CN117511810B - Bifidobacterium breve HY002 for enhancing immunoregulatory function, and application, product and method thereof - Google Patents
Bifidobacterium breve HY002 for enhancing immunoregulatory function, and application, product and method thereof Download PDFInfo
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- CN117511810B CN117511810B CN202311566033.8A CN202311566033A CN117511810B CN 117511810 B CN117511810 B CN 117511810B CN 202311566033 A CN202311566033 A CN 202311566033A CN 117511810 B CN117511810 B CN 117511810B
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Abstract
The invention relates to bifidobacterium breve HY002 for enhancing immunity and application, a product and a method thereof, belonging to the technical field of microorganisms. The invention provides a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC NO.26838. Based on the strain HY002, the invention also provides the application of the strain in preparing medicines for enhancing the immunity regulating function, health-care foods with the immunity regulating function or bacteriostats, medicines for enhancing the immunity regulating function, health-care foods with the immunity regulating function and bacteriostats. The bifidobacterium breve HY002 provided by the invention can effectively enhance immunity, promote immune cell proliferation and immune factor level, and has broad-spectrum antibacterial performance.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to bifidobacterium breve HY002 for enhancing an immunoregulatory function, and application, a product and a method thereof.
Background
The immune system can protect the host from antigens in the environment or in vivo, malnutrition, drug or chemical exposure can cause immune system abnormalities, cause immunosuppression, affect immune system function, lead to reduced body defense mechanisms, and increase the incidence of allergic immune responses, autoimmune diseases, infectious diseases, and neoplasia.
Macrophages play a variety of roles in host defense, which can limit the pathogenesis of infectious organisms and affect the process of degenerative diseases. Macrophages and splenocytes, including T lymphocytes, B lymphocytes, etc., are activated to produce various inflammatory mediators and pro-inflammatory cytokines such as NO, TNF- α, IL-1β, IL-6, etc., thereby further protecting the host from foreign pathogens.
Currently, levamisole hydrochloride is a drug for treating immune dysfunction, and has serious side effects, so that the development of a new and safer immunomodulator is one of the most effective methods for preventing and treating immunosuppressive diseases.
The intestinal microbiota of the human body consists of hundreds of bacteria, and colonization of the intestinal microbiota plays an important role in host health. The main functions of the microbiota are protection, metabolism and nutritional activities. Lactic acid bacteria are the dominant flora in the intestinal resident flora, which can bind pattern recognition receptors expressed by immune cells and many other tissues including intestinal epithelium, generating immunomodulating signals, triggering the host's innate and adaptive immune response to better cope with pathogen or cancer cell attack. In terms of immunomodulation, lactic acid bacteria regulate the intestinal microbiota, thereby interacting with the host immune system.
The bifidobacterium breve has been reported to have the functions of repairing skin barrier, resisting aging, preventing oxidative stress, relieving depression anxiety and the like as one of lactic acid bacteria, and the bifidobacterium breve provided on the market at present can not meet the multiple selection demands of industrial production, market, consumers and the like, has smaller selectable space, and is especially in the aspect of enhancing immunity.
In view of the fact that the bifidobacterium breve strain provided by the prior art has fewer resources and single functions, and cannot meet the diversified demands of production and consumption, more beneficial novel bifidobacterium breve strains with abundant functions still need to be developed in the field.
Disclosure of Invention
In order to solve the problems existing in the prior art: the invention provides bifidobacterium breve HY002 for enhancing immunity, and an application and a product thereof in preparing an immunity enhancing drug or preparing a health-care food or a bacteriostatic agent with an immunity enhancing function, aiming at developing more beneficial novel bifidobacterium breve strain with rich functions.
The technical scheme of the invention is as follows:
A bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC NO.26838 for enhancing the immunoregulatory function.
The application of bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 in preparing medicines for enhancing the immunoregulation function or preparing health-care foods with the immunoregulation function or preparing bacteriostats.
The enhancing immunity comprises: promoting immune cell proliferation and immune factor production;
preferably, the promoting immune factor production means: promoting immune cells to secrete immune factors;
Preferably, the immune cells are RAW264.7 cells;
Preferably, the immune factor comprises: TNF-alpha, IL-10, IL-6, IL-1β, IL-17;
Preferably, the bacteriostasis spectrum of the bacteriostat comprises: staphylococcus aureus, escherichia coli, salmonella, klebsiella pneumoniae, escherichia coli, bacteroides fragilis, streptococcus angina, and streptococcus pneumoniae.
