CN114574390A

CN114574390A - Bifidobacterium longum subspecies of infant for relieving colitis and application

Info

Publication number: CN114574390A
Application number: CN202210224209.0A
Authority: CN
Inventors: 杨波; 陈卫; 李明洁; 陈海琴; 刘小鸣; 崔树茂; 赵建新; 张灏
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2022-03-09
Filing date: 2022-03-09
Publication date: 2022-06-03
Anticipated expiration: 2042-03-09
Also published as: CN114574390B

Abstract

The invention discloses a bifidobacterium longum subspecies neonatorum for relieving colitis and application thereof, belonging to the technical field of biology. The bifidobacterium longum subspecies of infant CCFM1210 provided by the invention has the immunoregulation capability, and can improve the intestinal barrier and relieve colitis. Animal experiments prove that the tissue damage of a mouse can be obviously relieved by the intervention of bifidobacterium longum subsp. infantis CCFM1210, the myeloperoxidase level is obviously reduced, the content of tight junction protein in colon tissue is obviously increased, the levels of proinflammatory cytokines IL-1 beta and IL-6 are obviously reduced, the level of an inflammation-inhibiting cytokine IL-10 is obviously increased, the concentration of butyric acid is obviously increased, the diversity of intestinal flora is obviously improved, and the disorder of flora is relieved. The medicament or probiotic preparation prepared by adopting the bifidobacterium longum subspecies infantis CCFM1210 has certain advantages compared with the traditional medicament for treating colitis, and has wide market prospect.

Description

Bifidobacterium longum subspecies of infant for relieving colitis and application

Technical Field

The invention relates to a bifidobacterium longum subspecies neonatorum for relieving colonitis and application thereof, belonging to the technical field of microorganisms.

Background

Inflammatory Bowel Disease (IBD) is a chronic inflammatory disease of the intestinal tract, Ulcerative Colitis (UC) is one of the major forms of IBD, which is mainly characterized by a reduced intestinal barrier function and a dysregulated intestinal immune system, with the most common symptoms in patients with UC being diarrhoea, rectal bleeding, urgency and heaviness of the exterior, mucus discharge, abdominal pain. The exact pathogenesis of ulcerative colitis is unclear and several factors are currently thought to be involved, including immune response disorders, changes in intestinal microbes, genetic susceptibility, and environmental factors. The life quality of ulcerative colitis is directly influenced, and the selection of an appropriate treatment method is very important for improving the life quality of patients with ulcerative colitis. The conventional therapeutic drugs currently used are: sulfasalazine salicylic acid preparations such as mesalamine and the like; the corticosteroid is prednisone or dexamethasone, which is commonly used, however, the long-term use of the above drugs can cause side effects, including hypertension, diabetes, osteoporosis, etc.

In view of the various problems with existing treatment regimens, alternative to conventional approaches for colitis alleviation, new treatment regimens including biologics, prebiotics, probiotics for cytokines and adhesion molecules, some microbial metabolites such as unsaturated fatty acids, short chain fatty acids, etc. are of particular importance. The feasibility of the above-mentioned therapies has also prompted us to continue to search for dietary supplements with broader application and greater potential, and at the same time, with a colitis-alleviating effect.

Disclosure of Invention

The invention provides an application of bifidobacterium longum subsp infantis in preparing a product for preventing and/or treating colitis, and the bifidobacterium longum subsp infantis can be used for preparing the product for preventing and/or treating colitis.

The first purpose of the invention is to provide a Bifidobacterium longum subsp. infarnatum CCFM1210 strain, wherein the Bifidobacterium longum subsp. infarnatum CCFM1210 is preserved in Guangdong province collection center of microbial strains with the preservation number of GDMCC No: 62234, with a preservation date of 2022, 1 month, 23 days.

The bifidobacterium longum subspecies infantis CCFM1210 was isolated from a stool sample of an infant originating in the state of yangzhou, jiangsu, and had the following properties:

(1) bacterial colonies on the MRS solid culture medium are milky white, smooth in surface and round and convex;

(2) helps prevent and/or treat colitis;

(3) is helpful for increasing the content of short chain fatty acid in intestinal tract of mammal.

