CN117264826A - Bifidobacterium longum capable of regulating abundance of intestinal flora and intestinal health and application thereof - Google Patents
Bifidobacterium longum capable of regulating abundance of intestinal flora and intestinal health and application thereof Download PDFInfo
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- CN117264826A CN117264826A CN202311237766.7A CN202311237766A CN117264826A CN 117264826 A CN117264826 A CN 117264826A CN 202311237766 A CN202311237766 A CN 202311237766A CN 117264826 A CN117264826 A CN 117264826A
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- bifidobacterium longum
- ccfm5871
- bacteroides
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Abstract
The invention discloses bifidobacterium longum capable of regulating abundance of intestinal flora and intestinal health and application thereof, and belongs to the technical field of microorganisms. The bifidobacterium longum (Bifidobacterium longum) CCFM5871 is screened, and the bifidobacterium longum CCFM5871 can remarkably improve the relative abundance of the rikenella in the intestinal tract of the mice, promote the growth of the bacteroides norborni, improve the health of the intestinal tract and prolong the service life of nematodes. The bifidobacterium longum CCFM5871 has the advantages of remarkably improving the abundance of the rhizoctonia and the rogowski bacteria in the intestinal tract and promoting the growth of the bacteroides northly and has wide application prospect in the directions of foods and microecological preparations.
Description
Technical Field
The invention relates to bifidobacterium longum capable of regulating abundance of intestinal flora and intestinal health and application thereof, and belongs to the technical field of microorganisms.
Background
The genus rimellina (Rikenella), gram-negative, obligate anaerobe. Rikenella can ferment propionate, produce energy in normal cells, and promote gluconeogenesis. Rikenella can also ferment carbohydrates (e.g., glucose) to promote growth. Rikenella is a potential probiotic and plays an important role in maintaining metabolic balance in the body, the immune system of the body and intestinal health. In recent years, a process for producing a plastic film, there are many studies showing abundance of the genus Agrimonia closely related to the immune system of the host. For example, one patent showed that the relative abundance of the genus rimonia was useful as a microbial biomarker for diagnosing food allergy (application publication No. CN 202210216093.6), and another study showed that the relative abundance of the genus rimonia was significantly reduced in food allergy mice. In addition, studies have shown that the genus rimonia is associated with another autoimmune disease, rheumatoid arthritis, and that the abundance of the genus rimonia is significantly reduced in rheumatoid arthritis volunteers compared to normal volunteers. Another animal experiment showed that the abundance of the genus rimonia was significantly reduced in mice with autoimmune hepatitis (AIH). Therefore, the regulation of the abundance of the rhizoctonia in the intestinal tract can play a certain role in protecting the health of the organism and relieving diseases.
At present, some patents relate to health-care food with the function of regulating intestinal flora and a preparation method thereof, for example, patent CN108018248A discloses lactobacillus casei which can remarkably recover the intestinal flora change caused by antibiotics in mice; patent CN111004731a discloses a bacillus coagulans composition which significantly increases the abundance of allobacilum bacteria in the intestinal tract. However, there is no report on the design of bifidobacterium for regulating the content of genus joint grinding (Rikenella). In addition, bacteroides (Bacteroides) is an important basic stone fungus in the intestinal tract, is an important participant for maintaining the stable state of the intestinal flora, and Bacteroides northwest (Bacteroides nordii) is an important species in Bacteroides, so that the growth of Bacteroides northwest is promoted to be beneficial to improving the abundance of Bacteroides northwest in the intestinal tract, and has important significance for maintaining the stable state of the intestinal flora, and no report on the promotion of the growth of Bacteroides northwest by bifidobacterium exists at present.
Therefore, the technical problem to be solved is to provide a method for improving the structural disturbance of intestinal flora by up-regulating the relative abundance of Rikenella (Rikenella) in the intestinal tract and promoting the growth of North bacteroides (Bacteroides nordii).
Disclosure of Invention
The invention aims to solve the technical problem of providing bifidobacterium longum (Bifidobacterium longum) and provides a method capable of remarkably improving the relative abundance of Rikenella (Rikenella) in intestinal tracts, promoting the growth of bacteroides northeast, regulating the health of the intestinal tracts and prolonging the service life of nematodes.
The invention provides a bifidobacterium longum (Bifidobacterium longum) CCFM5871, wherein the bifidobacterium longum (Bifidobacterium longum) is preserved in the Guangdong province microorganism strain preservation center, the preservation number is GDMCC No. 63537, and the preservation date is 2023, 06 and 07.
The bifidobacterium longum (Bifidobacterium longum) CCFM5871 is isolated from a 38 year old male fecal sample from the forward head village of mountain pond in 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 longum is 99.48%, which indicates that the strain is bifidobacterium longum and is named as bifidobacterium longum (Bifidobacterium longum) CCFM5871.