A medicament for enhancing immunomodulatory function comprising: a pharmaceutically active ingredient; the pharmaceutically active ingredients include: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
The medicine for enhancing immunity further comprises: pharmaceutically acceptable auxiliary materials.
A health food having an enhanced immune function, comprising: a functional active ingredient; the functional active ingredients include: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
The health food with the function of enhancing immunity further comprises: food auxiliary materials.
A bacteriostatic agent comprising: a bacteriostatic active ingredient; the antibacterial active ingredients comprise: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
The bacteriostatic agent further comprises: auxiliary materials.
An in vitro antibacterial method adopts a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 for antibacterial.
The beneficial effects of the invention are as follows:
The bifidobacterium breve (Bifidobacterium breve) strain HY002 provided by the invention is screened from infant feces, and experiments prove that the strain has higher surface hydrophobicity, which proves that the strain has higher potential of colonizing intestinal tracts; meanwhile, the immunity enhancing effect of the strain HY002 is also proved, and the specific expression is as follows: can obviously promote the proliferation of immune cell RAW264.7 and effectively promote immune cell RAW264.7 to secrete various immune factors such as TNF-alpha, IL-10, IL-6, IL-1 beta, IL-17 and the like. Meanwhile, the strain HY002 has broad-spectrum antibacterial performance and has good inhibition effect on pathogenic bacteria such as staphylococcus aureus, escherichia coli, salmonella, klebsiella pneumoniae, friesen escherichia, bacteroides fragilis, streptococcus angina, streptococcus pneumoniae and the like. Moreover, the strain HY002 is sensitive to various common antibiotics, does not produce biogenic amine, and is safe to human bodies, and has the condition of being used as probiotics for industrial mass production.
The preservation information of bifidobacterium breve (Bifidobacterium breve) strain HY002 of the invention is as follows:
preservation number: CGMCC No.26838
Classification naming: bifidobacterium breve Bifidobacterium breve
Preservation date: 2023, 03, 17
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Preservation address: the korean district beichen, beijing city, part No. 1, no. 3.
Drawings
FIG. 1 shows the results of an experiment of acid resistance and bile salt resistance of bifidobacterium breve HY002 in experimental example 4 of the present invention.
FIG. 2 shows the results of the experiment of the antibacterial zone of the bifidobacterium breve HY002 antagonistic to pathogenic bacteria in experimental example 5 of the present invention. In the figure, the results of the antibacterial circle of bifidobacterium breve HY002 antagonizing escherichia coli, staphylococcus aureus, salmonella, klebsiella pneumoniae, escherichia coli frailter, streptococcus pneumoniae and streptococcus angina are sequentially shown from left to right and from top to bottom.
FIG. 3 shows the effect of bifidobacterium breve HY002 in experimental example 7 of the present invention on the proliferation potency of RAW264.7 cells.
FIG. 4 is a graph showing the results of experiment of the production of biogenic amine by bifidobacterium breve HY002 in experimental example 10, the results of experiment of the production of biogenic amine by the strain HY002 in the first behavior, and the results of experiment of the production of biogenic amine by the strain E.coli in the second behavior positive control.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described below by way of specific examples. The following experimental examples are illustrative of the present invention, but are not intended to limit the scope of the present invention.
Sources of biological materials
1. Escherichia coli ATCC25922, salmonella enteritidis ATCC14028, staphylococcus aureus ATCC25923, bacteroides fragilis ATCC25285 and Streptococcus pneumoniae ATCC49619 used in Experimental example 5 of the present invention were purchased from China general microbiological culture Collection center.
2. The Klebsiella pneumoniae used in experimental example 5 of the present invention is Klebsiella pneumoniae strain 587, which has been registered with NCBI and has a registration number of MT573143; the Friesen strain is Friesen strain 7610, the strain is registered with NCBI, and the registration number is MT516163; the streptococcus angina is streptococcus angina 10378, the strain is registered in NCBI, and the registration number of the strain is MW090399; currently kept by applicant laboratories, applicant promises to deliver to the public within 20 years from the filing date of the present application for verifying the technical effect of the present invention.
3. RAW264.7 cells used in experimental examples 7 and 8 of the present invention were purchased from the chinese collection of typical cultures.
4. The E.coli strain used in Experimental example 10 of the present invention was named Escherichia coli ATCC25922, which was purchased from China general microbiological culture Collection center.
Group 1 example, strain HY002 of the present invention
The embodiment of the group provides a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC NO.26838.
