microorganisms, thereby preventing and / or treating colitis.

[0023] In summary, this application has the following beneficial effects:

[0024] 1. The Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002 and Lactobacillus plantarum UJS003 of the present application are all isolated and screened from sauerkraut samples in Zhenjiang City, Jiangsu Province, and have the potential for maintaining intestinal barrier function and excellent anti-colitis application.

[0025] 2. In the present application, Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 can relieve the symptoms of weight loss, colon shortening, etc. caused by colitis, and have excellent anti-colitis effects. Lactobacillus plantarum UJS001 has the best effect, and can promote the enrichment of butyrate-producing bacteria in intestinal microorganisms, promote the secretion of butyric acid and its derivatives, and improve the intestinal barrier function.

[0026] 3. In the present application, Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002 and Lactobacillus plantarum UJS003 can be applied to the fields of food, health care products and medicine, which broadens the application scope of Lactobacillus plantarum, improves the application value of Lactobacillus plantarum, and provides a new option for the daily treatment and health care of patients with colitis. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It is a diagram of the bacterial identification test results in Example 1 of the present application; wherein A is the Gram staining result; B is the agarose gel electrophoresis test result; and C is the phylogenetic tree;

[0028] Figure 2 The growth curves of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 in Example 1 of the present application;

[0029] Figure 3This is a graph showing the effects of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on the body weight and DAI scores of mice with colitis in Example 2 of the present application; wherein A is a graph showing the effects of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on the body weight of mice with colitis; and B is a graph showing the effects of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on the DAI scores of mice with colitis;

[0030] Figure 4 These are the evaluation results of intestinal permeability of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on colitis mice in Example 3 of the present application; wherein A is the result of in vivo imaging of mice; and B is the statistical result of serum FD4 concentration of mice;

[0031] Figure 5 This is a graph showing the effects of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on the length of the colon of colitis mice in Example 3 of the present application; wherein A is a photo of the mouse colon; and B is a statistical graph of the length of the mouse colon;

[0032] Figure 6 The HE staining results of the colon tissues of mice in the Control group, DSS group, LP-UJS001 group, LP-UJS002 group, and LP-UJS003 group in Example 4 of the present application;

[0033] Figure 7 These are the AB staining results of the colon tissues of mice in the Control group, DSS group, LP-UJS001 group, LP-UJS002 group, and LP-UJS003 group in Example 4 of the present application;

[0034] Figure 8 This is a diagram of the abundance of different bacterial species in the intestines of mice in the Control group, DSS group, and LP-UJS001 group in Example 5 of the present application;

[0035] Fig. 9 This is a graph showing the 3D-PCA analysis results of mouse fecal metabolites in the Control group, DSS group, and LP-UJS001 group in Example 6 of the present application;

[0036] Fig.10 These are the metabolic pathway analysis and butyrate and related metabolite abundance analysis result diagrams in Example 6 of the present application; wherein A is the metabolic enrichment pathway analysis result diagram; and B is the metabolite abundance diagram of mice in the DSS group and the LP-UJS001 group. DETAILED DESCRIPTION

[0037] In the present application, a dextran sulfate sodium salt (DSS)-induced acute colitis mouse model was constructed, and the plant lactobacillus with the best inhibitory effect on colitis mice was screened by observing the relevant indicators of mouse enteritis (weight changes, DAI scores, colon shortening) and intestinal permeability (serum FD4 concentration).

[0038] Lactobacillus plantarum UJS001 was deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​October 31, 2023, with the deposit number CGMCC No: 28821;

[0039] Lactobacillus plantarum UJS002 (Lactobacillus. plantarum UJS002) was deposited on October 31, 2023 at the General Microbiology Center of China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No: 28822.

[0040] Lactobacillus plantarum UJS003 (Lactobacillus. plantarum UJS003) was deposited on October 31, 2023 at the General Microbiology Center of China Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No: 28823.

