CN116286439A - Bifidobacterium bifidum from infant intestinal tracts and application thereof - Google Patents

Bifidobacterium bifidum from infant intestinal tracts and application thereof Download PDF

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CN116286439A
CN116286439A CN202210991241.1A CN202210991241A CN116286439A CN 116286439 A CN116286439 A CN 116286439A CN 202210991241 A CN202210991241 A CN 202210991241A CN 116286439 A CN116286439 A CN 116286439A
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程如越
沈曦
何方
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Abstract

The invention discloses bifidobacterium bifidum from infant intestinal tracts and application thereof, and belongs to the technical field of biology. The invention provides a bifidobacterium bifidum from infant intestinal tracts, which has a preservation number of CGMCC NO.24517. The bifidobacterium bifidum provided by the invention is applied to the preparation of medicines or foods with the function of regulating immunity. Experiments show that the bifidobacterium bifidum has stronger adhesion to Caco-2 cells and also specifically stimulates the Caco-2 cells to obviously secrete IL-8.

Description

Bifidobacterium bifidum from infant intestinal tracts and application thereof
Technical Field
The invention belongs to the technical field of biology, and particularly relates to bifidobacterium bifidum from infant intestinal tracts with a preservation number of CGMCC No.24517 and application thereof.
Background
Bifidobacteria are the major components of the human intestinal microbiota and are one of the most important dominant microbiota in the infant intestinal tract. The first week after birth, bifidobacteria begin to colonise the neonatal gut, forming a protective barrier against potential pathogens and stimulating the immune system. Bifidobacterium bifidum (Bifidobacterium bifidum, b.bifidum) is one of the representative species of bifidobacteria in the infant intestinal tract. The study shows that b.bifidum can prevent infantile diarrhea caused by rotavirus and significantly reduce stress-related behavior, and is likely to treat stress-related diseases. There are studies indicating that bifidobacteria promote the development of thymus in infants, thereby enhancing their response to oral administration and injection of vaccines.
The low content of bifidobacteria in the intestinal tract is an important characteristic of abnormal intestinal flora of infants suffering from allergic diseases. The phenomenon that the quantity of intestinal bifidobacteria is too low in infants suffering from allergic diseases occurs even before the infants start to suffer from the allergic diseases, which indicates that the too low content of the intestinal bifidobacteria may be one of the factors inducing the allergic diseases in the infants. In recent years, it has been found that there are significant differences in the species composition of bifidobacteria in addition to differences in the number of intestinal bifidobacteria between healthy children and infants suffering from allergic diseases. It has been found that infants suffering from allergic diseases may have fewer b.longum and b.breve and other infant bifidobacteria colonization and more b.adolescence and other adult flora colonization than infants not suffering from allergic diseases. Thus, the species composition of bifidobacteria infantis may be closely related to allergic diseases, and the composition and function of bifidobacteria enterica at different age stages should be studied in more detail.
Disclosure of Invention
The invention aims to provide bifidobacterium bifidum from infant intestinal tracts, which has strong adhesion to Caco-2 cells and can also specifically stimulate the Caco-2 cells to obviously secrete IL-8.
The invention also provides application of the bifidobacterium bifidum in preparing medicines or foods with the immunity regulating effect.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
the invention provides a bifidobacterium bifidum from infant intestinal tracts, which has a preservation number of CGMCC NO.24517.
The bifidobacterium bifidum provided by the invention is applied to the preparation of medicines or foods with the function of regulating immunity.
In some embodiments of the invention, the pharmaceutical or food product comprises a pharmaceutical or food product having intestinal epithelial cell adhesion function; preferably, it comprises a pharmaceutical or food product having an adhesion to Caco-2 cells.
In some embodiments of the invention, the pharmaceutical or food product comprises a pharmaceutical or food product that stimulates secretion of IL-8; preferably, a pharmaceutical or food product is included that stimulates IL-8 secretion by Caco-2 cells.
In some embodiments of the invention, the strain in the pharmaceutical or food product is an active bacterium.
In some embodiments of the invention, the strain in the pharmaceutical or food product is an inactivated strain.
In some embodiments of the invention, the strain in the pharmaceutical or food product is a heat-inactivated bacterium.
In some embodiments of the invention, the pharmaceutical or food product comprises bifidobacterium bifidum with a collection number of CGMCC No.24517, and one or more pharmaceutically acceptable carriers.
In some embodiments of the invention, the pharmaceutical or food product is an oral formulation.