Any of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, reforming, selling, offering to sell the behavior of a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838, and/or the behavior of combining a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 with other probiotics, and/or antagonizing various pathogenic bacteria with a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838, and/or preparing an antibacterial product, and/or preparing an immunity-enhancing medicament, and/or preparing a health-care food with an immunity-enhancing function falls within the scope of the present invention.
Such other probiotics include, but are not limited to: lactobacillus plantarum, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus delbrueckii subspecies bulgaricus, lactobacillus delbrueckii subspecies lactis, lactobacillus helveticus, lactobacillus casei, lactobacillus crispatus, lactobacillus fermentum, lactobacillus gasseri, lactobacillus johnsonii, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus salivarius, saccharomyces cerevisiae, torulopsis delbrueckii, candida, wilhelminth, pichia, saccharomyces brueckii, candida, schwannoma, rhodotorula, schizosaccharomyces pombe, saccharomyces bauhini, bacillus thuringiensis, bacillus laterosporus, bacillus megaterium, bacillus mucilaginosus, bacillus azotemonis, bacillus sphaericus, clostridium butyricum, bifidobacterium adolescentis, bifidobacterium animalis, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium infantis (i.e., bifidobacterium longum subspecies infantis), bifidobacterium lactis (i.e., bifidobacterium animalis subspecies lactis), bifidobacterium longum, bifidobacterium pseudocatenulatum, bifidobacterium thermophilum, and bifidobacterium acidophilus.
According to the actual production requirement, the person skilled in the art can combine the conventional technical means or the common general knowledge of the production process in the pharmaceutical field (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like) to perform conventional selection or adjustment on the pharmaceutical auxiliary materials, so that a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 is prepared into products with different dosage forms, different storage conditions and different shelf lives, which has no technical obstacle to the person skilled in the art and can be easily achieved.
Group 2 example, use of the inventive Strain HY002
The embodiment of the group provides application of bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 in preparing medicines for enhancing immunity or preparing health-care foods with the function of enhancing immunity or preparing bacteriostats.
In particular embodiments, the enhancing immunity comprises: promoting immune cell proliferation and immune factor production;
preferably, the promoting immune factor production means: promoting immune cells to secrete immune factors;
Preferably, the immune cells are RAW264.7 cells;
Preferably, the immune factor comprises: TNF-alpha, IL-10, IL-6, IL-1β, IL-17;
Preferably, the bacteriostasis spectrum of the bacteriostat comprises: staphylococcus aureus, escherichia coli, salmonella, klebsiella pneumoniae, escherichia coli, bacteroides fragilis, streptococcus angina, and streptococcus pneumoniae.
Group 3 examples, immunopotentiators of the invention
The present set of embodiments provides a medicament for enhancing immunity. All embodiments of this group share the following common features: the immunity enhancing medicine comprises: a pharmaceutically active ingredient; the pharmaceutically active ingredients include: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
In a further embodiment, the immunity enhancing drug further comprises: pharmaceutically acceptable auxiliary materials.
In a more specific embodiment, the pharmaceutically acceptable excipients are selected from the group consisting of: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.
According to the invention, in view of different requirements in practical production and application, in combination with conventional technical means in the field of medicine preparation (for example, encyclopedia of preparation technology, technology of pharmaceutical preparation, research and application of microbial agent technology, etc.), a person skilled in the art can select and blend the pharmaceutically acceptable auxiliary materials, and make bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 into different dosage forms, such as powder, tablets, injection, oral liquid, suppository, gel, patch, spray, lotion, granule, etc.
In a specific embodiment, the dosage form of the drug is selected from the group consisting of: one or more of powder, tablet, liquid and capsule.
Group 4 examples, health foods of the present invention
The embodiment of the group provides a health food with the function of enhancing immunity. All embodiments of this group share the following common features: the health food with the immunity enhancing function comprises the following components: a functional active ingredient; the functional active ingredients include: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
In a further embodiment, the health food with immunity enhancing function further comprises: food auxiliary materials.
In a more specific embodiment, the edible auxiliary is selected from the group consisting of: food additives, solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-binding agents, integration agents, permeation enhancers, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.
According to the invention, the person skilled in the art can select and blend the edible auxiliary materials and prepare the bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 into different dosage forms, such as powder, tablets, injection, oral liquid, suppositories, gel, patch, spray, lotion, granule and the like, according to different requirements in practical production and application and in combination with conventional technical means in the field of medicine preparation (for example, food technology, new technology for food processing, experimental course for food technology, and the like).
In a specific embodiment, the dosage form of the drug is selected from the group consisting of: one or more of powder, tablet, liquid and capsule.
