CN118126894A

CN118126894A - Probiotic capable of antagonizing enterobacteriaceae and relieving colonitis

Info

Publication number: CN118126894A
Application number: CN202410348881.XA
Authority: CN
Inventors: 翟齐啸; 高晓翔; 张程程; 陈卫; 于雷雷; 陆文伟; 田丰伟; 崔树茂; 王刚; 赵建新
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Filing date: 2024-03-26
Publication date: 2024-06-04

Abstract

The invention discloses a bifidobacterium breve capable of regulating and controlling the relative abundance of enterobacteriaceae, belonging to the technical field of microorganisms. According to the invention, a bifidobacterium breve (Bifidobacterium breve) CCFM1369 is screened out, and the bifidobacterium breve CCFM1369 can remarkably inhibit the relative abundance of enterobacteriaceae in the intestinal tract of a mouse. Therefore, the bifidobacterium breve CCFM1369 has great application prospect in preparing products (such as foods or medicines and the like) for adjusting the abundance of strains of the enterobacteriaceae and further preventing and/or treating related diseases including colonitis.

Description

Probiotic capable of antagonizing enterobacteriaceae and relieving colonitis

Technical Field

The invention relates to a bifidobacterium breve capable of antagonizing enterobacteriaceae and relieving colonitis, belonging to the technical field of microorganisms.

Background

Enterobacteriaceae, including the symbiotic bacteria Escherichia coli, klebsiella and Proteus, is a large family of gram-negative facultative bacteria belonging to the class Gamma-Proteus and Proteus. Enterobacteriaceae are present in relatively low levels in the intestinal tract and are located near mucosal epithelium due to their relatively high tolerance to oxygen diffusion from the epithelium. Enterobacteriaceae are the most common overgrown symbiota in inflammatory diseases, such as inflammatory bowel disease, obesity, colorectal cancer, celiac disease and antibiotic therapy. Environmental and nutritional changes caused by intestinal inflammation may give growth advantages to enterobacteriaceae bacteria. In many cases, intestinal inflammation appears to provide a favorable environment for the expansion of enterobacteriaceae bacteria, whether it is pathogen infection, chemically induced colitis or host immunodeficiency. For example, in patients with Crohn's disease or ulcerative colitis, the prevalence of Enterobacteriaceae bacteria, including Adherent Invasive E.coli (AIEC), has also increased. The mass production of enterobacteria often has a negative effect on the host's defense against pathogens or injury. For example, in mice receiving a dose of clindamycin treatment, enterobacteria breeding may also enhance susceptibility to clostridium difficile induced colitis. Coli pathogens present in antibiotic or Dextran Sulfate Sodium (DSS) treated mice can lead to bacteremia and ultimately death of the mice. Lipopolysaccharide from the enterobacteriaceae family has been shown to exacerbate intestinal damage caused by non-steroidal anti-inflammatory drugs and to increase intestinal permeability of celiac disease.

Intestinal microorganisms play a key role in preventing colonization by pathogens and inhibiting growth, with probiotics playing a major beneficial role, including immune system regulation, enhancement of intestinal epithelial barriers, and competition with pathogens. Several studies have shown that probiotics reduce adhesion of Salmonella typhimurium, clostridium sporogenes and enterococcus faecalis to Caco-2 cells. Lactobacillus plantarum 299v competes for mucin with listeria monocytogenes to reduce colonization of listeria monocytogenes. In addition, probiotics can secrete mutans, streptococcus lactis, lactic acid bacteria and the like to inhibit the growth of harmful bacteria. Nevertheless, the relationship between probiotics and enteric pathogens is still unclear due to the complexity of the human gut.

For screening probiotics capable of antagonizing specific harmful bacteria from a huge strain resource library, a great deal of funds and manpower are generally consumed, and the traditional mode is to co-culture the probiotics and the harmful bacteria in an in-vitro manner, so that the probiotics potentially targeting the specific harmful bacteria are screened out. At present, the genome scale metabolism model is gradually matured through continuous iteration and updating, and the construction of the 733 intestinal tract bacteria model facilitates the simulation and prediction of the metabolic capacity of the strain by people. The 733 strain is simulated in pairs one by one, and a technical basis is provided for screening probiotics of harmful bacteria so as to obtain a probiotic strain for regulating intestinal microorganisms.

Disclosure of Invention

Aiming at the defects of the prior art, the invention obtains the bifidobacterium breve capable of antagonizing enterobacteriaceae and relieving colonitis by screening, and aims to solve the technical problem of the existing probiotic strain for antagonizing enteropathogenic microorganisms and improving colonitis.