The growth of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 thalli on an MRS culture medium is characterized in that: the colony had a round shape (FIG. 1), a diameter of 1-2 mm, a convex shape on the side, a milky white color, a wet and smooth surface, a blue-violet gram stain (FIG. 2), a gram positive bacterium, and no spore formation (FIG. 1).
Growth characteristics of the bifidobacterium longum CCFM 5871: the obligate anaerobe is very sensitive to oxygen, and can grow optimally at 37-41 ℃ with optimal initial growth pH of 6.5-7.0.
The invention also provides a microbial preparation containing the bifidobacterium longum (Bifidobacterium longum) CCFM5871.
In one embodiment of the present invention, the viable count of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
The invention also provides a product containing the bifidobacterium longum (Bifidobacterium longum) CCFM5871 or the microbial preparation.
In one embodiment of the invention, the product comprises a food, a pharmaceutical or a nutraceutical.
In one embodiment of the present invention, the viable count of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products containing bifidobacterium longum.
In one embodiment of the present invention, the pharmaceutical product comprises the above bifidobacterium longum (Bifidobacterium longum) CCFM5871, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise fillers, binders, wetting agents, disintegrants, lubricants and/or flavoring agents.
The invention also provides a method for promoting the proliferation of the bacteroides northbound (Bacteroides nordii), which comprises the step of co-culturing the bifidobacterium longum (Bifidobacterium longum) CCFM5871 or the microbial preparation and the bacteroides northbound.
The invention also provides a product for promoting the proliferation of the bacteroides northbound (Bacteroides nordii), which contains the bifidobacterium longum (Bifidobacterium longum) CCFM5871 or the microbial preparation.
In one embodiment of the invention, the product comprises a food, a pharmaceutical or a nutraceutical.
In one embodiment of the present invention, the viable count of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products containing bifidobacterium longum.
In one embodiment of the present invention, the pharmaceutical product comprises the above bifidobacterium longum (Bifidobacterium longum) CCFM5871, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise fillers, binders, wetting agents, disintegrants, lubricants and/or flavoring agents.
The invention also provides a method for improving the abundance of the genus Rikenella (Rikenella) or the genus Rocibutipes (Roseburia), which is characterized in that the method adopts CCFM5871 containing the bifidobacterium longum (Bifidobacterium longum) or adds the microbial preparation to an environment containing the genus Rikenella (Rikenella) or the genus Rocibutipes (Roseburia) for co-cultivation.
In one embodiment of the invention, the environment is an in vivo environment.
In one embodiment of the invention, the environment is an in vitro environment.
The invention also provides a product for improving the genus Rikenella (Rikenella) or the genus Roche (Roseburia), wherein the product contains the bifidobacterium longum (Bifidobacterium longum) CCFM5871 or the microbial preparation.
In one embodiment of the invention, the product comprises a food, a pharmaceutical or a nutraceutical.
In one embodiment of the present invention, the viable count of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products containing bifidobacterium longum.
In one embodiment of the present invention, the pharmaceutical product comprises the above bifidobacterium longum (Bifidobacterium longum) CCFM5871, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise fillers, binders, wetting agents, disintegrants, lubricants and/or flavoring agents.
The invention also provides application of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 or the microbial preparation in preparation of products for promoting the proliferation of the bacteroides norborni or improving the genus Rikenella or the genus Rosiburia (Roseburia).
In one embodiment of the invention, the product comprises a food, a pharmaceutical or a nutraceutical.
In one embodiment of the present invention, the viable count of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products containing bifidobacterium longum.
In one embodiment of the present invention, the pharmaceutical product comprises the above bifidobacterium longum (Bifidobacterium longum) CCFM5871, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise fillers, binders, wetting agents, disintegrants, lubricants and/or flavoring agents.
The invention also provides a product, which contains the bifidobacterium longum (Bifidobacterium longum) CCFM5871 and bacteroides northbound.
In one embodiment of the invention, the product comprises a food, a pharmaceutical or a nutraceutical.
In one embodiment of the present invention, the viable count of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products containing bifidobacterium longum.
In one embodiment of the present invention, the pharmaceutical product comprises the above bifidobacterium longum (Bifidobacterium longum) CCFM5871, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise fillers, binders, wetting agents, disintegrants, lubricants and/or flavoring agents.
The invention also provides application of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 or the microbial preparation in preparation of products for improving intestinal health or in preparation of probiotic health products or in preparation of probiotic foods.
In one embodiment of the invention, the product comprises a food, a pharmaceutical or a nutraceutical.
In one embodiment of the present invention, the viable count of the bifidobacterium longum (Bifidobacterium longum) CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
In one embodiment of the invention, the food product comprises fermented fruit and vegetable, fermented milk, cheese, milk-containing beverages, milk powder or other food products containing bifidobacterium longum.