Group 5 examples, bacteriostats of the invention
The present set of embodiments provides a bacteriostatic agent. All embodiments of this group share the following common features: the bacteriostatic agent comprises: a bacteriostatic active ingredient; the antibacterial active ingredients comprise: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
In a further embodiment, the bacteriostatic agent further comprises: auxiliary materials.
In a more specific embodiment, the adjuvant is selected from: solvents, propellants, solubilizing agents, co-solvents, emulsifiers, colorants, binders, disintegrants, fillers, lubricants, wetting agents, osmotic pressure modifiers, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, integration agents, permeation promoters, pH modifiers, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants, deflocculants, filter aids, release retarders, and the like.
According to the invention, the person skilled in the art can select and blend the auxiliary materials and prepare the bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 into different dosage forms, such as powder, tablets, injection, oral liquid, suppositories, gel, application, spray, lotion, granules and the like, according to different requirements in practical production and application and in combination with conventional technical means in the field of medicine preparation (for example, encyclopedia of preparation technology, technology of medicine preparation, technical research and application of microbial agents and the like).
In specific embodiments, the bacteriostatic agent is in a dosage form selected from the group consisting of: one or more of powder, tablet, liquid and capsule.
Group 6 example, in vitro method of inhibiting bacteria of the invention
The present set of embodiments provides an in vitro bacteriostasis method. All embodiments of this group share the following common features: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 is adopted for bacteriostasis.
In a specific embodiment, the bacteriostatic spectrum of the bacteriostasis includes: staphylococcus aureus, escherichia coli, salmonella, klebsiella pneumoniae, escherichia coli, bacteroides fragilis, streptococcus angina, and streptococcus pneumoniae.
In some embodiments, the in vitro bacteriostasis refers to: the external bacteria of the human body or animal body are not contacted.
In other embodiments, a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the accession number CGMCC No.26838 can be used by those skilled in the art to inhibit bacteria in combination with other probiotics having conventional technical implications known to those skilled in the art, for example, lactobacillus plantarum, lactobacillus acidophilus, lactobacillus rhamnosus, lactobacillus delbrueckii subspecies bulgaricus, lactobacillus delbrueckii subspecies lactis, lactobacillus helveticus, lactobacillus casei, lactobacillus crispatus, lactobacillus fermentum, lactobacillus gasseri, lactobacillus johnsonii, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus salivarius, saccharomyces cerevisiae, torulopsis delbrueckii, candida, wilhelminth, pichia, saccharomyces brueckii, candida, schwannoma, rhodotorula, schizosaccharomyces pombe, saccharomyces pombe, bacillus bauhinensis, bacillus thuringiensis, bifidobacterium laterosporus, bacillus megaterium, bacillus mucilaginosus, bifidobacterium azotemlobus, bifidobacterium adolescentis, bifidobacterium bigeminum, bifidobacterium animalis, bifidobacterium bifidum, bifidobacterium infantis (i.e, bifidobacterium longum infantis), bifidobacterium thermophilum, bifidobacterium lactis, bifidobacterium thermobifidum, and the like.
The technical effects of the present invention are described and verified in further detail below in connection with experimental examples.
The culture medium used in the experimental example of the present invention:
MRS solid medium: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 1mL of Tween 80, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate, 15g of agar and 1000mL of distilled water, sterilizing for 20min at 121 ℃, and adjusting the pH to 6.8.
MRS liquid medium: 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 20g of glucose, 1mL of Tween 80, 5g of sodium acetate, 2g of dipotassium hydrogen phosphate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and 1000mL of distilled water, sterilizing for 20min at 121 ℃, and adjusting the pH to 6.8.
LB medium, TSA medium, columbia medium are all commercial media.
LMRS medium: 0.05% L-cysteine hydrochloride was added on the basis of MRS medium.
Unless otherwise specified, various reagent consumables used in the examples and experimental examples of the invention are commercially available, and related experimental steps are common operations in the field and have technical meanings which can be conventionally understood by those skilled in the art.
Experimental example 1, isolation and screening of Strain
The collected infant faeces sample is transported in an ice box in a sterile sampling tube, diluted with 0.85% physiological saline in a gradient manner under sterile conditions, a proper dilution gradient is selected and coated on an MRS agar plate added with 5% (V/V) mupirocin lithium salt and containing 0.05% L-cysteine hydrochloride, and the infant faeces sample is cultured for 48-72 hours under anaerobic conditions at 37 ℃. The suspected single colony is picked up by observing the colony morphology by naked eyes, and the suspected single colony is observed by microscopic examination and is subjected to preliminary screening and purification culture. Culturing for 8-12 hr at 37deg.C with MRS liquid anaerobic tube, centrifuging to remove supernatant, and re-suspending in sterile 30% glycerol water solution, and preserving in ultra-low temperature refrigerator.