The first technical scheme provided by the invention is that a bifidobacterium breve (Bifidobacterium breve) CCFM1369, the bifidobacterium breve CCFM1369 is preserved in the microorganism strain preservation center of Guangdong province at the 1 st day of 2024, the preservation number is GDMCC No.64317, and the preservation address is the No. 59 building 5 of the university 100 in Guangzhou martyr.

The bifidobacterium breve (Bifidobacterium breve) CCFM1369 is isolated from a fecal sample from a 83 year old male in Shunfu village in mountain pond, lian, guangdong, and the strain is sequenced and analyzed, the 16SrDNA sequence of the strain is shown as SEQ ID NO.1, and BLAST nucleic acid sequence alignment is carried out on the sequence obtained by sequencing in NCBI, so that the matching degree with the bifidobacterium breve is 99.83%, which indicates that the strain is bifidobacterium breve and is named as bifidobacterium breve (Bifidobacterium breve) CCFM1369.

The bacterial cells of the bifidobacterium breve (Bifidobacterium breve) CCFM1369 grow on an MRS culture medium and are characterized in that: the bacterial colony has round front surface shape, diameter of 0.3-2 mm, raised side surface shape, milky white color, wet and smooth surface, gram negative dyeing and no spore formation.

Growth characteristics of the bifidobacterium breve CCFM 1369: strictly anaerobic, sensitive to oxygen, and optimal in growth at 30-37 ℃ and optimal in initial growth pH of 7.0.

The second technical scheme provided by the invention is a microbial preparation containing the bifidobacterium breve (Bifidobacterium breve) CCFM1369 according to the first technical scheme.

In certain embodiments, the viable count of the bifidobacterium breve CCFM1369 in the microbial preparation is not less than 1×10 ⁶ CFU/mL or 1×10 ⁶ CFU/g.

Further, the viable count of the bifidobacterium breve CCFM1369 in the microbial preparation is not less than 1X 10 ⁹ CFU/mL or 1X 10 ⁹ CFU/g.

The third technical scheme provided by the invention is a product containing the bifidobacterium breve CCFM1369 according to the first technical scheme or the microbial preparation according to the second technical scheme.

In certain embodiments, the product comprises a food product or a pharmaceutical product.

In certain embodiments, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products containing bifidobacterium breve.

In certain embodiments, the pharmaceutical product contains bifidobacterium breve CCFM1369, a pharmaceutical carrier and/or a pharmaceutical adjuvant as described above.

In certain embodiments, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and/or liposomes.

In certain embodiments, the pharmaceutical excipients comprise fillers, binders, wetting agents, disintegrants, lubricants and/or flavoring agents.

In certain embodiments, the pharmaceutical product is in the form of a powder, granule, capsule, tablet, pill, or oral liquid.

In certain embodiments, the food product is a health food, beverage, or snack.

The fourth technical scheme provided by the invention is the application of the bifidobacterium breve CCFM1369 or the microbial preparation of the second technical scheme in the preparation of products for reducing the relative abundance of Enterobacteriaceae (Enterobacteriaceae) in intestinal tracts.

In certain embodiments, the enterobacteriaceae include E.coli, E.coli

In certain embodiments, the product comprises a food product, a pharmaceutical product, or a nutraceutical product.

In certain embodiments, the food product is a health food, beverage, or snack.

The fifth technical scheme provided by the invention is the application of the bifidobacterium breve CCFM1369 of the first technical scheme or the microbial preparation of the second technical scheme in preparing products for relieving colonitis and/or improving intestinal health.

In certain embodiments, the application includes at least one of the following:

(1) Inhibiting dilation of the enterobacteriaceae family of the individual;

(2) Stabilizing the body weight of the individual suffering from colitis;

(3) Stabilizing the colon length of the individual suffering from colitis.

In certain embodiments, the food product is a health food, beverage, or snack.

The invention has the beneficial effects that:

1. The invention screens out a bifidobacterium breve (Bifidobacterium breve) CCFM1369 which has the effect of regulating intestinal pathogens and is specifically characterized in that: the relative abundance of enterobacteriaceae in the intestinal tract is significantly reduced. Therefore, the bifidobacterium breve CCFM1369 has great application prospect in preparing products (such as foods or medicines and the like) which antagonize the abundance of the enterobacteriaceae and further prevent and/or relieve related diseases.