[0041] The present application has found through experimental research that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 have a significant alleviating effect on weight loss, colon shortening, and reduced intestinal permeability caused by colitis mice, among which the alleviating effect of Lactobacillus plantarum UJS001 is particularly obvious. In addition, Hematoxylin-Eosinstaining (HE) staining shows that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 improve the integrity of colon tissue structure. Alcian Blue (AB) staining shows that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 can significantly increase the colon goblet cells of colitis mice, and the apical exocytosis and secretion of mucin particles in mice on the mucosal surface are also enhanced; among them, Lactobacillus plantarum UJS001 has the best effect. It can be seen that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 all have good preventive effects on colitis and can be used to prepare food or medicine for preventing colorectal cancer. Therefore, Lactobacillus plantarum UJS001 is further preferred to explore its preventive effect on colitis.

[0042] By detecting the 16S rRNA sequencing of mouse feces, it was shown that Lactobacillus plantarum UJS001 could promote a significant increase in Lactobacillus in mouse fecal metabolites, and based on the functional potential to predict the species composition of the metabolic pathway, it was found that the bacteria that produce butyrate are enriched. It was found that Lactobacillus plantarum UJS001 can affect a specific microbial community in the process of preventing colitis - the bacteria that produce beneficial butyrate, thereby promoting the production of butyrate metabolites.

[0043] Further metabolomics detection of mouse feces showed that after preventive treatment with Lactobacillus plantarum UJS001, the results of 3D principal component analysis of PCA showed that there were certain differences in the metabolic profiles of each group of mice, indicating that the composition of fecal metabolites of colitis mice changed after intervention with Lactobacillus plantarum UJS001. The differential metabolites were further subjected to KEGG enrichment analysis, and the results showed that the enriched pathways mainly involved lipid metabolism (sphingolipid metabolism, fatty acid biosynthesis, unsaturated fatty acid biosynthesis), amino acid metabolism (arginine and ornithine metabolism, phenylalanine metabolism), etc. Combined with the above-mentioned flora composition analysis results, butyrate-producing bacteria were significantly enriched after intervention with Lactobacillus plantarum UJS001. At the same time, it was found that the content of butyric acid (Butyric acid) and its derivative 4-phenylbutyrate sodium salt (4-Phenylbutyrate) in the differential metabolites in the fecal metabolites of mice after intervention with Lactobacillus plantarum UJS001 was significantly increased. The above results show that butyric acid and its derivatives play an important role in improving the intestinal barrier of colitis mice with Lactobacillus plantarum UJS001.

[0044] To further help understand the technical solution of the present invention, the technical solution of the present invention is described in more detail below by providing several specific implementation examples and combining with the accompanying drawings. All of the described embodiments are only partial embodiments of the present invention, not all;

[0045] The following specific embodiments may be combined with each other, and the same or similar concepts or processes may not be repeated in some embodiments; and the reaction devices, reaction reagents, etc. involved in the following embodiments are all commercially available.

[0046] The following specific embodiments may be combined with each other, and the same or similar concepts or processes therein may not be described in detail in some embodiments.

[0047] The following examples are further explanations of the present invention, but the present invention is not limited thereto. Unless otherwise specified in the examples, the percentages are all by mass.

[0048] Example

[0049] Example 1

[0050] This embodiment discloses a Lactobacillus plantarum composition, comprising live bacteria and / or dead bacteria of Lactobacillus plantarum UJS001 and their metabolites, and / or live bacteria and / or dead bacteria of Lactobacillus plantarum UJS002 and their metabolites, and / or live bacteria and / or dead bacteria of Lactobacillus plantarum UJS003 and their metabolites.

[0051] Lactobacillus plantarum UJS001 (L. plantarum UJS001), deposited in China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28821;

[0052] Lactobacillus plantarum UJS002 (L. plantarum UJS002) was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28822.

[0053] Lactobacillus plantarum UJS003 (L. plantarum UJS003) was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28823.

[0054] Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 were all isolated and screened from fermented sauerkraut samples in Zhenjiang. The three strains of Lactobacillus plantarum isolated and purified from the sauerkraut samples were frozen for future use and identified as follows:

[0055] (1) Observation of bacterial morphology

[0056] The three strains were inoculated on MRS plates by streaking, and then appropriate amounts of monoclonal colonies on the plates were picked and evenly smeared on glass slides. Gram staining of the bacteria was performed according to the instructions of the kit and then observed and photographed under an oil microscope.