Experimental example 2, strain identification
The screened target strain is subjected to liquid amplification, thalli are collected, genome DNA is extracted, a 16S rDNA sequence is amplified by adopting universal primers 27F and 1492R described in Chinese patent No. 202210478937.4, PCR amplification products are detected by agarose gel electrophoresis, and the PCR products are sequenced. Wherein the PCR reaction system comprises: 10 Xbuffer 10. Mu.L, 10mM dNTP 2. Mu.L, 1. Mu.L each of the upper and lower primers, 2. Mu.L of DNA template, 0.5. Mu.L of Taq enzyme, and 34.5. Mu.L of ddH 2 O. Pre-denaturation at 95℃for 10min; then, the temperature is 94 ℃ for 30s, 60 ℃ for 30s and 72 ℃ for 1min for 35 cycles, and the temperature is 72 ℃ for 5min after the completion of the cycle. The PCR products were detected by gel electrophoresis and sent to the Wuhan Jin Kairui Bio-engineering Co., ltd for sequencing. The identified gene sequences were aligned in NCBI database using BLAST tool, and based on molecular biological identification, the strain was identified as Bifidobacterium breve under the Latin name Bifidobacterium breve. The strain is named as bifidobacterium breve HY002 and is preserved, and the preservation information is as follows:
preservation number: CGMCC No.26838
Classification naming: bifidobacterium breve Bifidobacterium breve
Preservation date: 2023, 03, 17
Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)
Preservation address: the korean district beichen, beijing city, part No. 1, no. 3.
Experimental example 3, physiological and Biochemical identification of Bifidobacterium breve HY002
The physiological and biochemical characteristic results of bifidobacterium breve HY002 strain are shown below: the strain can utilize D-arabinose, D-ribose, D-galactose, D-glucose, D-fructose, D-mannose, mannitol, sorbitol, alpha-methyl-D-glucoside, N-acetyl-glucosamine, amygdalin, esculin, salicin, cellobiose, maltose, lactose, melibiose, raffinose, starch, glycogen, gentiobiose, D-melissose and L-fucose.
Experimental example 4 acid and bile salt resistance of Bifidobacterium breve HY002
(1) Simulated artificial gastric juice preparation: naCl 0.5% (w/v), pepsin 0.3% (w/v) was added, the pH was adjusted to 3.0 with 1M HCl, and after complete dissolution, the solution was sterilized by filtration through a sterile microporous membrane of 0.22. Mu.m.
(2) Preparation of simulated artificial bile salt solution: adding 0.2% (w/v) sodium thioglycolate into LMRS liquid culture medium, adding bile salt with final concentration of 0.1%, dissolving completely, and filtering with 0.22 μm sterile microporous membrane for sterilization.
(3) Tolerance test: artificial gastric juice and bile salt resistance experiment: 0.1mL of the bacterial suspension is added into 0.9 mL artificial gastric juice (pH=3.0) and bile salt (0.1%) respectively, and incubated for 3 hours at 37 ℃; the treatment solution was subjected to gradient dilution at 0h and 3h, respectively, and then applied to LMRS plates for viable count. The survival rate of the strain in artificial gastric juice and bile salt is calculated according to the following calculation formula:
wherein N1 represents the number of viable bacteria in the treated strain system, lg (CFU/mL); n0 represents the initial viable count in the strain system, i.e., the viable count measured at 0h, lg (CFU/mL).
As shown in the experimental result in FIG. 1, the survival rate of the bifidobacterium breve HY002 in the artificial gastric juice with the pH value of 3.0 is 100.65 percent, and the survival rate of the bifidobacterium breve HY002 in the artificial bile salt solution with the pH value of 0.1 percent is 100.19 percent, which proves that the bifidobacterium breve HY002 has stronger gastrointestinal tract tolerance.
Experimental example 5, ability of Bifidobacterium breve HY002 to inhibit pathogenic bacteria
Preparation of escherichia coli ATCC25922 bacterial suspension: escherichia coli ATCC25922 was inoculated into LB liquid medium at 2% (v/v), and after culturing at 37℃for 16 to 20 h, the concentration of the bacterial liquid was adjusted to 10 8 CFU/mL.