The invention also provides a microbial preparation containing the bifidobacterium longum subspecies infantis CCFM 1210.

In one embodiment, the microbial preparation is a liquid microbial preparation or a solid microbial preparation.

In one embodiment, the microbial preparation has a viable count of Bifidobacterium longum subspecies infantis of not less than 1X 10⁹CFU/g or 1X 10⁹CFU/mL。

In one embodiment, the microbial preparation is prepared by: inoculating the seed solution of the bifidobacterium longum subspecies infantis CCFM1210 into an MRS culture medium added with cysteine according to the inoculation amount accounting for 2-4% of the total mass of the culture medium, and carrying out anaerobic culture at 37 ℃ for 24-36 h to obtain a culture solution; centrifuging the culture solution to obtain thalli; and washing the thalli with normal saline for 3-4 times, and then resuspending to obtain the microbial preparation.

In one embodiment, the microbial preparation further comprises a cytoprotective agent; the cell protective agent includes but is not limited to skim milk, trehalose, sucrose one or more of the mixture.

In one embodiment, the cytoprotective agent comprises 130g/L skim milk, 20g/L trehalose, and 20g/L sucrose.

The invention also provides application of the bifidobacterium longum subspecies infantis CCFM1210 in preparing a product for treating or relieving colitis.

In one embodiment, the colitis is ulcerative colitis.

In one embodiment, the application includes at least one of the following functions:

(1) relieving weight loss;

(2) reducing damage to colon tissue;

(3) inhibit the reduction of Claudin-1, Claudin-1 and Occludin;

(4) reducing the concentration of proinflammatory cytokines IL-1 beta and IL-6 and increasing the concentration of anti-inflammatory cytokines IL-10;

(5) increasing the concentration of butyric acid in the intestinal tract;

(6) improving the diversity of intestinal flora and improving the disturbance of the intestinal flora.

In one embodiment, the product has a viable count of not less than 1 × 10 of Bifidobacterium longum subspecies infantis⁹CFU/g or 1X 10⁹CFU/mL。

In one embodiment, the product includes, but is not limited to, a food product or a pharmaceutical.

In one embodiment, the medicament comprises the bifidobacterium longum subspecies infantis CCFM1210 as described above, a pharmaceutical carrier and/or a pharmaceutical excipient.

In one embodiment, the food product includes, but is not limited to, a fermented food or a fermented beverage product comprising bifidobacterium longum subsp. Or fermented food produced by using microbial preparation containing the Bifidobacterium longum subspecies infantis CCFM 1210.

The invention also provides application of the bifidobacterium longum subspecies infantis CCFM1210 or the microbial preparation in producing fermented food.

Has the beneficial effects that:

(1) the bifidobacterium longum subsp. infantis (B.longum subsp.infantis) CCFM1210 provided by the invention is separated from intestinal flora of healthy infants, and the strain has no toxic or side effect on human bodies, so that the medicament prepared by adopting the bifidobacterium longum subsp.infantis (B.longum subsp.infantis) CCFM1210 provided by the invention has certain advantages compared with the traditional medicament for treating colitis, and the strain can be used for preparing probiotic preparations and the like, thereby having wide market prospect.

(2) By adopting the bifidobacterium longum subsp. infantis CCFM1210, the disease activity index of a mouse during DSS induced colitis can be obviously reduced, the weight reduction and the colon shortening are reduced, and the damage of colon tissues is obviously reduced.

(3) Intervention of bifidobacterium longum subsp. infantis (B.longum subsp. infantis) CCFM1210 can obviously inhibit reduction of zonulin ZO-1, Claudin-1 and Occludin.

(4) Intervention of bifidobacterium longum subsp. infantis (B.longum subsp. infantis) CCFM1210 remarkably reduces the concentration of proinflammatory cytokines IL-1 beta and IL-6, and remarkably increases the concentration of an anti-inflammatory cytokine IL-10.

(5) Bifidobacterium longum subsp. infantis CCFM1210 intervention significantly increased butyric acid concentrations.