In one embodiment of the present invention, the pharmaceutical product comprises the above bifidobacterium longum (Bifidobacterium longum) CCFM5871, a pharmaceutical carrier and/or a pharmaceutical adjuvant.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles and/or liposomes.
In one embodiment of the invention, the pharmaceutical excipients comprise fillers, binders, wetting agents, disintegrants, lubricants and/or flavoring agents.
Advantageous effects
1. The invention screens out a bifidobacterium longum (Bifidobacterium longum) CCFM5871 which has the functions of regulating the abundance of the genus of joint grinding bacteria and the genus of rochanterium, promoting the growth of bacteroides northeast and prolonging the service life of nematodes, improving the defecation frequency, the fecal water content, the fecal acetic acid content and the small intestine propulsion rate and reducing the intestinal transit time. .
Therefore, the bifidobacterium longum CCFM5871 has great application prospect in preparing products (such as foods or medicines and the like) for regulating the abundance of the joint ground bacteria, promoting the growth of the bacteroides northeast, improving the health of intestinal tracts and further preventing and/or treating related diseases.
2. Bifidobacterium longum (Bifidobacterium longum) CCFM5871 is an edible probiotic. The bifidobacterium longum CCFM5871 and the effective component of the invention are bifidobacterium longum CCFM5871.
Preservation of biological materials
Bifidobacterium longum (Bifidobacterium longum) CCFM5871, taxonomic designation Bifidobacterium longum, was deposited at the Cantonese China center for type culture Collection of microorganisms, accession No. GDMCC No. 63537, and was deposited at floor 5 of the university of Mitsui 100, guangzhou, md.
Drawings
Fig. 1: bifidobacterium longum (Bifidobacterium longum) CCFM5871 dilution coating results.
Fig. 2: bifidobacterium longum (Bifidobacterium longum) CCFM5871 gram microscopy results.
Fig. 3: effect of bifidobacterium longum (Bifidobacterium longum) CCFM5871 on relative abundance of riken bacteria in the mouse gut.
Fig. 4: effect of bifidobacterium longum (Bifidobacterium longum) CCFM5871 on bacteroides northbound growth.
Fig. 5: bifidobacterium longum (Bifidobacterium longum) CCFM5871 affects nematode longevity.
Fig. 6: bifidobacterium longum (Bifidobacterium longum) CCFM5871 affects intestinal physiological index.
Detailed Description
The invention is further illustrated below in conjunction with specific embodiments and figures.
MRS, BHI, GMM medium referred to in the examples below was purchased from Qingdao high tech industrial garden Haibo biotechnology Co., ltd; the Fast DNA Spin Kit for Feces kit referred to in the examples below was purchased from MP Biomedicals company.
Bacterial powder for clinical test: the probiotics and placebo products in the experimental products are food grade and are manufactured by Jiangsu microorganism and technology limited company entrusted by food biotechnology center of food college of Jiangnan university, and the bacteria powder production license is: SC10632050900407. All the products are powder, the appearance and the package are the same, and each bacterial powder has a net weight of 2g and contains 5 multiplied by 10 9 CFU live bacteria and maltodextrin, placebo 2g maltodextrin, all experimental products during the test were stored in a refrigerator at 4 ℃.
The following examples relate to Balb/c mice purchased from Beijing Vitolihua laboratory animal technologies Co., ltd; the caenorhabditis elegans referred to in the examples below are: n2 wild caenorhabditis elegans (Caenorhabditis elegans) which are given away by the food college of the university of Nanjing and are preserved in the food biotechnology center of the university of Jiangnan.
The following examples relate to the following media:
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.
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.
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.
BHI broth (1L): dissolving 38.5g of brain-heart leaching solution culture medium powder in 1L of water, adding 10.002g/L vitamin K, 0.01g/L hemin and 1g/L cysteine hydrochloride, and adjusting pH to 7.0.
S liquid medium: naCl 5.85g, K 2 HPO 4 1g，KH 2 PO 4 6g, fixing the volume to 1L, aseptically adding 1mL of 5mg/mL cholesterol ethanol solution to prepare 1L amount of S basic culture medium; caCl 1mol/L 2 Solution, 1mol/L MgSO 4 3mL to S basic culture medium are added into the solutions respectively; 1mol/L potassium citrate solutionLiquid, pH 6.0 (citric acid. H) 2 O20 g, potassium citrate H 2 O293.5 g to 1L) plus 10mL; trace metal solution (EDTA.2Na 1.86g, feSO) 4 ·7H2O0.69g，MnCl 2 ·4H 2 O 0.2g，ZnSO 4 ·7H 2 O 0.29g，CuSO 4 ·5H 2 O0.025 g, constant volume to 1L, autoclaving, and storing in a dark place) and 10mL.
The preparation method of the bifidobacterium longum suspension referred in the following examples is as follows:
streaking bifidobacterium longum 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 longum thalli;
washing Bifidobacterium longum cells with physiological saline, and re-suspending in 30% glycerol solution (containing 1g/L cysteine hydrochloride) to give a bacterial concentration of 5×10 9 CFU/mL, stored in-80℃refrigerator.