[0057] Reference Figure 1 A. The Gram staining results show that the three strains are all short rod-shaped, arranged in pairs or short chains, and stained purple, that is, they are all Gram-positive rods, which conform to the morphological characteristics of the genus Lactobacillus.

[0058] (2) Identification of bacterial 16S rDNA

[0059] 16S rDNA sequencing primers 27F and 1492R were used to perform PCR amplification using the extracted bacterial genomic DNA as a template. The PCR primer sequences are as follows:

[0060] 27F:5-AGAGTTTGATCCTGGCTCAG-3;

[0061] 1492R:5-CTACGGCTACCTTGTTACGA-3.

[0062] After the reaction was completed, agarose gel electrophoresis was performed to confirm the PCR amplification fragment and the gel with the band was cut out and recovered using a DNA recovery kit. The DNA was sent to Shanghai Shenggong Biotechnology Co., Ltd. for sequencing and the sequencing results were submitted to NCBI for comparison and construction of a phylogenetic tree.

[0063] The sequencing result of the 16S rDNA sequence of Lactobacillus plantarum UJS001 is shown in SEQ ID NO.1; the sequencing result of the 16S rDNA sequence of Lactobacillus plantarum UJS002 is shown in SEQ ID NO.2; and the sequencing result of the 16S rDNA sequence of Lactobacillus plantarum UJS003 is shown in SEQ ID NO.3.

[0064] Reference Figure 1 B, The results of electrophoresis and gel imaging showed that the target band appeared at the position of about 1500 bp in the agarose gel.

[0065] Reference Figure 1 C, The sequencing results of the amplified products were compared for sequence homology in NCBI and a phylogenetic tree was constructed. It can be seen that the three strains are located in the same branch as Lactobacillus plantarum.

[0066] (3) Growth curve determination

[0067] The activated bacterial solution was inoculated into MRS liquid medium at a 3% inoculation ratio and cultured at 37°C. Samples were taken every 2 hours to detect the OD600 value until 24 hours. A growth curve was prepared with time as the horizontal axis and the absorbance OD600 value as the vertical axis.

[0068] Reference Figure 3 , it can be seen from the growth curve that all three strains were successfully revived and cultured.

[0069] In summary, from the morphological characteristics and molecular biological characteristics, it can be seen that the three strains isolated by the inventor from the Zhenjiang sauerkraut sample are all Lactobacillus plantarum, which are respectively deposited in the China Center for the Collection of General Microbiological Cultures and named Lactobacillus plantarum UJS001 (L. plantarum UJS001), Lactobacillus plantarum UJS002 (L. plantarumUJS002), and Lactobacillus plantarum UJS003 (L. plantarumUJS003).

[0070] Example 2

[0071] This example studies the effects of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on disease characterization indicators of colitis mice. The test method is as follows:

[0072] (1) Preparation of Lactobacillus plantarum UJS001 powder, Lactobacillus plantarum UJS002 powder, and Lactobacillus plantarum UJS003 powder

[0073] Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 were inoculated into liquid MRS medium, and cultured anaerobically at 37°C for 22-24 h. The bacterial solution was added with secondary activation medium (skim milk 100 g + yeast extract 1.0 g + double distilled water 1000 ml) and cultured again for 12 h (final concentration of about 1×10 11 CFU / mL); add 10% trehalose, 1% L-glutamic acid, 2% vitamin C, 2% proline, 0.5% glycine, 15% skim milk powder, and then freeze-dry; viable count ≥1.0×10 10 CFU / mL. The product is in powder form, with no visible impurities, and is white to light yellow in color. They are named LP-UJS001 bacterial powder, LP-UJS002 bacterial powder, and LP-UJS003 bacterial powder.

[0074] (2) Mouse preparation

[0075] 5-6 week old C57B / 6 male mice weighing (18±2) g were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were housed in an SPF laminar flow cabinet with a 12h:12h cycle, an ambient temperature of 23-25°C, and an ambient humidity of 40%-60%. The experiment was officially started after one week of adaptive feeding.