Salmonella enteritidis ATCC14028 bacterial suspension preparation: salmonella enteritidis ATCC14028 is inoculated into LB liquid medium according to 2% (v/v), and after culturing at 37 ℃ for 16-20 h, the concentration of the bacterial liquid is regulated to 10 8 CFU/mL.
Preparation of staphylococcus aureus ATCC25923 bacterial suspension: staphylococcus aureus ATCC25923 is inoculated into LB liquid culture medium according to 2% (v/v), and after culturing at 37 ℃ for 16-20 h, the concentration of bacterial liquid is regulated to 10 8 CFU/mL.
Preparation of Bacteroides fragilis ATCC25285 bacterial suspension: bacteroides fragilis ATCC25285 is inoculated into TSA blood plates containing 5% defibrinated sheep blood, and after 16-20 h are cultured at 37 ℃, the concentration of bacterial liquid is adjusted to 10 8 CFU/mL.
Preparation of Streptococcus pneumoniae ATCC49619 bacterial suspension: streptococcus pneumoniae ATCC49619 is inoculated into TSA blood plates containing 5% defibrinated sheep blood, and after culturing at 37 ℃ for 16-20 h, the bacterial liquid concentration is regulated to 10 8 CFU/mL.
Preparation of streptococcus angina 10378 MW090399 bacterial suspension: streptococcus angina is inoculated into TSA blood plates containing 5% defibrinated sheep blood, and after 16-20 h is cultured at 37 ℃, the concentration of bacterial liquid is regulated to 10 8 CFU/mL.
Preparation of klebsiella pneumoniae 587 m 573143 suspension: klebsiella pneumoniae is inoculated into TSA blood plates containing 5% defibrinated sheep blood, and after culturing at 37 ℃ for 16-20 h, the concentration of bacterial liquid is regulated to 10 8 CFU/mL.
Preparation of a suspension of the bacterium escherichia fraGlen 7610 MT516163: the Friesen strain is inoculated into LB liquid culture medium according to 2% (v/v), and after culturing at 37 ℃ for 16-20 h, the concentration of the strain liquid is regulated to 10 8 CFU/mL.
Preparing a bacterial strain fermentation liquid: bifidobacterium breve HY002 was inoculated at 1% (v/v) into LMRS liquid medium and cultured at 37℃for 12h.
Bacteriostasis experiment: MRS broth culture medium (TSA culture medium containing 5% defibrinated sheep blood for Bacteroides fragilis, streptococcus pneumoniae and Streptococcus angina) containing 1.5% agar is cooled to about 55deg.C, and mixed with indicator bacteria suspension at a certain ratio to make the number of viable bacteria of indicator bacteria be on the order of 10 6 CFU/mL, then poured into a plate in which oxford cup is placed in advance rapidly, after the culture medium is cooled and solidified, the oxford cup is taken out, 200 μl of strain fermentation liquor (number of viable bacteria is on the order of 10 8 CFU) is poured into each hole, and after overnight culture at 37deg.C, the diameter of the antibacterial circle is measured.
As shown in FIG. 2 and Table 1, the bifidobacterium breve HY002 has obvious inhibiting effect on staphylococcus aureus, escherichia coli, salmonella, klebsiella pneumoniae, fuglen escherichia, bacteroides fragilis and streptococcus angina.
Experimental example 6, bifidobacterium breve HY002 surface hydrophobicity experiment
Activation of bifidobacterium breve: taking out the strain from a refrigerator at the temperature of minus 80 ℃, inoculating the strain into LMRS anaerobic tube liquid culture medium according to the inoculum size of 2% (v/v), and standing and culturing at the temperature of 37 ℃ to obtain a first-generation bifidobacterium breve liquid; inoculating the strain into LMRS anaerobic tube liquid culture medium according to inoculum size of 2% (v/v), and standing and culturing at 37 ℃ for 16h to obtain second-generation bifidobacterium breve liquid for later use;
Transferring the activated bacterial liquid into a 50mL sterile centrifuge tube, centrifuging at 5000rmp/min for 5min to collect bacterial bodies, washing the bacterial bodies twice with an equal amount of PBS buffer, re-suspending the bacterial liquid in the PBS buffer, adjusting the OD600 value of the bacterial liquid suspension to be about 1.0, and simultaneously measuring the absorbance value (A0) of the bacterial liquid suspension at 600 nm; 12 mL bacterial suspension and 4mL of xylene are taken and fully mixed in a test tube, after being placed at room temperature for 10: 10min, the mixture is vortexed and mixed, 20: 20 min layered transparent lower water layer and upper solvent layer containing cells are placed at room temperature, and the absorbance value (A1) of the water phase at 600: 600 nm is measured by taking down the water phase. Buffer was used as a blank. The surface hydrophobicity is expressed as a percentage of bacterial adhesion organic solvent and is calculated as:
surface hydrophobicity (%) = (1-A1/A0) ×100%
The cell surface hydrophobicity of the strain is examined by taking LGG as a control, and the results are shown in table 2, and the experimental results show that the surface hydrophobicity of the HY002 strain is up to 70.69 percent and superior to that of the star strain LGG. On the other hand, the surface hydrophobicity of the strain can reflect the field planting condition of the strain in human intestinal tracts, and higher surface hydrophobicity reflects higher field planting rate, so that HY002 has the potential of adhesion field planting in gastrointestinal tracts.