(6) Intervention of bifidobacterium longum subsp. infantis (B.longum subsp.infantis) CCFM1210 remarkably improves diversity of intestinal flora of colitis mice, and relieves disturbance of intestinal flora.

Biological material preservation

A Bifidobacterium longum subsp.sp.infanis CCFM1210, which is classified and named as Bifidobacterium longum subsp.infanis, is deposited in Guangdong province microorganism culture collection center at 23.1.2022, and has the deposit number of GDMCC No.62234 and the deposit address of No. 59 building of Dazhou institute No. 100, Piezhi, Guangzhou.

Drawings

FIG. 1: weight change during DSS modeling of different groups of mice;

FIG. 2: disease activity index DAI index changes for different groups of mice;

FIG. 3: colon length of different groups of mice;

FIG. 4: colon tissues of different groups of mice are subjected to H & E staining;

FIG. 5: histopathological scoring of mice of different groups;

FIG. 6: the content of myeloperoxidase in colon tissues of different groups of mice;

FIG. 7: the content of ZO-1 in colon tissues of mice of different groups;

FIG. 8: the content of Occludin in colon tissues of mice of different groups;

FIG. 9: the content of Claudin-1 in colon tissues of mice of different groups;

FIG. 10: the content of IL-1 beta in colon tissues of different groups of mice;

FIG. 11: the content of IL-6 in colon tissues of different groups of mice;

FIG. 12: the content of IL-10 in colon tissues of different groups of mice;

FIG. 13: the concentration of butyric acid in the caecum contents of mice of different groups;

FIG. 14: the chao1 diversity index of intestinal flora of different groups of mice;

FIG. 15: shannon diversity index of intestinal flora of mice of different groups;

FIG. 16: the simpson diversity index of intestinal flora of different groups of mice;

FIG. 17: analyzing main coordinates of intestinal flora beta diversity indexes of mice of different groups;

in fig. 1 to 16, "" and "all" indicate significant differences from the DSS group, and the more, the greater the significant difference, the error is presented in the form of Mean ± SEM.

Detailed Description

The mice referred to in the examples below were male SPF (Specific pathogen free) grade C57BL/6N mice 6 weeks old, purchased from witnessee laboratories ltd; the tight junction protein ELISA kit related in the following examples was purchased from Nanjing Senega Biotechnology, Inc., and the inflammatory factor ELISA kit was purchased from R & D Systems, Inc.; sodium dextran sulfate (DSS) referred to in the following examples was purchased from MP Biomedicals, usa. Bifidobacterium longum subsp. infantis ATCC15697 was purchased from China general microbiological culture Collection center (accession number CGMCC 1.2202).

The media involved in the following examples are as follows:

MRS liquid medium: 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 5g/L of anhydrous sodium acetate, 0.5g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate monohydrate, 2g/L of diammonium hydrogen citrate, 2.6g/L, Tween 801 mL/L of dipotassium hydrogen phosphate trihydrate and 0.5g/L of cysteine hydrochloride.

MRS solid medium: 10g/L of tryptone, 10g/L of beef extract, 5g/L of yeast powder, 20g/L of glucose, 5g/L of anhydrous sodium acetate, 0.5g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate monohydrate, 2g/L of diammonium hydrogen citrate, 2.6g/L, Tween 801 mL/L of dipotassium hydrogen phosphate trihydrate, 0.5g/L of cysteine hydrochloride and 20g/L of agar.

Method for detecting Disease Activity Index (DAI):

the DAI score was based on Murthy's scoring system and included three aspects of weight change, hematochezia status and fecal characteristics (specific scoring criteria are shown in Table 1). During modeling, the weight of the mice was measured every day, the hematochezia condition and the stool characteristics of the mice were measured and scored according to table 1, and DAI ═ weight change score + hematochezia condition score + stool characteristics score. The fecal occult blood condition is measured by a Pirami hole fecal occult blood reagent, the specific operation is carried out according to a reagent instruction, and if reddish brown or bright red blood can be seen by naked eyes in the feces, the feces are bloody by naked eyes. Stool traits are divided into three grades: normal, loose and loose feces, and normal feces of mice are formed into granules; stool is loose if it has increased viscosity and is easily loosened but does not adhere to the anus; if the feces are unformed or watery and adhere to the anus, it is loose stool.