The detection method involved in the following examples:
detecting the abundance of the bacteroides northeast strain:
1mL of the culture broth was taken to extract DNA, and qPCR was performed using specific primers. The qPCR mixture (total volume 20. Mu.L) consisted of TapAlse Mix, 2. Mu.L DNA template and 0.2. Mu.M each primer. The standard curve was calculated using ten-fold dilution series of DNA extracted from each strain to absolute quantify the strain. qPCR experimental procedure, first step: 94 ℃ for 10s; and a second step of: 94 ℃ for 20s; and a third step of: 55 ℃,20s, fourth step: the second to fourth steps were performed for 40 cycles at 72℃for 50 s. Longum (F: 5'-TTCCAGTTGATCGCATGGTCTTCTA-3', R: 5'-GGCTACCCGTCGAAGCCACG-3'), nordii (F: 5'-GCGGGGAACAGAATCAGACA-3', R: 5'-ATTCCACCAAATGTAGGCGGGACGTTTAAT-3')
Example 1: separation and screening of bifidobacterium longum CCFM5871
1. Sample collection
Collecting 38-year-old male feces sample of Shunjiao village in mountain pond of Lianzhou 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 into 10mL centrifuge tube containing 4.5mL physiological saline under aseptic condition to obtain 10 -1 And (3) diluting the solution, repeating the diluting steps to sequentially obtain 10 -2 、10 -3 、10 -4 、10 -5 、10 -6 A dilution liquid;
(2) Coating and culturing: 100 mu L of the above 10 were sucked respectively -4 、10 -5 、10 -6 The three gradient dilutions are used for selecting culture medium species in the bifidobacterium specificity, uniformly coated by a coating rod, and cultured for 48 hours under the anaerobic condition of 37 ℃;
(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 60% 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;
table 1: primer name
| 16S rDNA PCR primer name | Sequence(s) |
| 27F | 5’-AGAGTTTGATCCTGGCCTCA-3’ |
| 1492R | 5’-GGTTACCTTGTTACGACTT-3’ |
Table 2: bacterial strain identification 25 mu L16S 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 |
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.
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 CCFM5871 is a bifidobacterium longum strain, and the 16S rDNA amplification sequence is shown as SEQ ID NO. 1.
Example 2: effect of Bifidobacterium longum CCFM5871 on the composition of the intestinal flora of mice
The method comprises the following specific steps:
24 healthy male BALB/c mice with the age of 6-8 weeks and the age of 20-22 g are randomly divided into 4 groups of 6 mice each, and the 4 groups are respectively: blank, model and intervention groups of bifidobacterium longum CCFM5871 and CCFM1114 (described in chinese patent publication No. CN 113943682B), respectively.
The experimental process is as follows:
blank control group: irrigating the stomach twice a day with 0.2mL of physiological saline;
model set (LOP): gastric lavage 0.2mL loperamide solution (10 mg/kg b.w), gastric lavage 0.2mL saline after 1 h;
CCFM5871 group: the loperamide solution (10 mg/kg b.w) was filled with 0.2mL of the loperamide solution per day, and the bacterial concentration was 5X 10 at 0.2mL after 1h 9 CFU/mL of Bifidobacterium longum CCFM5871 bacterial suspension;
CCFM1114 group: stomach is irrigated daily with 0.2mL loperamide solution (10 mg/kg b.w)After 1h, the gastric lavage is carried out for 0.2mL with a bacterial concentration of 5X 10 9 CFU/mL of Bifidobacterium longum CCFM1114 bacterial suspension;
the method comprises the steps of collecting mouse faeces on the 14 th day, extracting bacterial genomes of each group of mouse faeces by using a Fast DNA Spin Kit for Feces kit, carrying out PCR amplification on sequences of 16s V3-V4 regions, and carrying out composition differences of intestinal flora in faeces samples by a second-generation sequencer, wherein the relative abundance of main flora in the intestinal tract of each group of mice is shown in Table 3 and figure 3.