Experimental example 7 influence of Bifidobacterium breve HY002 on RAW264.7 cell proliferation potency
RAW264.7 cells were seeded at a density of 1X 10 5 cells/mL in 96-well cell culture plates (100. Mu.L per well) and cultured for 24 hours, after which cells were attached, the stock culture was replaced by the bacterial DMEM cell culture. Experiments were performed in blank (DMEM broth) and probiotic groups (live bacteria were washed twice with PBS and resuspended in DMEM) with 5 replicates each. Culturing for 24h at 37 ℃ in a 5% CO 2 incubator. After the cultivation is finished, the old culture medium is sucked, 100 mu L of DMEM culture medium containing 10% of CCK-8 is added into each hole, and the mixture is placed into a CO 2 incubator to be continuously cultivated for 2 hours in a dark place; the OD of each well was measured at 450nm, and the relative proliferation rate of cells was calculated according to the following formula.
Cell relative proliferation% = experimental OD/blank OD
As shown in FIG. 3, the bifidobacterium breve HY002 can promote proliferation of RAW264.7 cells with a proliferation rate up to 150.71%, and the effect is superior to that of the control strain LGG.
Experimental example 8 influence of Bifidobacterium breve HY002 on cytokine secretion from RAW264.7 cells
Experimental example 8 influence of Bifidobacterium breve HY002 on cytokine secretion from RAW264.7 cells
RAW264.7 cells were adjusted to 1X 10 6 cells/mL, and 0.5mL per well was inoculated into 24-well plates and cultured for 24 hours. The experiments were divided into a blank group and a probiotic group, the blank group: sucking out the supernatant, washing with PBS for 2 times, adding 0.5mL of DMEM medium into the plate holes, and culturing in a culture box with 5% CO 2 at 37 ℃ for 24 hours without any other treatment; probiotic group: the supernatant was aspirated, washed 2 times with PBS, and 0.5mL of the bacterial suspension (after washing twice with PBS, the second-cultured bifidobacterium breve HY002 was resuspended in DMEM and diluted in a ratio of bacterial mass to cell mass of 10:1) was added to the well plate, followed by culturing for 24 hours. After the completion of the culture, the supernatant was removed, and centrifuged at 1000rpm for 5 minutes, and the contents of TNF- α, IL-10, IL-6, IL-1. Beta. And IL-17 in the supernatant were measured using the kit.
The experimental results are shown in Table 3, and the results show that the bifidobacterium breve HY002 can promote the production of immune factors TNF-alpha, IL-10, IL-6, IL-1 beta and IL-17 compared with the CK group, which shows that the strain HY002 has an immunoregulatory effect.
Experimental example 9, bifidobacterium breve HY002 sensitivity test to antibiotics
After marking and activating bifidobacterium breve HY002 on an MRS solid plate, selecting lawn into normal saline to prepare bacterial suspension, adjusting the concentration of the bacterial suspension to 10 8 CFU/mL, taking 100 mu L of bacterial suspension, uniformly coating the bacterial suspension on the MRS solid plate by using a sterile cotton swab, orderly placing antibiotic drug-sensitive test paper sheets on the surface of the plate, placing the plate under anaerobic condition, culturing at 37 ℃ for 24-36h, and measuring the diameter of a bacteriostasis ring by using a vernier caliper.
The experimental results are shown in Table 4, and the bifidobacterium breve HY002 is sensitive to 13 antibiotics tested, and the experiment shows that the bifidobacterium breve HY002 is a safe probiotic strain with development and application potential.