TABLE 1 disease Activity index Scoring criteria

Body weight loss (%)	Occult/macroscopic bloody stool	Stool characteristics	Score of
				0	Negative in occult blood	Is normal	0
1～5	Negative in occult blood	Loosening						1
				6～10	Positive occult blood	Loosening			2
11～15	Positive occult blood	Thin stool	3
				>15	Bloody stool with naked eyes	Thin stool	4

The detection method of the colon length comprises the following steps:

after the mice were sacrificed, the entire colon (end of cecum to anus) was removed and the length was measured.

The detection method of the colon histopathology characteristics comprises the following steps:

a1 cm distal colon (1 cm from the anus) was taken for subsequent fixation, embedding, sectioning and H & E staining. H & E colon sections were scanned using a panoramic MIDI digital section scanner and groups of colon tissue sections were scored for tissue damage using the scoring system of Dieleman, the tissue damage score including four aspects of inflammation, lesion depth, crypt destruction and lesion extent (see table 2 for specific criteria).

TABLE 2 tissue damage score criteria

And (3) determination of colon tissue biochemical indexes:

colon tissue is prepared according to the following steps of 1: 9 adding PBS, crushing the colon tissue by a high-throughput crusher to obtain homogenate, then centrifuging at 12000g and 4 ℃ for 15min, and collecting the supernatant to obtain the colon tissue supernatant.

The colon tissue tight junction protein content is detected by an ELISA kit (Nanjing Senega Biotechnology Co., Ltd.); the concentrations of cytokines IL-1. beta., IL-6 and IL-10 were determined by ELISA kits (R & D Systems, Inc.); the concentration of total protein in the colon tissue supernatant was measured using BCA kit (siemer feishell science).

And (3) detection of butyric acid concentration:

butyric acid in the mouse cecal contents is extracted and detected by GC-MS (the specific method refers to the influence and mechanism of the functional oligosaccharide on intestinal bacteria, 2015. university of Jiangnan, doctor academic paper).

Analysis of the diversity of the intestinal flora:

genomic DNA in feces is extracted by a FastDNA Spin Kit (MP biomedicine company in America), a V3-V4 region of the extracted genomic DNA is subjected to specific PCR amplification, 16S rDNA sequencing, fecal flora alpha diversity (Chao 1, Shannon and Simpson) and fecal flora beta diversity are analyzed on an online website, and main coordinate analysis of intestinal flora beta diversity is carried out based on weighted UniFrac distance.

Example 1: screening, identification, culture, observation and preservation of Bifidobacterium longum subsp

1. Screening

Taking 0.5g of a healthy infant feces sample from Yangzhou region of Jiangsu preserved in 30% (v/v) glycerol, coating the feces sample on an MRS solid culture medium (containing 0.5g/L cysteine) after gradient dilution, carrying out anaerobic culture at 37 ℃ for 72h, and observing and recording the colony morphology; selecting a milky white colony with a smooth surface and a circular bulge, streaking the milky white colony on an MRS solid culture medium (containing 0.5g/L cysteine), performing purification culture at 37 ℃ under an anaerobic condition, and repeating the operation for 3 times to obtain a purified single colony; and (3) selecting a purified colony, inoculating the colony into an MRS liquid culture medium (containing 0.5g/L cysteine), and performing anaerobic culture at 37 ℃ for 36 hours to obtain a strain CCFM 1210.

2. Identification

Extracting and screening a genome of the strain, amplifying and sequencing the 16S rDNA of the strain (the nucleotide sequence of the 16S rDNA obtained by amplification is shown as SEQ ID NO. 1), comparing the obtained sequence with the nucleotide sequence in NCBI-Blast, and then further confirming that the strain is a Bifidobacterium longum subspecies neonatorum through genome draft sequencing and phylogenetic analysis, wherein the strain is named as the Bifidobacterium longum subspecies neonatorum (B.longum subsp.infantis) CCFM 1210.