2. The bifidobacterium breve (Bifidobacterium breve) CCFM1369 can effectively relieve colonitis, thereby improving intestinal health, and is specifically characterized in that: (1) inhibiting the distension of enterobacteriaceae; (2) stabilizing the weight of the individual suffering from colitis; (3) stabilizing colon length in an individual with colitis. Has great application prospect in preparing products for relieving colonitis and/or improving intestinal health. .

Preservation of biological materials

A bifidobacterium breve CCFM1369, taxonomic designation Bifidobacterium breve, was deposited at the Cantonese province microorganism strain collection at day 1 and day 25 of 2024 under accession number GDMCC No.64317 and at accession number Guangzhou city martyr at floor 5, college 100.

Drawings

FIG. 1 bifidobacterium breve CCFM1369 plate count;

morphological observation and microscopic identification of bifidobacterium breve CCFM1369 of FIG. 2;

FIG. 3 in vitro screening assay to screen for probiotics that inhibit E.coli; bib6: bifidobacterium breve FZJHZ-M2, bib14: bifidobacterium breve HuNan-2016 49-7, bib9: bifidobacterium breve CJ1041, bib2: bifidobacterium breve CJ951, bib11: bifidobacterium breve JSWX M4; bia5: bifidobacterium animalis FSDHZD m6_t72, bia6: bifidobacterium animalis FFJNDD7m3_t73, bia12: bifidobacterium animalis SDJN M ② 1032, bia11: bifidobacterium animalis AHWH M1, bia9: bifidobacterium animalis FBJHD m6_t74); la: lactobacillus acidophilus FFJND L5;

FIG. 4 effect of Bifidobacterium breve CCFM1369 on the relative abundance of enterobacteriaceae in colon inflammatory mice;

FIG. 5 effect of Bifidobacterium breve CCFM1369 on absolute abundance of Enterobacteriaceae in colon inflammatory mice;

FIG. 6 effect of Bifidobacterium breve CCFM1369 on body weight of mice with colitis;

FIG. 7 effect of Bifidobacterium breve CCFM1369 on colon length in colon inflammatory mice.

Detailed Description

The invention is further illustrated below in conjunction with specific embodiments and figures.

MRS broth medium referred to in the following examples was purchased from Qingdao high tech Industrial Yuan Haibo Biotechnology Co., ltd; the FAST DNA SPIN KIT for Feces kit referred to in the examples below was purchased from MP Biomedicals company.

The following examples relate to the following media:

1. Bifidobacterium selective medium (1L): 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 0.1g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate monohydrate, distilled water: 1000mL; pH:6.2-6.4; sterilizing at 115 deg.C for 20min. When preparing the solid culture medium, 15g of agar is added, and before the plate is poured, sterile mupirocin and sterile nystatin are added according to the volumes of 1 per mill and 0.5 per mill of the culture medium respectively, namely, the final concentrations are 100 mug/mL mupirocin and 25U/mL nystatin respectively.

2. MRS liquid Medium (1L): 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 0.1g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate monohydrate, distilled water: 1000mL; pH:6.2-6.4; sterilizing at 115 deg.C for 20min.

3. MRS solid Medium (1L): 10g of peptone, 10g of beef extract, 5g of yeast extract, 20g of glucose, 5g of sodium acetate, 1mL of Tween 80, 2g of dipotassium hydrogen phosphate, 2g of diammonium citrate, 0.1g of magnesium sulfate heptahydrate, 0.05g of manganese sulfate monohydrate, distilled water: 1000mL; pH:6.2-6.4; sterilizing at 115 deg.C for 20min. When preparing the solid medium, 15g of agar was added.

The preparation method of the bifidobacterium breve suspension in the following examples is as follows:

Streaking the bifidobacterium breve onto an MRS solid culture medium, and carrying out anaerobic culture for 48 hours at 37 ℃ to obtain a single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activated liquid into MRS liquid culture medium (inoculum size is 2% -4% (v/v)), and performing anaerobic culture for 18h at 37 ℃ to obtain bacterial liquid; centrifuging 10000g of the bacterial liquid for 5min to obtain bifidobacterium breve bacterial cells; the bifidobacterium breve cells were washed with normal saline and resuspended in 20% glycerol solution (containing 1g/L cysteine hydrochloride) to a cell concentration of 1X 10 ¹⁰ CFU/mL and stored in a-80℃refrigerator.