Experimental example 10, experiment of Bifidobacterium breve HY002 production of biogenic amine
Amino decarboxylase test medium ratio: 5g of peptone, 3g of yeast extract, 1g of glucose, 1000ml of distilled water, 1ml of 1.6% bromocresol purple-ethanol solution, 1.8% of agar, pH6.8 and sterilizing at 115 ℃ for 20min. (the added amino acids are L-arginine, L-lysine, L-tryptophan, and L-histidine, respectively).
Inoculating the cultured bacterial liquid into 7mL anaerobic tube culture liquid without adding precursor amino acid for overnight culture; each filter paper piece is dripped with the activated and cultured bacterial liquid, soaked, placed in an improved amino decarboxylase detection agar medium added with precursor amino acid, and cultured for 2 days at 37 ℃. The transparent ring around the filter paper was observed for color change. If the color around the filter paper turns purple, this indicates that biogenic amine is produced, whereas if not, this indicates that biogenic amine is not produced.
As shown in FIG. 4, the experiment results showed that there was no color change around HY002, but E.coli produced alkaline biogenic amine, and the medium around the filter paper sheet was changed from yellow to purple. The result shows that HY002 does not produce biogenic amine and has better safety.
Claims (14)
1. A bifidobacterium breve (Bifidobacterium breve) strain HY002 with the function of enhancing immune regulation is characterized in that the preservation number is CGMCC NO.26838.
2. The application of bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No.26838 in preparing medicines for enhancing the immunoregulation function or preparing health-care foods with the immunoregulation function or preparing bacteriostats.
3. The use of a bifidobacterium breve (Bifidobacterium breve) strain HY002 with a accession number of CGMCC No.26838 in the preparation of a medicament for enhancing immune modulating function or in the preparation of a health food having an immune enhancing function or in the preparation of a bacteriostatic agent according to claim 2, wherein the immune enhancing comprises: promote immune cell proliferation and immune factor production.
4. The use of a bifidobacterium breve (Bifidobacterium breve) strain HY002 with a preservation number of CGMCC No.26838 for preparing a medicament for enhancing an immunoregulatory function or for preparing a health food having an immunoregulatory function or for preparing a bacteriostatic agent according to claim 3, wherein the means for enhancing the immune factor production: promote immune cells to secrete immune factors.
5. The use of a bifidobacterium breve (Bifidobacterium breve) strain HY002 with a preservation number of CGMCC No.26838 in the preparation of a medicament for enhancing an immunoregulatory function or in the preparation of a health food having an immunoregulatory function or in the preparation of a bacteriostatic agent according to claim 4, wherein the immune cells are RAW264.7 cells.
6. The use of a bifidobacterium breve (Bifidobacterium breve) strain HY002 with a accession number of CGMCC No.26838 in the preparation of a medicament for enhancing immune modulating function or in the preparation of a health food having an enhanced immune function or in the preparation of a bacteriostatic agent according to claim 3 or 4, wherein the immune factor comprises: TNF-alpha, IL-10, IL-6, IL-1β, IL-17.
7. Use of a bifidobacterium breve (Bifidobacterium breve) strain HY002 with a accession number of CGMCC No.26838 in the preparation of a medicament for enhancing immune modulating function or in the preparation of a health food having an enhanced immune function or in the preparation of a bacteriostatic agent according to any one of claims 2 to 5, wherein the bacteriostatic spectrum of the bacteriostatic agent comprises: staphylococcus aureus, escherichia coli, salmonella, klebsiella pneumoniae, escherichia coli, bacteroides fragilis, streptococcus angina, and streptococcus pneumoniae.
8. A medicament for enhancing an immunomodulatory function, comprising: a pharmaceutically active ingredient; the pharmaceutically active ingredients include: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
9. The medicament for enhancing an immunomodulatory function according to claim 8, further comprising: pharmaceutically acceptable auxiliary materials.
10. A health food having an enhanced immune function, comprising: a functional active ingredient; the functional active ingredients include: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
11. The health food with immunity-enhancing function as claimed in claim 10, further comprising: food auxiliary materials.
12. A bacteriostatic agent, comprising: a bacteriostatic active ingredient; the antibacterial active ingredients comprise: a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC No. 26838.
13. A bacteriostatic agent according to claim 12, further comprising: auxiliary materials.
14. An in vitro bacteriostasis method is characterized in that a bifidobacterium breve (Bifidobacterium breve) strain HY002 with the preservation number of CGMCC NO.26838 is adopted for bacteriostasis.
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| 短双歧杆菌的生物学特性及益生功能研究进展;王元弛;黄玲;张旭光;张学荣;;农产品加工;20200330(第06期);115-120 * |
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