In some embodiments of the invention, the oral formulation comprises a solid oral formulation or a liquid oral formulation.
By "pharmaceutically acceptable carrier" as used herein is meant a diluent, adjuvant, excipient or vehicle with which the therapeutic agent is administered, and which is suitable for contacting the tissues of humans and/or other animals without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable benefit/risk ratio, within the scope of sound medical judgment.
The bifidobacterium bifidum BD-1 of the present invention was isolated from the intestinal tract of a healthy infant of one year old and identified as bifidobacterium bifidum. Experiments show that the bifidobacterium bifidum BD-1 has stronger adhesion to Caco-2 cells, and can also specifically stimulate the Caco-2 cells to obviously secrete IL-8, thereby improving the immunity of organisms.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates bifidobacteria from intestinal tracts of healthy infants of one year to obtain bifidobacteria BD-1. Animal experiments show that the bifidobacterium bifidum BD-1 has stronger adhesion to Caco-2 cells, and can also specifically stimulate the Caco-2 cells to obviously secrete IL-8.
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FIG. 1 is a view of strain BD-1 under a microscope (1000X).
FIG. 2 is a plate of colony morphology of strain BD-1.
FIG. 3 is a phylogenetic tree of strain BD-1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
This example discloses a method for separating and screening bifidobacterium bifidum BD-1 of the present invention.
1. Isolation of strains from crowd sources
Normal term newborns born at the university of Sichuan Hua Xifu child-producing hospital between 3 months 2013 and 4 months 2013 were selected as subjects. Inclusion criteria: residing in five urban areas of Chengdu city; the birth weight is 2500-4000 g after 37-42 weeks, and the infant has no congenital abnormality or birth defect. Exclusion criteria: the mother used antibiotics within one month before birth; parents have infectious diseases such as AIDS, tuberculosis, hepatitis B and the like; the neonate cannot collect the feces due to severe diseases such as neonatal pneumonia. The study followed the Helsink principle, and infants incorporated into the study were given informed consent by their father or mother on their behalf. The collection of faeces from a follow-up infant aged 1 facilitates a sterilized faeces cassette. Immediately after sampling, the sample was stored at 4℃and sent to a laboratory at low temperature by a researcher for dilution culture of the stool sample, and if the sample could not be immediately handled, the sample was stored at 4℃anaerobically and cultured on the same day.
2. Separation of dominant bifidobacteria in specimens
0.5g of excrement is weighed, 4.5mL of excrement diluent is added, and the mixture is stirred and mixed uniformly for serial 10-time dilution. 100 mu L of fecal mixed solution with proper concentration is respectively and evenly coated on a BL plate containing 5% horse blood by an L-shaped glass rod, and cultured for 48-72 h under anaerobic condition at 37 ℃. The total number of anaerobes was counted and suspicious colonies with bifidobacteria characteristics were picked from the high dilution BL plates and inoculated on selective medium TOS plates and placed in anaerobic conditions at 37℃for 48h, based on the remarkable characteristics of bifidobacteria growth on BL plates (brownish in central brown or marginal portion, smooth and moist surface, rounded, bulging, clean edge, diameter about 3-5 mm). The bacteria growing well on TOS plate are selected for gram staining, the form of the bacteria is observed under microscope, microscopic examination is gram positive bacillus, the staining is uneven, one end or two ends are enlarged or forked, no spore exists, and the bacteria are inoculated on MRS plate, after aerobic culture for 48 hours at 37 ℃, the bacteria are completely free of growing, the bacteria are primarily judged to be bifidobacterium, and the bacteria are preserved at-80 ℃ by taking skimmed milk powder as solvent.
Example 2
The bifidobacterium bifidum BD-1 of the invention is sent to China center for identification,
1. the strain is shown in figure 1 and figure 2.
2. And (3) carrying out 16SrRNA identification, wherein the identification sequence of the 16SrRNA of the strain is shown as SEQ ID NO: 1.
3. The results of BLAST sequencing of the 16SrRNA gene of this strain are shown in Table 1:
TABLE 1
Figure BDA0003744760830000041
4. The Neighbor-training phylogenetic tree constructed by taking Scardovia inopinata DSM 10107 (D89332) as an outer branch based on the 16S rRNA gene sequence comparison result is shown in the figure 3.
5. The strain of the invention is subjected to physiological and biochemical characteristic detection, the enzyme activity detection result is shown in table 2, and the acid production result by using a carbon source is shown in table 3.
TABLE 2 physiological and biochemical Properties of strain BD-1-enzyme Activity
Figure BDA0003744760830000051
Figure BDA0003744760830000061
+: a positive reaction; -: a negative reaction; w: weak positive reaction
TABLE 3 physiological and biochemical characteristics of strain BD-1-acid production Using carbon sources
Figure BDA0003744760830000062