Table 3: relative abundance of the main flora in the gut of each group of mice (%)
| Genus of bacteria | Blank space | Loperamide | CCFM5871 | CCFM1114 |
| g__Rikenella | 0.9502 | 0.4605 | 5.9804 | 1.1959 |
| g__Acinetobacter | 0.0037 | 0.1766 | 0.0910 | 0.0029 |
| g__Adlercreutzia | 1.8517 | 1.7793 | 1.5720 | 1.4590 |
| g__AF12 | 0.4783 | 0.1269 | 0.1758 | 0.7420 |
| g__Bacteroides | 11.3595 | 4.1614 | 36.8205 | 52.9094 |
| g__Bifidobacterium | 0.0000 | 0.0016 | 0.0145 | 0.0000 |
| g__Coprococcus | 0.1534 | 0.0205 | 0.2006 | 0.1559 |
| g__Corynebacterium | 0.0135 | 0.0262 | 0.0623 | 0.0083 |
| g__Dorea | 0.2015 | 0.0100 | 0.0738 | 0.0876 |
| g__Enterococcus | 1.1812 | 4.2490 | 0.9890 | 0.9028 |
| g__Helicobacter | 0.0856 | 0.0248 | 0.0923 | 0.1423 |
| g__Mucispirillum | 4.2623 | 1.6251 | 10.6890 | 1.7898 |
| g__Oscillospira | 1.1962 | 0.4542 | 0.6852 | 0.5927 |
| g__other | 2.4619 | 1.3905 | 2.2738 | 1.9034 |
| g__Parabacteroides | 0.3538 | 0.0927 | 0.0844 | 0.0102 |
| g__Proteus | 0.3867 | 0.0771 | 0.0087 | 0.0049 |
| g__Roseburia | 3.4547 | 0.3605 | 20.8645 | 7.0906 |
| g__Ruminococcus | 1.8232 | 0.8402 | 2.1574 | 2.5610 |
| g__Staphylococcus | 1.0174 | 2.6827 | 3.2503 | 0.2674 |
| g__Streptococcus | 0.1628 | 0.2691 | 0.1254 | 0.2294 |
| g__Turicibacter | 0.5101 | 1.0051 | 3.3741 | 3.2316 |
The genus of the riken plays an important role in maintaining intestinal health, the relative abundance of the riken in the intestinal tract of a blank group of mice is 0.95%, the relative abundance of the riken in the intestinal tract of a model group of mice is 0.46%, the relative abundance of the riken in the intestinal tract of a group of mice is 5.98% after intervention by bifidobacterium CCFM5871, which is 12.99 times that of a model group, and the relative abundance of the riken in the intestinal tract of a group of mice after intervention by bifidobacterium CCFM1114 is only 1.19%.
It has been reported that the genus rochanteria (Roseburia) can increase butyrate content in the intestinal tract, and the decrease of the genus rochanteria is significantly associated with the occurrence and development of chronic kidney disease. The genus Roche can modulate intestinal immune function by activating colon TLR-5 receptor, thereby alleviating Crohn's disease. In addition, the genus rochanteria has a function of alleviating alcoholic fatty liver disease. The relative abundance of the rogowski genus in the intestinal tract of the model group mice is 0.36%, the relative abundance of the rogowski genus in the intestinal tract of the model group mice is 20.86% after the intervention of bifidobacterium CCFM5871, which is 57.9 times of that of the model group mice, and the relative abundance of the riken genus in the intestinal tract of the mice after the intervention of bifidobacterium CCFM1114 is only 7.09%.
It can be seen that bifidobacterium longum CCFM5871 can significantly improve the intestinal flora structure and increase the relative abundance of beneficial intestinal bacteria of the genus Agrimonia and the genus Roche, while the other strain does not have the effect.
Example 3: effect of Bifidobacterium longum CCFM5871 on North Bacteroides growth
The method comprises the following specific steps:
(1) Cultivation of Bacteroides North
Respectively inoculating North Bacteroides CCFM1309 (GDMCC No. 63535) and North Bacteroides CCFM1246 (deposited in the biological technology center strain resource library of the university of Jiangnan food institute) into BHI liquid culture medium with an inoculum size of 2% (v/v), and culturing at 37deg.C for 18h; respectively preparing seed liquid;
(2) Culture of Bifidobacterium longum
Inoculating Bifidobacterium longum CCFM5871 and Bifidobacterium longum CCFM1114 respectively in an inoculum size of 2% (v/v) in MRS (0.05% [ w/v ] added cysteine) culture medium, culturing at 37deg.C for 18 hr for activation, and continuously activating for two generations according to the above method to obtain Bifidobacterium longum CCFM5871 seed solution and Bifidobacterium longum CCFM1114 seed solution respectively;
(3) Inoculating 2 kinds of Bacteroides North seed solution into GMM culture medium (containing 0.5% arabinan) to obtain final concentration of thallus of 10 5 CFU/mL, two seed solutions of Bifidobacterium longum were inoculated simultaneously to the same concentration (10 5 CFU/mL), co-culturing at 37℃for 24h;
periodic acquisitions at 0h, 3h, 6h, 9h, 12h, 18h and 24h, respectively, were used to analyze microbial growth (OD 600 ). The results are shown in table 4 and fig. 4; the data in the tables are for the growth OD values of Bacteroides North.
Table 4: effect of Bifidobacterium longum on North Bacteroides growth
The results show that:
after co-cultivation with bifidobacterium longum CCFM5871, OD of Bacteroides North CCFM1309 and Bacteroides North CCFM1246 600 The growth of the bacteroides norborni is not obviously improved after the co-culture of the bifidobacterium longum CCFM1114 and the bacteroides norborni is adopted.