3. Preservation of

Selecting a Bifidobacterium longum subspecies of infant CCFM1210 single colony, inoculating the single colony into an MRS liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 36 hours to obtain a bacterial liquid; placing the bacterial liquid in a freezing tube under the aseptic condition, centrifuging at 6000r/min for 3min, and collecting thalli; washing with sterile PBS buffer solution, centrifuging at 6000r/min for 3min to obtain washed thallus, and repeating the operation for 3 times; the cells were resuspended in sterile 30% (v/v) glycerol and the tubes were stored in a-80 ℃ freezer.

Example 2: preparation of Bifidobacterium longum subspecies infantis CCFM1210 bacterial suspension

(1) Streaking a bacterial liquid dipped with bifidobacterium longum subspecies of infant CCFM1210 from a freezing tube on an MRS solid culture medium, and carrying out anaerobic culture at 37 ℃ for 48h to obtain a single bacterial colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 36 hours to obtain an activation solution; inoculating the activated liquid into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), carrying out anaerobic culture at 37 ℃ for 24h, and repeating the activated culture for 3 times to obtain activated bacterial liquid.

(2) Inoculating the activated bacterial liquid obtained in the step (1) into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), culturing at 37 ℃ for 24h to obtain a fermentation liquid, centrifuging the fermentation liquid, collecting the thalli, washing twice with PBS (phosphate buffer solution) with the pH of 7.4, then resuspending the thalli with physiological saline, and adjusting the number of viable bacteria to be 1 multiplied by 10¹⁰CFU/mL to obtain bacterial suspension.

The preparation of a suspension of Bifidobacterium longum subspecies infant ATCC15697 is carried out in the same manner as described above.

Example 3: effect of bifidobacterium longum subspecies infantis CCFM1210 on relieving symptoms of DSS-induced colitis mice

The DSS-induced mouse colitis modeling method and the bifidobacterium longum infant subspecies intervention treatment steps are as follows:

(1) 2.7% Dextran Sodium Sulfate (DSS) solution was prepared: DSS was formulated with sterile water as a 2.7% (w/v) Dextran Sulfate Sodium (DSS) solution.

(2) 40 healthy male C57BL/6N mice at 6 weeks of age were randomly assigned to 5 groups of 8 mice each, 5 groups were: a blank control group, a DSS group, a bifidobacterium longum subspecies infantis ATCC15697 dry pretreatment group (ATCC15697+ DSS), a bifidobacterium longum subspecies infantis CCFM1210 dry pretreatment group (CCFM1210+ DSS), and a bifidobacterium longum subspecies infantis FJND2M2 dry pretreatment group (FJND2M2+ DSS), wherein bacterial suspensions of the bifidobacterium longum subspecies infantis ATCC15697 and FJND2M2 were prepared according to the method of example 2; the experimental protocol and the treatment pattern of each group of mice are shown in table 3.

(3) During the molding period, the body weight (see fig. 1) and disease activity index DAI (see fig. 2) of each group of mice were measured daily. After the molding is completed, the mice are sacrificed, the colon (the end of the cecum to the anus) is removed, and the length of the colon is measured (the test result is shown in fig. 3).

Table 3 experimental mouse treatment protocol

As can be seen from fig. 1, on day 7 of molding, the weight of mice in DSS group decreased by 13.40%, while the weight of mice in CCFM1210 intervention group, ATCC15697 intervention group and FJND2M2 intervention group decreased by 6.94%, 11.5% and 18.75%, respectively, indicating that the weight of mice decreased by intervention of FJND2M2, and the weight of mice decreased by DSS molding was alleviated by ATCC15697 and CCFM1210, and the alleviation effect of CCFM1210 was better than that of ATCC 15697. On day 7 of model creation, the DAI index of DSS group mice increased to 9.25, ATCC15697 intervention group was 9.5, FJND2M2 intervention group mice had a DAI index as high as 11.00, while CCFM1210 intervention group mice had a DAI index significantly lower than that of the model group, only 6.13 (see fig. 2). The colon length of the mice in the blank control group is 6.91cm on average, the colon length of the mice in the DSS group is only 5.00cm, the reduction of the colon length is accelerated by the ATCC15697 intervention, and the reduction of the colon length caused by the DSS is remarkably relieved by the CCFM1210 intervention, and reaches 5.76cm (as shown in figure 3).