Example 1: computer simulation screening of probiotics antagonizing escherichia coli

1. Downloading through Virtual Metabolic Human database to build enterobacteria genome scale metabolism model AGORA 1.03. Further downloading python, installing cobrapy software in python. Simulated co-cultivation was set according to cobra.9.0 documents online. The culture medium simulating co-cultivation is selected from nutrient files already configured in Virtual Metabolic Human database: comprises DACH,EU_average,Gluten_free,High_fat_low,High_fiber_VMH,High_protein_VMH,HighFiber_AGORA,Mediterranean_VMH,Type_2_diabetes_VMH,Unhealthy_VMH,Vegan_VMH,Vegetarian_VMH,Western_AGORA. selecting interaction mode as harmful commensal, and carrying out further in vitro screening verification on probiotics which can potentially inhibit escherichia coli, wherein the interaction mode is shown in table 1.

Table 1 computer simulation screening of probiotics against E.coli

As can be seen from Table 1, the probiotics which can antagonize the Escherichia coli are bifidobacterium animalis and bifidobacterium breve,

Example 2: separation and screening of bifidobacterium breve CCFM1369

1. Sample collection

Collecting 83-year-old male feces sample of Shunjiao village in mountain pond of Guangdong, placing the sample in a sampling tube filled with 30% glycerol, storing in a thermal insulation box filled with ice bag, taking back to laboratory, and rapidly placing in a refrigerator at-80deg.C for separation and screening.

2. Separation and purification of bifidobacteria

(1) And (3) dilution coating: taking about 0.5g of fecal sample, adding the fecal sample into a 10mL centrifuge tube filled with 4.5mL of physiological saline under a sterile environment to obtain 10 ^-1 of diluent, and repeating the dilution steps to sequentially obtain 10 ^-2、10^-3、10^-4、10^-5、10^-6 of diluent;

(2) Coating and culturing: respectively sucking 100 μl of the 10 ^-4、10^-5、10^-6 gradient dilutions on MRS screening culture medium, coating uniformly with a coating rod, and culturing at 37deg.C under anaerobic condition for 48 hr;

(3) Primary purification culture: taking a dilution coating flat plate with colony number in a range of 30-300, randomly selecting 10 single colonies which are milky white, smooth in surface and neat in edge from each sample, streaking the single colonies on an MRS solid culture medium, culturing the single colonies under anaerobic condition at 37 ℃ for 48 hours to obtain single colonies, and repeating the steps to obtain second streaked single colonies.

(4) Secondary purification culture: and (3) inoculating single colony on the second streaking plate in the step (3) into MRS liquid culture medium, and culturing for 12 hours under anaerobic condition at 37 ℃ to obtain secondary purified culture solution.

3. Preservation and identification of strains

(1) And (3) strain preservation:

Mixing the secondary purified culture solution by shaking, taking 700 mu L of bacterial solution (cultured for 18-24 h) to a 2mL clean strain preservation tube, adding 6 parts in parallel, adding 5 parts and 700 mu L of 40% glycerol to resuspend, standing for 30min, and then placing in a refrigerator at-80 ℃ for preservation; 1 part of bacterial liquid is used for identifying strains, 8000r/min is centrifuged for 3min, and the supernatant is discarded to obtain bacterial cells.

(2) And (3) strain identification:

adding 0.5mL of sterile water into a preservation tube for strain identification, blowing and washing thalli, centrifuging for 1min at 10000r/min, discarding the supernatant to obtain thalli, and adding 500 mu L of sterile water for resuspension to serve as a template of a PCR strain identification system;

Wherein, the primers and the system of the 16S rDNA PCR are shown in Table 1 and Table 2 respectively;

conditions of 16S rDNA PCR: the first step: 94 ℃ and 10min for the second step: 94 ℃,30s third step: 55 ℃,30s, fourth step: 72 ℃,2min, fifth step: the second to fourth steps were carried out for 30 cycles at 72℃for 10 min.

TABLE 1 primer names

16S rDNA PCR primer name	Sequence(s)
		27F	5’-AGAGTTTGATCCTGGCCTCA-3’
1492R	5’-GGTTACCTTGTTACGACTT-3’

TABLE 2 bacterial species identification 25. Mu.L 16S rDNA PCR reaction system

Component (A)	Addition (mu L)
		27F	0.25
1492R	0.25
		Taq enzyme Mix	12.5
Template	1
		Double distilled water	11

After the PCR product is confirmed to be a single bright band by nucleic acid electrophoresis, the PCR product is sent to the large gene for sequencing; the 27F and 1492R splice sequences returned from sequencing were uploaded to BLAST (http:// www.ncbi.nlm.nih.gov/BLAST) at NCBI for species validation; the comparison result shows that the strain with the number of CCFM1369 is a bifidobacterium breve strain, and the 16S rDNA amplification sequence is shown as SEQ ID NO. 1.