Figure BDA0003744760830000071
+: a positive reaction; -: a negative reaction;
based on the above-described detection results, the strain BD-1 of the present invention was identified as Bifidobacterium bifidum (bifidobacterium bifidum, great name: bacillus bifidus).
Test example 1
The test example discloses an in vitro adhesion test of the bifidobacterium bifidum BD-1 of the invention
Bifidobacterium bifidum BD-1 was grown on MRS medium (chinese road bridge technology ltd) containing 0.5% agar powder (beijing soleba technology ltd) and 0.03% l-cysteine (chinese korea). Anaerobic growth was carried out at 37℃for 48h, and the test bacteria were collected from the MRS agar surface and washed twice with sterilized 0.85% sodium chloride. The test bacterial solution was heat-inactivated in a water bath at 90℃for 15min to prepare heat-inactivated bacteria. Dissolving the test bacteriaThe absorbance of the solution at 625nm was adjusted to 0.200.+ -. 0.005 to normalize the bacterial count (about 2X 10) 8 cfu/mL)。
Caco-2 cell line (ATCCHTB-37) was purchased from the American type culture Collection ATCC (Rockville, malyland). Cells were cultured in 95% air and 5% carbon dioxide (Thermo U.S.) using dabber's essential minimal medium (DMEM, gibco, usa) supplemented with 10% fetal bovine serum (Gibco, usa), 2% penicillin and streptomycin (5000 IU/mL,5000 μg/mL).
Caco-2 cells were seeded onto sterile 6-wall plates (the company of biomedical engineering, inc. of beaver, suzhou) at a concentration of 2.0 mL/well. After Caco-2 cells became confluent monolayer and fully differentiated, they were washed twice with phosphate buffer (PBS, pH7.2, 10mmol/L phosphate). 100. Mu.L of the bacterial suspension prepared as above was added to the test wells simultaneously with 1.9mL of intact DMEM (Gibco Co., USA) at 37℃with 95% air and 5% CO 2 Is cultured in the environment of (Thermo U.S.). Incubate for 2h, wash all monolayers twice with PBS. Cells were digested with 500. Mu.L trypsin (HyClone America) and adherent bacteria were detached from the Caco-2 cell wall, and the reaction was stopped by adding 1.5mL of LHank's solution (Beijing Soy Co., ltd.). Cell and bacterial counts (24 h) were obtained by white blood cell count and plate count, and adhesion index was calculated.
The adhesion of bifidobacterium bifidum BD-1 to Caco-2 is shown in Table 4. The adhesion capacity of the bifidobacterium bifidum BD-1 to Caco-2 cells reaches 38.80 percent.
TABLE 4 adhesion index of Bifidobacterium bifidum BD-1 to Caco-2 cells
Species of type Adhesion index a
BD-1 38.80
a adhesion index = 100 bacteria number/total cell number.
Test example 2
This test example discloses an assay for the ability of bifidobacterium bifidum BD-1 to induce the secretion of pro-inflammatory and anti-inflammatory cytokines by Caco-2 cell lines. Cytokine concentrations in the co-culture supernatants were detected using an Enzyme-linked immunosorbent assay (Enzyme-Linked Immunosorbent Assay, ELISA).
The fused monolayer and fully differentiated Caco-2 cells were washed twice with 0.9 mM MEM medium PBS. 100. Mu.L of live/inactivated bacterial suspension was added at 37℃with 95% air and 5% CO 2 Is incubated in the environment of (2). After 24h, centrifugation was carried out at 3000r/min for 10min and the supernatant was collected and stored at-80 ℃. The preparation was carried out strictly according to the instructions of ELISA kit (Shanghai Amersham pharmacia). The results were averaged over three independent experiments.
The concentration of cytokine production by Caco-2 mediated by the activity and heat-inactivated bifidobacteria was examined (results are shown in Table 5). Under viable bacteria conditions, bifidobacterium bifidum BD-1 stimulated Caco-2 cells to significantly secrete IL-8. The ability of heat-inactivated bifidobacterium bifidum BD-1 to stimulate secretion of IL-8 by Caco-2 cells is reduced.
TABLE 5 adhesion index and concentration of cytokines to active and heat-inactivated bacteria in Caco-2 culture supernatants
Figure BDA0003744760830000081
b-means a concentration below the detection limit. Detection limit of cytokines: 8pg/mL (IL-6; IL-8; IL-10; TNF-. Alpha.); 16pg/mL (IL-12). The concentration of cytokine is expressed as pg/mL and represents mean ± standard deviation.
The results show that the bifidobacterium bifidum BD-1 has good adhesion performance on intestinal epithelial cells, and the bifidobacterium bifidum BD-1 with activity and heat inactivation can stimulate Caco-2 cells to obviously secrete IL-8, so that the immunity of the organism is regulated.
Finally, it should be noted that: the above embodiments are merely preferred embodiments of the present invention for illustrating the technical solution of the present invention, but not limiting the scope of the present invention; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the corresponding technical solutions; that is, even though the main design concept and spirit of the present invention is modified or finished in an insubstantial manner, the technical problem solved by the present invention is still consistent with the present invention, and all the technical problems are included in the protection scope of the present invention; in addition, the technical scheme of the invention is directly or indirectly applied to other related technical fields, and the technical scheme is included in the scope of the invention.