[0076] Forty mice were randomly divided into five groups, with 8 mice in each group, namely, normal control group (NC group), colitis model group (DSS group), DSS+LP-UJS001 group (LP-UJS001 group), DSS+LP-UJS002 group (LP-UJS002 group) and DSS+LP-UJS003 group (LP-UJS003 group).

[0077] (3) Establishment of colitis model

[0078] From the 1st to the 7th day of the experiment, mice in the NC group drank sterile water freely, while mice in the other groups drank 2% DSS aqueous solution to establish an acute colitis model; from the 8th to the 14th day, all the five groups drank sterile water. During the 14-day experiment, mice in the NC group and the DSS group were gavaged with 100 μL PBS solution every day, while the other three groups were fed with LP-UJS001 bacterial powder, LP-UJS002 bacterial powder, and LP-UJS003 bacterial powder (1×10 10 CFU / g), the corresponding bacterial solution concentration is 10 10 CFU / mL was administered in 100 μL.

[0079] During the experiment, the weight, stool characteristics, blood in stool, hair, and activity of each group of mice were measured and recorded every day, and the DAI score of the mice was calculated according to the scoring criteria in Table 1. DAI = (weight loss score + stool characteristics score + fecal occult blood / gross blood in stool score) / 3

[0080] Table 1 Disease activity index scoring criteria

[0081]

[0082] Reference Figure 3 A, The weight of mice in the DSS group continued to decrease from the 7th day, and the weight decreased significantly on the 14th day compared with the NC group (P<0.001); the weight loss rate of the LP-UJS001 group and the LP-UJS002 group was significantly slower than that of the DSS group, and the weight of mice in the DSS group was significantly higher on the 14th day (P<0.01); the weight loss of the LP-UJS003 group was more serious than that of the DSS group from the 6th to the 10th day, and the weight recovered slightly after the 11th day. The weight at the end of the experiment did not recover significantly compared with the DSS group (P>0.05). At the same time, the weight of mice in the LP-UJS001 group was significantly higher than that in the LP-UJS003 group (P<0.05).

[0083] Reference Figure 3 B. The DAI score results showed that all indicators of mice in the NC group were normal, so the DAI score remained at 0; the DSS group continued to rise from the 5th day and remained at a high level; the DAI scores of the LP-UJS001 group, LP-UJS002 group, and LP-UJS003 group on the 14th day were significantly lower than those of the DSS group (P<0.01), and the scores of the LP-UJS001 group and LP-UJS002 group were significantly lower than those of the LP-UJS003 group (P<0.01).

[0084] In summary, Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 all have certain alleviating effects on weight loss and increased DAI scores in colitis mice, and the alleviating effects of Lactobacillus plantarum UJS001 and Lactobacillus plantarum UJS002 on weight loss and increased DAI scores in colitis mice are more obvious than Lactobacillus plantarum UJS003.

[0085] Example 3

[0086] This example studies the effects of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on colonic permeability in colitis mice. The test method is as follows:

[0087] (1) Fluorescein tracer method to detect intestinal mucosal permeability

[0088] The night before the experiment, mice in the NC, DSS, LP-UJS001, LP-UJS002, and LP-UJS003 groups were deprived of food but not water for about 12 hours, and the hair on the abdomen and limbs of the mice was shaved in advance. The next day, the mice were gavaged with FD4 solution at a dose of 500 mg / kg, anesthetized 2 hours later, and a live imaging experiment was performed using a small animal live imaging device. After 4 hours, blood was collected from the eyeballs of the mice and serum was separated. The fluorescence intensity of FD4 in the serum was detected using a multifunctional microplate reader, and the corresponding FD4 concentration in the serum was calculated.

[0089] (2) Preparation of colon tissue pathology sections

[0090] Immediately after dissection of mice in the NC, DSS, LP-UJS001, LP-UJS002, and LP-UJS003 groups, colon tissues were removed for length measurement and photographing, and a portion of the colon was retained for fixation after rinsing with PBS. Colon tissues were fixed in 4% paraformaldehyde for 24-48 h, and then washed, dehydrated, transparent, wax-impregnated, embedded, sliced, spread, and baked to prepare histopathological sections of approximately 5 μm thickness.