Example 3: in vitro screening of probiotics against E.coli

And selecting 5 strains of bifidobacterium breve and bifidobacterium animalis in a Jiang Nada-way bacterial library (comprising bifidobacterium breve FZJHZ-M2, huNan-2016 49-7, CJ1041, CJ951 and CCFM1369; bifidobacterium animalis FSDHZD M6-T72, FFJNDD7M 3-T73, SDJN2M ②, AHWH4M1, FBJHDM4M 6-T74 and lactobacillus acidophilus FFJND L5), activating for two generations, and filtering with a sterile filter membrane of 0.22 mu M to obtain a fermentation supernatant for later use. The concentration of the activated two-generation escherichia coli suspension is adjusted to be 10 ⁷ CFU/mL, 100 mu L of the activated two-generation escherichia coli suspension is coated on an MRS flat plate, an oxford cup is placed and lightly pressed, 150 mu L of bifidobacterium breve fermentation supernatant is added, the mixture is placed in an anaerobic incubator for culturing for 72-96 hours, the diameter of a bacteria inhibition zone is measured, and an MRS culture medium is used as a negative control. The results of bacteriostasis are shown in figure 3.

As can be seen from fig. 3, bib11, among the eleven probiotics selected, is the bifidobacterium having the most bacteriostatic effect on escherichia coli, with a bacteriostatic diameter of 1.43±0.09cm on average.

Example 4: effect of Bifidobacterium breve CCFM1369 on Enterobacteriaceae mice

The method comprises the following specific steps:

24 healthy male BALB/c mice with the age of 6 weeks and 20-22 g are randomly divided into 4 groups of 6 mice each, and the 6 groups are respectively: blank, model and CCFM1369 and CCFM622 intervention groups of bifidobacterium intragastric respectively.

The experimental process is as follows: after one week of adaptation, mice were changed to 3% dss for 7 days. Blank control group, lavage 0.2mL physiological saline per day; DSS group (LOP), lavage 0.2mL of saline; the composition difference of intestinal flora in the faecal samples of the second generation sequencer after PCR amplification of the 16s V3-V4 region sequence after the extraction of the bacterial genome of each group of mouse faeces by using the FAST DNA SPIN KIT for Feces kit is shown in figures 4 and 5, wherein the composition difference of intestinal flora in the faecal samples of the second generation sequencer is obtained by respectively carrying out stomach infusion on 0.2mL of the solution of the strain containing 1X 10 ⁹ CFU of the bifidobacterium breve CCFM1369 and the bifidobacterium breve CCFM662 each day, and collecting the mouse faeces on 14 th day.

As shown in fig. 4 and 5, enterobacteriaceae produce abrupt expansion to occupy ecological niches when intestinal homeostasis is unbalanced, and the abundance of the model group mice enterobacteriaceae is significantly increased to 2434.33 ± 1753.48, and the absolute abundance is increased to 10 ^7.8 CFU. Bifidobacterium breve CCFM1369 (Bib 11) significantly inhibits the expansion of Enterobacteriaceae, reduces the relative abundance of Enterobacteriaceae to 923.67 + -407.49, and reduces the absolute abundance to 10 ^6.97 CFU. In fig. 6, mice from the dss+bifidobacterium breve CCFM1369 strain group had a weight loss of at least 80% of the initial weight on day 7, with subsequent weight gain, approaching the placebo group. In FIG. 7, the colon length of the model mice was 6.24.+ -. 0.39cm, and the colon lengths were 6.94.+ -. 0.4cm and 6.46.+ -. 0.78cm, respectively, after treatment with bifidobacterium breve CCFM1369 and bifidobacterium breve CCFM 662. Based on the results of fig. 6 and 7, it was found that bifidobacterium breve CCFM1369 has an effect of potentially inhibiting the weight and colon length of enterobacteriaceae-stable mice.

It can be seen that bifidobacterium breve CCFM1369 is capable of inhibiting enterobacteriaceae expansion and alleviating colitis in mice.