After the mice die, taking 1cm of distal colon tissue for fixing, dehydrating, embedding, HE staining, and observing HE staining of colon tissues of different groups of mice. As can be seen from fig. 4, after DSS induction and FJND2M2 dry prognosis, the colon tissues of mice were significantly damaged, including severe inflammatory cell infiltration, destruction of crypt and glandular structures, disappearance of goblet cells, and submucosal edema; the ATCC15697 intervention group had less damage to colonic tissue than the DSS group, but still had more inflammatory cell infiltration and submucosal edema; the colon tissue structure of the CCFM1210 intervention group is similar to that of the blank group, glands and crypts are more complete, goblet cells are more abundant, and only a small amount of inflammatory cell infiltration and submucosal edema exist.

The histopathological scoring criteria were referenced to relevant literature (see in particular J Agr Food Chem,2019,67(48): 13282-. Histopathological scores showed (fig. 5) that the pathology score of the DSS group reached 10.63, the FJND2M2 intervention group scored 11.63 higher than the DSS group and 8.67 for the ATCC15697 intervention group, with no significant difference from the DSS group, while the CCFM1210 intervention group scored only 6.38 significantly lower than the DSS group.

The results show that the bifidobacterium longum subsp. infantis CCFM1210 has better relieving effect on the symptoms of mice with DSS-induced colitis, and the relieving effect is better than that of the bifidobacterium longum subsp. infantis ATCC 15697.

Example 4: effect of Bifidobacterium longum subspecies infantis CCFM1210 on colonic myeloperoxidase-levels in colitis mice

The modeling method is the same as that in example 3, the mice are sacrificed after the modeling obtained in the step (3) in example 3 is finished, and the level of Myeloperoxidase (MPO) in the supernatant of the colon tissue is detected by taking the colon tissue according to the biochemical index measuring method of the colon tissue.

The experimental results show (as shown in fig. 6), compared with the blank control group, the intake of DSS causes the MPO level of the colon of the mouse to be remarkably increased to reach 73.11ng/mg protein; whereas intervention of CCFM1210 reduced colonic MPO levels to 45.85ng/mg protein. This indicates that bifidobacterium longum subsp.

Example 5: effect of Bifidobacterium longum subspecies infantis CCFM1210 on the content of intestinal claudin in colitis mice

The modeling method is the same as that in example 3, the mice are sacrificed after the modeling obtained in the step (3) in the example 3 is finished, and the colon tissues are taken to detect the content of the tight junction proteins including the tight junction proteins ZO-1, Occludin and Claudin-1 in the supernatant of the colon tissues according to the colon tissue biochemical index measuring method.

The experimental results show (as in fig. 7-9) that CCFM1210 intervention significantly increased the concentration of zon-1, Occludin and Claudin-1 as compared to DSS group, and that ATCC15697 and FJND2M2 also increased the concentration of these proteins, but did not have statistical significance. The intervention of bifidobacterium longum subsp.

Example 6: effect of Bifidobacterium longum subspecies infantis CCFM1210 on cytokine levels in colon of colitis mice

The modeling method is the same as that in example 3, the mouse is sacrificed after the modeling obtained in the step (3) in the example 3 is finished, and the colon tissue is taken to detect the biochemical indexes in the supernatant of the colon tissue according to the colon tissue biochemical index measuring method, wherein the biochemical indexes comprise the concentrations of IL-1 beta, IL-6 and IL-10 in the colon tissue.

As can be seen from FIGS. 10-12, DSS treatment significantly increased the concentration of proinflammatory cytokines IL-1 β and IL-6 in colon tissue, reaching 772.33pg/mg protein and 44.36pg/mg protein, respectively, and reduced the concentration of the anti-inflammatory cytokine IL-10 to 5.12pg/mg protein. Whereas bifidobacterium longum subspecies infantis CCFM1210 treatment significantly reduced the concentration of IL-1 β and IL-6 to 444.47pg/mg protein and 29.71pg/mg protein and significantly increased the concentration of IL-10 to 11.92pg/mg protein. The intervention of ATCC15697 and FJND2M2 also showed the same trend of change in mouse colon inflammatory factor levels, but only ATCC15697 significantly decreased the concentration of proinflammatory cytokine IL-1 β. Thus, intervention of bifidobacterium longum subspecies infantis CCFM1210 has a significant effect on colon cytokine levels.