[0091] Reference Figure 4 A, The results of animal in vivo imaging showed that the fluorescence diffusion range of mice in the NC group was small and the fluorescence intensity was weak; the fluorescence diffusion range of mice in the DSS group was significantly expanded and the intensity was significantly increased; compared with the DSS group, the fluorescence diffusion range of mice in the LP-UJS001 and LP-UJS002 groups was significantly reduced, and the fluorescence intensity was also significantly weakened, while the fluorescence diffusion range of mice in the LP-UJS003 group was not significantly reduced. Figure 4 B, Serum FD4 concentration results showed that compared with the DSS group, the serum FD4 concentration of mice in the LP-UJS001 group and the LP-UJS002 group decreased significantly (P<0.01), while the change in the LP-UJS003 group was not obvious (P>0.05), and the serum FD4 concentration of mice in the LP-UJS001 group was significantly lower than that in the LP-UJS003 group. In summary, Lactobacillus plantarum UJS001 has the best effect on improving the intestinal permeability of colitis mice, followed by Lactobacillus plantarum UJS002, and the improvement effect of Lactobacillus plantarum UJS003 is not obvious. This shows that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 have strain specificity in the ability to reduce intestinal permeability, among which Lactobacillus plantarum UJS001 has the best effect.

[0092] In addition, colitis can lead to a shortening of the colon length, so the change in the length of the mouse colon is also one of the indicators for evaluating the alleviating effect of Lactobacillus plantarum. Figure 5Compared with the NC group, the colon length of mice in the DSS group was significantly shortened (P<0.001), indicating the success of the colitis model; after intervention with Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003, the colon lengths of mice in the LP-UJS001, LP-UJS002, and LP-UJS003 groups were significantly longer than those in the DSS group (P<0.05), and the colon length of the LP-UJS001 group was significantly longer than that of the LP-UJS003 group (P<0.05). The results showed that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 could alleviate the shortening of the colon in mice with colitis, and Lactobacillus plantarum UJS001 had a better alleviating effect.

[0093] Example 4

[0094] This example studies the effects of Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 on the colon tissue structure of colitis mice. The test method is as follows:

[0095] (1) HE staining and scoring of mouse colon tissue

[0096] a. Drying: Dry the paraffin sections at 95℃ for 15 min.

[0097] b. Dewaxing and hydration: After drying, soak the slices in xylene while they are still hot for 10 minutes, repeat 3 times; soak them in 100% ethanol for 3 minutes, repeat 3 times; soak them in 95% ethanol for 3 minutes, repeat 2 times; soak them in 70% ethanol for 3 minutes, repeat 2 times; wash them with distilled water.

[0098] c. Hematoxylin staining: Place the sections in hematoxylin stain solution for 4-5 minutes and then wash with distilled water.

[0099] d. Eosin staining: Place the sections in eosin staining solution for 20-30 seconds and then wash with distilled water.

[0100] e. Dehydration, transparency and sealing: Soak the slices in 70% ethanol for 2 min; soak in 95% ethanol for 2 min; soak in 100% ethanol for 2 min, repeat twice; soak in xylene for 5 min, repeat three times; seal with neutral resin and let dry.

[0101] f. After microscopic examination, the samples were scanned and analyzed using a pathological section scanner, and the scanned images were scored histopathologically. The scoring criteria are shown in Table 2.

[0102] Table 2 Histological injury scoring criteria

[0103]

[0104] (2) AB staining of mouse colon tissue

[0105] a. Paraffin sections are dried, dewaxed and hydrated.

[0106] b. Alcian blue staining: Place the sections in Alcian blue staining solution for 10 minutes and then wash with distilled water.

[0107] c. Nuclear Fast Red Staining: Place the sections in Nuclear Fast Red staining solution for 3 minutes and then wash with distilled water.

[0108] d. Dehydrate, transparentize, seal and scan the slides.