Claims (10)

1. The bifidobacterium bifidum from the intestinal tract of infants is characterized in that the preservation number is CGMCC NO.24517.
2. The use of bifidobacterium bifidum according to claim 1, in the preparation of a medicament or food product having an immunoregulatory effect.
3. The use according to claim 2, wherein the medicine or food comprises a medicine or food having intestinal epithelial cell adhesion function; preferably, it comprises a pharmaceutical or food product having an adhesion to Caco-2 cells.
4. The use according to claim 2, wherein the pharmaceutical or food product comprises a pharmaceutical or food product that stimulates secretion of IL-8; preferably, a pharmaceutical or food product is included that stimulates IL-8 secretion by Caco-2 cells.
5. The use according to claim 4, wherein the strain in the pharmaceutical or food product is an active bacterium.
6. The use according to claim 4, wherein the strain in the pharmaceutical or food product is an inactivated bacterium.
7. The use according to claim 6, wherein the strain in the pharmaceutical or food product is a heat-inactivated bacterium.
8. The use according to any one of claims 2 to 7, wherein the medicament or food product comprises bifidobacterium bifidum with a collection number of cgmccno.24517 and one or more pharmaceutically acceptable carriers.
9. The use according to any one of claims 2 to 7, wherein the pharmaceutical or food product is an oral formulation.
10. The use according to claim 9, wherein the oral formulation comprises a solid oral formulation or a liquid oral formulation.
CN202210991241.1A 2022-07-13 2022-07-13 Bifidobacterium bifidum from infant intestinal tracts and application thereof Pending CN116286439A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116004416A (en) * 2022-07-13 2023-04-25 四川大学 Application of bifidobacterium bifidum from infant intestinal tract
CN116004416B (en) * 2022-07-13 2024-05-10 四川大学 Application of bifidobacterium bifidum from infant intestinal tract

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