Example 5: application of bifidobacterium breve CCFM1369

The bifidobacterium breve CCFM1369 can be used for preparing capsule products, and the specific preparation process of the capsule products is as follows:

The bifidobacterium breve CCFM1369 is streaked on MRS solid culture medium and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in an MRS improved liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously to obtain an activation solution; inoculating the activating solution into an MRS improved liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18 hours at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and then re-suspending the bacterial mud with a protective agent until the concentration is 1X 10 ¹⁰ CFU/mL to obtain bacterial suspension; adding the bacterial suspension into sodium alginate solution with the concentration of 30g/L until the concentration is 2X 10 ⁹ CFU/mL, and fully stirring to uniformly disperse cells of bifidobacterium breve CCFM1369 in the sodium alginate solution to obtain a mixed solution; extruding the mixed solution into a calcium chloride solution with the concentration of 20g/L to form colloidal particles; after the formed colloidal particles are stationary and solidified for 30min, filtering and collecting the colloidal particles; freeze-drying the collected colloidal particles for 48 hours to obtain powder; and (5) filling the powder into a medicinal capsule to obtain a capsule product.

Example 6: application of bifidobacterium breve CCFM1369

The bifidobacterium breve CCFM1369 can be used for preparing tablets, and the specific preparation process of the tablets is as follows:

The bifidobacterium breve CCFM1369 is streaked on MRS solid culture medium and cultured for 48 hours at 37 ℃ to obtain single colony; single colony is selected and inoculated in MRS liquid culture medium, and is cultured for 18 hours at 37 ℃ for activation, and the activation is carried out for two generations continuously, so as to obtain an activation solution; inoculating the activating solution into MRS liquid culture medium according to the inoculum size of 2% (v/v), and culturing for 18h at 37 ℃ to obtain bacterial solution; centrifuging 8000g of bacterial liquid for 10min to obtain bacterial mud; washing the bacterial mud with physiological saline for 3 times, and then re-suspending the bacterial mud with a protective agent until the concentration is 1X 10 ¹⁰ CFU/mL to obtain bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain bifidobacterium breve CCFM1369 bacterial powder;

Wherein the protective agent is a skimmed milk powder solution with the concentration of 130 g/L. Weighing 25.7 parts by weight of bifidobacterium breve CCFM1369 bacterial powder, 55.0 parts by weight of starch, 4.5 parts by weight of cellulose derivative, 12.0 parts by weight of carboxymethyl starch sodium, 0.8 part by weight of talcum powder, 1.0 part by weight of sucrose and 1.0 part by weight of water to obtain raw materials; mixing the raw materials to obtain wet particles; tabletting the wet granules by a tablet press of a pharmaceutical machinery factory in the middle south, and drying by a small-sized drug dryer of Yikang traditional Chinese medicine machinery Co., ltd.

While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and 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.

Claims

1. A strain of bifidobacterium breve (Bifidobacterium breve), wherein the strain of bifidobacterium breve is deposited at the cantonese province at the collection of microorganisms and cell cultures under accession number GDMCC No.64317 and at a date of deposition of 2024, day 1 and day 25.

2. A microbial preparation comprising the bifidobacterium breve CCFM1369 of claim 1.

3. The microbial preparation according to claim 2, wherein the viable count of the bifidobacterium breve CCFM1369 in the microbial preparation is not less than 1 x 10 ⁶ CFU/mL or 1 x 10 ⁶ CFU/g.

4. A product comprising the bifidobacterium breve CCFM1369 of claim 1 or the microbial agent of claim 2 or 3.

5. The product of claim 4, wherein the product comprises a food, pharmaceutical, or nutraceutical.

6. Use of a microbial preparation comprising bifidobacterium breve CCFM1369 as claimed in claim 1 or claim 2 or 3 for the manufacture of a product for use in reducing the relative abundance of Enterobacteriaceae (Enterobacteriaceae) in the gut.

7. The use according to claim 6, wherein the viable count of bifidobacterium breve CCFM1369 in the product is not lower than 1 x 10 ⁶ CFU/mL or 1 x 10 ⁶ CFU/g.

8. Use of a bifidobacterium breve CCFM1369 of claim 1 or a microbial agent of claim 2 or 3 in the manufacture of a medicament for alleviating colitis and/or improving intestinal health.

9. The use according to claim 8, characterized in that the use comprises at least one of the following actions:

(1) Inhibiting dilation of the enterobacteriaceae family of the individual;

(2) Stabilizing the body weight of the individual suffering from colitis;

(3) Stabilizing the colon length of the individual suffering from colitis.

10. The use according to claim 8, wherein the medicament comprises the above bifidobacterium breve CCFM1369, a pharmaceutical carrier and/or a pharmaceutical adjuvant.