[0109] Reference Figure 6 As shown by HE staining, the colon morphology of mice in the NC group (i.e., Control group) was good, the mucosal structure was clear and the epithelial structure was intact, the crypt structure was normal, the glands were neatly arranged and abundant, and almost no inflammatory cells were found; the colon mucosal structure of mice in the DSS group was destroyed, the crypt and glandular structures were lost, the inflammatory cell infiltration in the lamina propria was severe, and the histopathological score was significantly higher than that in the normal control group (P<0.001); the colon morphology of mice in the LP-UJS001 group, LP-UJS002 group, and LP-UJS003 group was significantly improved, and the histopathological score was significantly lower than that in the DSS group; this shows that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 can improve the integrity of the colon tissue structure, and Lactobacillus plantarum UJS001 has the best improvement effect.

[0110] Reference Figure 7 , AB staining showed that the colonic goblet cells of LP-UJS001, LP-UJS002, and LP-UJS003 increased significantly, and the apical exocytosis and secretion of mucin particles on the mucosal surface were also enhanced; among them, LP-UJS001 had the best relief effect. It can be seen that Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 all have good preventive effects on colitis and can be used to prepare food or medicine for preventing colorectal cancer, among which Lactobacillus plantarum UJS00 has the best effect.

[0111] Example 5

[0112] This example studies the effect of Lactobacillus plantarum UJS001 on the homeostasis of the colitis flora. The test method is as follows:

[0113] The mouse feces of the NC group (ie, Control group), DSS group, and LP-UJS001 group of Example 2 were collected into cryopreservation tubes, quickly frozen with liquid nitrogen, and stored at -80°C, and the following experiments were performed.

[0114] (1) Extraction of total microbial DNA

[0115] Total DNA was extracted from mouse fecal samples according to the instructions of the DNA extraction kit and stored at -80°C.

[0116] (2) Sample PCR amplification

[0117] Nanodrop was used to quantify DNA, and agarose gel electrophoresis was used to test the quality of DNA extraction. PCR primers were synthesized based on the highly variable V3-V4 region of the bacterial 16S rRNA gene, and PCR amplification was performed on the target fragment.

[0118] PCR reaction system: 0.25 μL of ultra-fidelity DNA polymerase, 5 μL each of 5× Reaction Buffer and 5× High GC Buffer, 2 μL each of dNTP (10 mM) and template DNA, 1 μL each of forward and reverse primers (10 μM), 8.75 μL of DEPC water;

[0119] The PCR amplification primer sequences are as follows:

[0120] 338F: 5'-barcode+ACTCCTACGGGAGGCAGCA-3';

[0121] 806R: 5'-GGACTACHVGGGTWTCTAAT-3'.

[0122] The PCR amplification program was set as follows: 98°C pre-denaturation for 30 s, 98°C denaturation for 15 s, 50°C annealing for 30 s, 72°C extension for 30 s, and the number of cycles was set to 25-27 times. Finally, the product was kept at 72°C for 5 min to complete the extension and stored at 4°C.

[0123] The amplified product was subjected to 2% agarose gel electrophoresis, and the target fragment was cut out and recovered and purified.

[0124] (3) Library construction and sequencing

[0125] The PCR products were subjected to fluorescence quantitative detection, and then the samples were mixed according to the data volume required for each sample to construct the library. The libraries that passed the quality inspection were sequenced with 2×250bp double-end sequencing using an Illumina NovaSeq sequencer.

[0126] Reference Figure 8, by detecting the 16S rRNA sequencing of mouse feces, it was shown that Lactobacillus in the LP-UJS001 group increased significantly, and the species composition of the metabolic pathway predicted by functional potential was found to be enriched in butyrate-producing bacteria, including Akkermansia and Butyricoccus; in addition, Ruminococcus, Clostridium, Butyricoccus, Alistipes, and Coprococcus were all important butyrate-producing bacteria, and their relative abundances also increased. This shows that Lactobacillus plantarum UJS001 can promote the relative abundance of beneficial butyrate-producing bacteria in the process of preventing colitis, thereby promoting the secretion of butyrate metabolites, and exerting the potential therapeutic effect of butyrate on diseases such as colitis.