Meanwhile, the abundance of bifidobacterium longum and bacteroides northleopardus in the co-culture process is detected by qPCR, the result is consistent with the conclusion, and bifidobacterium longum CCFM5871 can obviously promote the growth of bacteroides northleopardus CCFM1309 and bacteroides northleopardus CCFM1246, while bifidobacterium longum CCFM1114 does not have the effect (see figure 4).
Example 4: influence of Bifidobacterium longum CCFM5871 on the intestinal tract of healthy mice
The method comprises the following specific steps:
15 healthy male BALB/c mice with the age of 6-8 weeks and the age of 20-22 g are randomly divided into 3 groups of 5 mice each, and the 3 groups are respectively: blank, bifidobacterium longum CCFM5871 and bifidobacterium longum CCFM1114 intervention.
The experimental process is as follows:
control group, lavage 0.2mL physiological saline per day;
CCFM5871 group: the content of 0.2mL strain for each day of gastric lavage is 1×10 9 Bifidobacterium longum CCFM5871 bacterial liquid of CFU;
CCFM1114 group: the content of 0.2mL strain for each day of gastric lavage is 1×10 9 Bifidobacterium longum CCFM1114 bacterial liquid of CFU.
And continuously filling the stomach for 14 days until the last day of the experiment, and then, all mice are fasted for 24 hours, and then, filling gastric ink, recording the discharge time of the first black stool of the mice, and discharging the number of the black stools within 6 hours.
The frequency of defecation, intestinal transit time, small intestine propulsion rate, fecal water content, and fecal acetic acid content of each group of mice during the experimental period are shown in fig. 6.
The results show that: the bifidobacterium longum CCFM5871 remarkably improves the acetic acid content, defecation frequency, fecal water content and small intestine propulsion rate in the feces of healthy mice, remarkably reduces the intestinal transit time, and the other bifidobacterium longum CCFM1114 does not have the effect.
Example 5: effect of Bifidobacterium longum CCFM5871 on caenorhabditis elegans longevity
The method comprises the following specific steps:
120 caenorhabditis elegans (N2) (Caenorhabdit elegans N2) cultured synchronously to stage L4 were randomly grouped into 4 groups of 30, 4 groups each: blank, fed with e.coli OP50; CCFM5871 group (bifidobacterium longum fed CCFM 5871); group CCFM1232 (bifidobacterium longum fed CCFM 1232); group CCFM1114 (bifidobacterium longum fed CCFM 1114).
The experimental process is as follows:
(1) Inoculating Escherichia coli OP50 into 5mL LB liquid medium, and shake culturing at 37deg.C and 150rpmCulturing for 12h when OD 600 When the temperature is 1.0-1.2, 200 mu L of bacteria are absorbed after shaking and mixing evenly and are dripped on an NGM flat plate and smeared evenly (about 60% of the whole flat plate), cultured for 12h at 37 ℃ and stored for standby at 4 ℃.
Inoculating Bifidobacterium longum into 5mL LB liquid medium, shake culturing at 37deg.C and 150rpm for 12 hr, and standing at OD 600 When the temperature is 1.0-1.2, 200 mu L of bacteria are absorbed after shaking and mixing evenly and are dripped on an NGM flat plate and smeared evenly (about 60% of the whole flat plate), cultured for 12h at 37 ℃ and stored for standby at 4 ℃.
E.coli OP50 plates and long bifidobacterium plates were prepared separately.
(2) Placing the frozen and thawed nematodes on an NGM culture medium plate with escherichia coli OP50, and placing the plate at 15 ℃ to revive the nematodes to obtain the nematodes containing a large amount of spawning periods. 3.5mL of sterile water suspension containing nematodes is added into a centrifuge tube, 0.5mL of 5M sodium hydroxide solution and 1mL of 5% sodium hypochlorite solution are sucked and fully mixed, M9 buffer solution is repeatedly washed for 2-3 times, S culture medium solution is used for resuspension, S culture medium solution containing eggs is placed at 20 ℃ for 8-12h,1500 Xg is centrifuged for 3min to collect the nematodes in L1 phase, and the nematodes are transferred into NGM culture plates of E.coli OP50 growing with nematode foods, and are cultured at 25 ℃ for 48h to obtain the nematodes in L4 phase.
(3) 180 nematodes of stage L4 were placed on plates containing E.coli OP50 and different bifidobacterium longum plates (30 per plate), the nematodes were transferred to new plates each day, the number of deaths was recorded and the mortality was calculated (the tip of the picker was gently touched to the body during observation, and the nematodes were considered to have died without reaction).
The calculation formula is as follows: survival (%) = (number of nematodes surviving/total number of worms) ×100%. The results are shown in Table 5 and FIG. 5.