Example 7: effect of Bifidobacterium longum subspecies of infant CCFM1210 on butyric acid content in cecal intestine content of colitis mice

The molding method was the same as in steps (1) to (3) of example 3; after the completion of the molding obtained in step (3) in example 3, the mice were sacrificed, and the cecal contents were taken, weighed, dried, and the content of butyric acid was determined.

Short chain fatty acids are metabolites of the gut flora and are involved in host immune regulation and enhance gut barrier function. Among them, butyric acid plays a particularly important role in alleviating ulcerative colitis. Research has shown that butyric acid can inhibit the expression of proinflammatory factors and promote the expression of tight junction protein, and the use of butyric acid preparation can help to relieve ulcerative colitis. As can be seen from FIG. 13, the intervention of Bifidobacterium longum subspecies CCFM1210 significantly increased the content of butyric acid in the cecal contents compared to the DSS group, from 14.16. mu. mol/g to 20.61. mu. mol/g.

Example 8: effect of Bifidobacterium longum subspecies of infants CCFM1210 on intestinal flora diversity of colitis mice

The molding method was the same as in steps (1) to (3) of example 3; collecting the feces of the mice after the molding obtained in the step (3) in the example 3, extracting the genome DNA in the feces, sequencing the 16S rDNA, and analyzing the alpha diversity and the beta diversity of the flora as shown in the figures 14-17.

It has been shown that DSS modeling causes disorder of intestinal flora in mice, and thus the effect of intervention of bifidobacterium longum subsp. As can be seen from fig. 14, in terms of the Chao1 index, the blank control group, the bifidobacterium longum subspecies infantis ATCC15697 group, and the FJND2M2 group did not have significant difference (p >0.05) compared to the DSS group, but the bifidobacterium longum subspecies infantis CCFM1210 could significantly improve the species abundance of the intestinal community of the mice; as can be seen from fig. 15 and 16, with respect to the shannon diversity index and the simpson diversity index, DSS modeling reduced the diversity of microorganisms in the sample, CCFM1210 significantly increased the diversity of intestinal flora in the mouse fecal sample, while bifidobacterium longum subspecies infantis ATCC15697 group and FJND2M2 group did not have significant differences (p >0.05) compared to the model group.

As can be seen from fig. 17, the intestinal flora of DSS group mice was different from that of the blank control group mice; intestinal flora of FJND2M2 group mice is very similar to that of the model building group, and after intervention of Bifidobacterium longum subspecies CCFM1210, intestinal flora of the mice moves to a blank control group, and the intestinal flora of ATCC15697 group also moves to a certain extent but does not change greatly.

The results show that the bifidobacterium longum subspecies CCFM1210 can effectively improve the diversity of intestinal flora of mice with colitis and improve the intestinal flora disorder caused by DSS modeling.

Example 9: preparation of bifidobacterium longum subspecies infantis CCFM1210 microbial preparation

The method comprises the following specific steps:

(1) activation of the strain: streaking a bacterial liquid dipped with bifidobacterium longum subspecies infantis CCFM1210 from a freezing tube on an MRS solid culture medium, and culturing for 48 hours at 37 ℃ in an anaerobic environment to obtain a single colony; and selecting a single colony, inoculating the single colony in an MRS liquid culture medium, carrying out anaerobic culture at 37 ℃ for 24h for activation, and repeating the operation for 3 times to obtain activated bacterial liquid.

(2) Inoculating the bacterial liquid obtained in the step (1) into an MRS liquid culture medium according to the inoculation amount of 5%, carrying out anaerobic culture at 37 ℃ for 24h to obtain a fermentation liquid, centrifuging the obtained fermentation liquid at 8000g for 20min, collecting bacterial sludge, washing the bacterial sludge for 3 times by using PBS buffer solution with the pH of 7.4 for later use, and adjusting the viable count to be 1 multiplied by 10⁹CFU/mL to obtain liquid microbial preparation.