[0127] Example 6

[0128] This example studies the effect of Lactobacillus plantarum UJS001 on fecal metabolites in colitis mice. The test method is as follows:

[0129] (1) Preparation of Lactobacillus plantarum UJS001 powder

[0130] Lactobacillus plantarum UJS001 was inoculated into liquid MRS medium and cultured anaerobically at 37°C for 22-24 h. The bacterial solution was added with secondary activation medium (skim milk + yeast extract + double distilled water) and cultured again for 12 h (final concentration was about 1×10 11 CFU / mL); add 10% trehalose, 1% L-glutamic acid, 2% vitamin C, 2% proline, 0.5% glycine, 15% skim milk powder, and then freeze-dry; viable count ≥1.0×10 10 CFU / mL.

[0131] (2) Construction of mouse model

[0132] 5-6 week old C57B / 6 male mice weighing (18±2) g were purchased from Changzhou Cavens Experimental Animal Co., Ltd. The mice were housed in an SPF laminar flow cabinet with a 12h:12h cycle, an ambient temperature of 23-25°C, and an ambient humidity of 40%-60%. The experiment was officially started after one week of adaptive feeding.

[0133] Fifteen mice were randomly divided into three groups, with 5 mice in each group, namely the normal control group (Control group), the colitis model group (DSS group), and the DSS+LP-UJS001 group (LP-UJS001 group).

[0134] From the 1st to the 7th day of the experiment, except for the mice in the Control group, which drank sterile water freely, the mice in the other groups drank 2% DSS aqueous solution to establish the acute colitis model; from the 8th to the 14th day, all the three groups drank sterile water. During the 14-day experiment, the mice in the Control group and the DSS group were gavaged with 100 μL PBS solution every day, while the LP-UJS001 group was fed with LP-UJS001 bacterial powder (1×1010 CFU / g) every day, and the corresponding bacterial solution concentration was 1010 CFU / mL and 100 μL was given.

[0135] (3) Metabolomics testing

[0136] a. Metabolite Extraction

[0137] An appropriate amount of mouse fecal metabolite sample was added to a precooled methanol / acetonitrile / water solution (2:2:1, v / v), subjected to low-temperature ultrasound for 30 min, allowed to stand at -20 °C for 10 min, centrifuged at 14000 g, 4 °C for 20 min, and the supernatant was vacuum dried.

[0138] b. High performance liquid chromatography-mass spectrometry (LC-MS / MS)

[0139] 100 μL of acetonitrile solution (acetonitrile: water = 1:1, v / v) was added, centrifuged at 4°C for 15 min, and the supernatant was sampled for analysis. Agilent 1290 Infinity LC HILIC column was used for separation, mobile phase A: water, 25 mM ammonium acetate and 25 mM ammonia, mobile phase B: acetonitrile.

[0140] The gradient elution process was: 95% B, 0.5 min; 95%-65% B, 6.5 min; 65%-40% B, 1 min; 40% B, 1 min; 40%-95% B, 0.1 min; 95% B, 2.9 min. The sample was automatically injected at 4°C with a volume of 2 μL.

[0141] (2) Non-targeted metabolomics analysis

[0142] Pattern recognition was performed using SIMCA-P 14.1 software. After Pareto-scaling preprocessing, principal component analysis (PCA) was used to reduce the dimension of multivariate raw data and analyze the similarities, differences, and outliers within and between groups. Partial least squares-discriminant analysis (PLS-DA), variable projection importance analysis (VIP), variability analysis, difference fold analysis, and Student t-test were used to screen differential metabolites. The screening criteria were P < 0.05 and VIP > 1. Metabolic pathway enrichment analysis of differential metabolites was performed based on the KEGG database.

[0143] Reference Fig. 9The results of 3D principal component analysis of PCA analysis of mouse fecal metabolites showed that the samples of each group of mice had a separation trend, indicating that there were certain differences in the metabolic profiles of each group of mice, suggesting that the composition of fecal metabolites of colitis mice changed after intervention with Lactobacillus plantarum UJS001.