Table 5: effect of Bifidobacterium longum strains on nematode longevity
The results show that: the bifidobacterium longum CCFM5871 remarkably improves the average life span of the caenorhabditis elegans by 18.3 percent and the longest life span of the caenorhabditis elegans by 21.6 percent, and experiments show that the bifidobacterium longum CCFM5871 can remarkably improve the life span of the caenorhabditis elegans, but other strains do not have the effect.
Example 6: bifidobacterium longum CCFM5871 for preparing fermented cow milk
The bifidobacterium longum CCFM5871 can be used for preparing fermented cow milk, and the specific preparation process of the cow milk is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM5871 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; the bacterial sludge is washed 3 times with phosphate buffer solution of pH7.2 and then resuspended to a concentration of 1X 10 with a protective agent 10 CFU/mL, obtain suspension; pre-culturing the suspension at 37 ℃ for 60min, and freeze-drying to obtain a starter;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 100g/L skimmed milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose, 10g/L L-sodium glutamate;
(2) Sterilizing the skimmed milk at 95deg.C for 20min, and cooling to 4deg.C to obtain raw materials; adding the starter culture obtained in the step (1) into the raw material until the concentration is not less than 1X 10 6 CFU/mL, cow milk (cow milk is stored under refrigeration at 4deg.C).
Example 7: bifidobacterium longum CCFM5871 for preparing fermented soybean milk
The bifidobacterium longum CCFM5871 can be used for preparing soymilk, and the specific preparation process of the soymilk is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM5871 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; the bacterial sludge is washed 3 times with phosphate buffer solution of pH7.2 and then resuspended to a concentration of 1X 10 with a protective agent 10 CFU/mL, obtain suspension; pre-culturing the suspension at 37deg.C for 60min, and lyophilizing to obtainTo a starter;
the preparation method of the culture medium comprises the following steps: dissolving 10% of skim milk, 0.5% of glucose, 1.5% of tryptone and 0.3% of yeast extract by using 87.7% of water based on the total weight of the culture medium, and then adjusting the pH value to 6.8 to obtain the culture medium;
the components of the protective agent comprise: 100g/L skimmed milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose, 10g/L L-sodium glutamate;
(2) Soaking soybean at 80deg.C for 2 hr, removing soybean hull to obtain peeled soybean; removing the soaking water from peeled soybean, adding boiling water, and pulping to obtain soybean milk; maintaining the temperature of the soybean milk at a temperature higher than 80 ℃ for 12min to obtain cooked soybean milk; filtering cooked soybean milk with 150 mesh sieve, and centrifuging to obtain coarse soybean milk; heating the crude soymilk to 140-150 ℃, then rapidly introducing the heated crude soymilk into a vacuum cooling chamber for vacuumizing, so that the peculiar smell substances in the crude soymilk are rapidly discharged along with water vapor to obtain cooked soymilk; cooling cooked soybean milk to 37deg.C, adding the starter obtained in step (1) to cooked soybean milk until the concentration is not less than 1×10 6 CFU/mL to give soymilk (soymilk is stored cold at 4deg.C).
Example 8: bifidobacterium longum CCFM5871 for preparing fruit and vegetable beverage
The bifidobacterium longum CCFM5871 can be used for preparing vegetable beverages, and the specific preparation process of the vegetable beverages is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM5871 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; the bacterial sludge is washed 3 times with phosphate buffer solution of pH7.2 and then resuspended to a concentration of 1X 10 with a protective agent 10 CFU/mL, obtain suspension; pre-culturing the suspension at 37 ℃ for 60min, and freeze-drying to obtain a starter;
the preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 100g/L skimmed milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose, 10g/L L-sodium glutamate;
(2) Cleaning fresh vegetables, and squeezing to obtain vegetable juice; sterilizing the vegetable juice at 140 ℃ for 2 seconds to obtain sterilized vegetable juice; cooling the sterilized vegetable juice to about 37deg.C, adding the starter prepared in step (1) to the sterilized vegetable juice until the concentration is not less than 1×10 6 CFU/mL, vegetable beverage (vegetable beverage is stored at 4deg.C).