Alternatively, the preparation of the solid microbial preparation may be continued with the completion of the above steps as follows:

(3) preparing a freeze-drying protective agent: 130g/L of skim milk, 20g/L of trehalose, 20g/L of sucrose and the balance of water are mixed to obtain the freeze-drying protective agent.

(4) And (3) adding the freeze-drying protective agent prepared in the step (3) into the bacterial sludge obtained in the step (2), wherein the weight of the freeze-drying protective agent is 2 times of that of the bacterial sludge, uniformly mixing, performing vacuum freeze drying, and finally performing vacuum packaging on the preparation obtained by freeze drying.

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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gtccggtgtg aaagtccatc gcttaacggt ggatccgcgc cgggtacggg cgggcttgag 540

tgcggtaggg gagactggaa ttcccggtgt aacggtggaa tgtgtagata tcgggaagaa 600

caccaatggc gaaggcaggt ctctgggccg ttactgacgc tgaggagcga aagcgtgggg 660

agcgaacagg attagatacc ctggtagtcc acgccgtaaa cggtggatgc tggatgtggg 720

gcccgttcca cgggttccgt gtcggagcta acgcgttaag catcccgcct ggggagtacg 780

gccgcaaggc taaaactcaa agaaattgac gggggcccgc acaagcggcg gagcatgcgg 840

attaattcga tgcaacgcga agaaccttac ctgggcttga catgttcccg acgatcccag 900

agatggggtt tcccttcggg gcgggttcac aggtggtgca tggtcgtcgt cagctcgtgt 960

cgtgagatgt tgggttaagt cccgcaacga gcgcaaccct cgccccgtgt tgccagcgga 1020

ttgtgccggg aactcacggg ggaccgccgg ggttaactcg gaggaaggtg gggatgacgt 1080

cagatcatca tgccccttac gtccagggct tcacgcatgc tacaatggcc ggtacaacgg 1140

gatgcgacgc ggcgacgcgg agcggatccc tgaaaaccgg tctcagttcg gatcgcagtc 1200

tgcaactcga ctgcgtgaag gcggagtcgc tagtaatcgc gaatcagcaa cgtcgcggtg 1260

aatgcgttcc cgggccttgt acacaccgcc cgtcaagtca tgaaagtggg cagcacccga 1320

agccggtggc ctaacccctt gtgggatgga gccgtctaag gtgaggctcg tgattgggac 1380

taagtcgtaa caaggtagcc gtaccggaag gtgcggctgg atcacctcct tt 1432

Claims

1. Bifidobacterium longum subsp. infantis CCFM1210, deposited in Guangdong province collection of microorganisms with the deposit number GDMCC No: 62234, preservation date is 2022, 1 month, 23 days.

2. Food product comprising a bifidobacterium longum subsp.

3. The food product of claim 2, comprising a fermented food product or a fermented beverage product.

4. A medicament containing bifidobacterium longum subsp.

5. The medicament according to claim 4, wherein the Bifidobacterium longum subspecies of infantis CCFM1210 has a viable count of not less than 1 x 10⁹CFU/g or 1X 10⁹CFU/mL。

6. The medicament according to claim 4 or 5, further comprising a pharmaceutical carrier and/or a pharmaceutical excipient.

7. Use of a bifidobacterium longum subsp.

8. The use of claim 7, wherein the treatment or amelioration of colitis comprises at least one of the following functions:

(1) relieving weight loss;

(2) reducing damage to colon tissue;

(3) inhibit the reduction of Claudin-1, Claudin-1 and Occludin;

(5) increasing the concentration of butyric acid in the intestinal tract;

(6) increasing the diversity of the intestinal flora and/or improving the intestinal flora disturbance.

9. A microbial preparation comprising Bifidobacterium longum subspecies infantis CCFM1210 according to claim 1.

10. Use of a microbial preparation of bifidobacterium longum subsp.