[0144] Based on pathway analysis and abundance maps of butyrate and related metabolites, the differential metabolites were further subjected to KEGG enrichment analysis. Fig.10 A, the results show that the enriched pathways mainly involve lipid metabolism (sphingolipid metabolism, fatty acid biosynthesis, unsaturated fatty acid biosynthesis), amino acid metabolism (arginine and ornithine metabolism, phenylalanine metabolism), etc. Fig.10 B. Combined with the above-mentioned flora composition analysis results, butyrate-producing bacteria were significantly enriched in the fecal metabolites of mice in the LP-UJS001 group. At the same time, it was found that the content of butyric acid and its derivative 4-phenylbutyrate in the differential metabolites was significantly increased in the LP-UJS001 group (P<0.05). This shows that butyric acid and its derivatives play an important role in improving the intestinal barrier of mice with colitis treated with Lactobacillus plantarum UJS001.

[0145] In combination with Examples 1-6, the three plant lactobacilli provided by the present application, namely Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003, can all significantly restore the intestinal barrier function of colitis, among which the effect of Lactobacillus plantarum UJS001 is more significant; in addition, the present application proposes a new idea of ​​using a specific strain of Lactobacillus plantarum to promote the secretion of butyrate metabolites, that is, by affecting a specific microbial community in the process of preventing colitis through Lactobacillus plantarum UJS001 - a community that produces beneficial butyric acid, so as to promote the secretion of butyric acid and derivatives. Lactobacillus plantarum UJS001, Lactobacillus plantarum UJS002, and Lactobacillus plantarum UJS003 can be widely used in improving the function of intestinal flora and related development diseases, as well as in preventing and / or treating colitis, bringing more personalized and effective daily health care plans to patients with colitis, and also providing new perspectives and ideas for the study of the relationship between intestinal microbiota and intestinal health.

[0146] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites, characterized in that: Including live bacteria and / or dead bacteria of Lactobacillus plantarum UJS001 and their metabolites, and / or live bacteria and / or dead bacteria of Lactobacillus plantarum UJS002 and their metabolites, and / or live bacteria and / or dead bacteria of Lactobacillus plantarum UJS003 and their metabolites; The Lactobacillus plantarum UJS001 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28821; The Lactobacillus plantarum UJS002 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28822; The plant lactobacillus UJS003 was deposited in the China Center for Microbiological Culture Collection on October 31, 2023, with the deposit number CGMCC No: 28823.

2. The composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites according to claim 1, characterized in that: The 16s rDNA sequence of the Lactobacillus plantarum UJS001 is shown in SEQ ID NO.

1.

3. The composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites according to claim 1, characterized in that: The 16s rDNA sequence of the Lactobacillus plantarum UJS002 is shown in SEQ ID NO.

2.

4. The composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites according to claim 1, characterized in that: The 16s rDNA sequence of the Lactobacillus plantarum UJS003 is shown in SEQ ID NO.

3.

5. The composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites according to claim 1, characterized in that: The composition containing plant lactobacillus that promotes the secretion of butyrate metabolites is a pharmaceutical composition, a health product composition, and / or a food composition.

6. The composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites according to claim 5, characterized in that: The composition containing plant lactobacillus that promotes the secretion of butyrate metabolites also includes pharmaceutical excipients, health care product excipients, and / or food excipients.

7. Use of a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites as claimed in claims 1 to 6, characterized in that: Used to promote the enrichment of butyrate-producing bacteria in intestinal microorganisms.

8. The use of the composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites according to claim 7, characterized in that: The butyrate-producing bacteria group includes one or more of the genera Akkermansia, Butyricoccus, Ruminococcus, Clostridium, Butyricoccus, Alternaria, and Coprococcus.

9. Use of a composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites as claimed in any one of claims 1 to 6 in preventing and / or treating colitis.

10. Use of the composition containing Lactobacillus plantarum that promotes the secretion of butyrate metabolites according to claim 9 in preventing and / or treating colitis, characterized in that: The composition containing plant lactobacillus that promotes the secretion of butyrate metabolites improves the intestinal barrier function of mice by promoting the secretion of butyrate and its derivatives by intestinal microorganisms, thereby preventing and / or treating colitis.

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