Example 9: bifidobacterium longum CCFM5871 for preparing fermented dairy products
The bifidobacterium longum CCFM5871 can be used for preparing fermented milk, and the specific preparation process of the fermented milk is as follows:
(1) Inoculating the secondary purified culture solution of the bifidobacterium longum CCFM5871 obtained in the example 1 into a culture medium with an inoculum size of 3% (v/v), and culturing for 18 hours at 37 ℃ to obtain a bacterial solution; centrifuging the bacterial liquid to obtain bacterial mud; the bacterial sludge is washed 3 times with phosphate buffer solution of pH7.2 and then resuspended to a concentration of 1X 10 with a protective agent 10 CFU/mL, obtain suspension; pre-culturing the suspension at 37deg.C for 60min, and lyophilizing to obtain lyophilized powder with thallus concentration of 1×10 9 CFU/g；
The preparation method of the culture medium comprises the following steps: dissolving 10% enzyme hydrolyzed skim milk, 0.5% glucose, 1.5% tryptone and 0.3% yeast extract with 87.7% water based on the total weight of the culture medium, and adjusting pH to 6.8 to obtain culture medium;
the components of the protective agent comprise: 100g/L skimmed milk powder, 30mL/L glycerol, 100g/L maltodextrin, 150g/L trehalose, 10g/L L-sodium glutamate;
(2) Lactobacillus bulgaricus (thallus concentration 1×10) as a commercial dry powder starter 9 CFU/g) and commercial dry powder starter Streptococcus thermophilus (cell concentration 1X 10) 9 CFU/g) are mixed according to the mass ratio of 1:1:1 to obtain a starter, wherein the bacterial concentration of lactobacillus bulgaricus and streptococcus thermophilus in the starter is 2%;
(3) Adding sugar into fresh milk until the concentration is 5% to obtain a mixed solution; homogenizing the mixed solution at 65deg.C and 20MPa, and sterilizing at 95deg.C for 5min to obtain fermentation raw material; cooling the fermentation raw material to 35 ℃, inoculating the starter prepared in the step (2) into the fermentation raw material with an inoculum size of 0.03% (v/v), and fermenting at 35 ℃ for 16 hours to obtain fermented milk; and (3) after the fermented milk is curdled at 42 ℃, refrigerating at 4 ℃ for 24 hours for after-ripening, thus obtaining a fermented milk finished product.
Example 10: application of bifidobacterium longum CCFM5871
The bifidobacterium longum CCFM5871 can be used for preparing capsule products, and the specific preparation process of the capsule products is as follows:
the bifidobacterium longum CCFM5871 is streaked on an MRS solid culture medium and cultured 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 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 re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; the bacterial suspension is added into sodium alginate solution with the concentration of 30g/L to the concentration of 2 multiplied by 10 9 After CFU/mL, fully stirring to uniformly disperse cells of bifidobacterium longum CCFM5871 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 11: application of bifidobacterium longum CCFM5871
Bifidobacterium longum CCFM5871 can be used for preparing tablets, and the specific preparation process of the tablets is as follows:
the bifidobacterium longum CCFM5871 is streaked on an MRS solid culture medium and cultured for 48 hours at 37 ℃ to obtain a single colony; picking single colony, inoculating into MRS liquid culture medium, culturing at 37deg.C for 18 hr for activation, and continuously activating for two generations to obtain activating solutionThe method comprises the steps of carrying out a first treatment on the surface of the 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 re-suspending with protective agent to a concentration of 1×10 10 CFU/mL, obtaining bacterial suspension; pre-culturing the bacterial suspension at 37 ℃ for 60min, and freeze-drying to obtain bifidobacterium longum CCFM5871 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 longum CCFM5871 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 (10)
1. A bifidobacterium longum (Bifidobacterium longum) CCFM5871 is characterized in that the bifidobacterium longum CCFM5871 is deposited with the Guangdong province microorganism strain collection with the deposition number of GDMCC No. 63537 and the deposition date of 2023, 06 and 07.
2. A microbial preparation comprising bifidobacterium longum CCFM5871 of claim 1.
3. The microbial preparation according to claim 2, wherein the viable count of the bifidobacterium longum CCFM5871 in the microbial preparation is not less than 1X 10 6 CFU/mL or 1X 10 6 CFU/g。
4. A product comprising the bifidobacterium longum CCFM5871 of claim 1 or the microbial agent of claim 2 or 3.
5. The product of claim 4, wherein the product comprises a food, a pharmaceutical, or a nutraceutical.
6. A method for promoting proliferation of bacteroides northsonii (Bacteroides nordii) or increasing abundance of rhizoctonia (Rikenella) or rochanteria (Roseburia), comprising co-culturing bifidobacterium longum CCFM5871 according to claim 1 or a microbial preparation according to claim 2 or 3 in an environment containing bacteroides northsonii or rhizoctonia or rochanteria.
7. Use of bifidobacterium longum CCFM5871 of claim 1, or a microbial agent of claim 2 or 3, in the manufacture of a product capable of promoting proliferation of bacteroides northeast or increasing abundance of rhizoctonia or rocera.
8. The product of claim 7, wherein the product comprises a food, a pharmaceutical, or a nutraceutical.
9. A product, which is characterized in that the product contains bifidobacterium longum CCFM5871 and bacteroides northbound according to claim 1; preferably, the product comprises a food, a pharmaceutical or a health product.
10. Use of bifidobacterium longum CCFM5871 of claim 1, or the microbial preparation of claim 2 or 3 in the preparation of a product for improving intestinal health, or in the preparation of a probiotic health care product, or in the preparation of a probiotic food.
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