CN117106628A - Lactobacillus acidophilus LA15 with immunoregulation capability and application, product and method thereof - Google Patents
Lactobacillus acidophilus LA15 with immunoregulation capability and application, product and method thereof Download PDFInfo
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- CN117106628A CN117106628A CN202310825497.XA CN202310825497A CN117106628A CN 117106628 A CN117106628 A CN 117106628A CN 202310825497 A CN202310825497 A CN 202310825497A CN 117106628 A CN117106628 A CN 117106628A
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Abstract
The invention relates to lactobacillus acidophilus with immunity regulating capability, and application, a product and a method thereof, belonging to the technical field of microorganisms. The invention provides a lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with immunoregulation capability, and the preservation number is CCTCCNO: M2023768. The invention also provides application of the strain LA15 in preparing antibacterial products and/or preparing immune regulation products and/or preparing products for regulating intestinal flora in vitro, and products and methods based on the strain LA15. The strain LA15 has a strong inhibition effect on escherichia coli, staphylococcus aureus, salmonella and streptococcus mitis, and can effectively play roles in immunoregulation and intestinal flora regulation.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to lactobacillus acidophilus LA15 with immunity regulating capability, and application, a product and a method thereof.
Background
The immune system includes innate immunity and adaptive immunity. It is a tightly regulated network that maintains steady state under normal physiological conditions. Lymphocytes and macrophages play an important role in both immunizations. Lymphocytes and macrophages in the activated state produce a range of inflammatory mediators, such as Nitric Oxide (NO), tumor necrosis factor TNF- α, interferon IFN- γ, and interleukin IL-12, to combat the disease. Immunosuppression is a state of immune dysfunction that reduces the immune response to antigens, rendering individuals more susceptible to disease. Depending on the immunosuppression, patients may be at risk for different types of infections and complications, delaying diagnosis and therapeutic outcome. Immunodeficiency conditions such as infant colic, gastrointestinal infections, irritable bowel disease and cancer often result in low antibody levels or ineffective treatment. In order to control viral infectious diseases and prevent infections, both vaccines and immunostimulants, a large amount of the drugs is required to be used for a long period of time, which often causes serious side effects. Thus, the discovery and development of novel immunostimulants is the most effective way to treat and prevent immunosuppressive diseases.
Probiotics are living microorganisms that provide various health benefits to the host, and in particular the immunomodulatory effects of probiotics have been of great research interest. The probiotics play an important role in maintaining balance of intestinal flora, regulating immunity, preventing gastrointestinal infection, improving blood indexes and the like. Furthermore, studies have shown that probiotics can stimulate the immune response in immunocompromised mice. Therefore, the probiotics strain which has no side effect and can play the role of immunostimulation is developed, and the impaired immune function of the immunodeficiency patient can be improved. Lactic acid bacteria, such as lactobacillus acidophilus, are known as common probiotic species. However, their effects on the immune system vary greatly even among strains within the same species. Therefore, it is necessary to characterize the effects of specific strains and determine their dosages to verify their immunomodulatory effects.
Lactobacillus acidophilus (Lactobacillus acidophilus) belongs to the genus Lactobacillus, gram-positive bacilli, the ends of the rods are rounded, mainly in the small intestine, releasing lactic acid, acetic acid and some antibiotics acting on harmful bacteria, but the bacteriostatic effect is weak.
Therefore, there is a need in the art to develop novel strains of lactobacillus acidophilus which have a strong bacteriostatic action and an immunomodulatory action.
Disclosure of Invention
In order to solve the above-mentioned needs in the art, the present invention provides lactobacillus acidophilus LA15 with immunoregulatory capability, its application in enhancing immunocyte activity and immunoregulation, and intestinal flora regulating products and methods thereof.
The aim of the invention is achieved by the following technical scheme:
a lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with immunoregulation capability is characterized in that the preservation number is CCTCC NO: M2023768.
The Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a preservation number of CCTCC NO: M2023768 is applied to the preparation of antibacterial products and/or the preparation of immune regulation products and/or in vitro antibacterial products and/or the preparation of products for regulating intestinal flora.
The bacteriostasis refers to inhibiting one or more than two of the following bacteria: coli, staphylococcus aureus, salmonella, streptococcus mitis;
preferably, the immunomodulation is selected from: increasing proliferation rate of immune cells, regulating cytokines, increasing body weight and organ index of immunocompromised animals, and regulating one or more of immunoglobulins;
preferably, the modulating intestinal flora is selected from: improving the microbial community diversity index of the intestinal tracts of the immunocompromised animals, reducing the abundance of harmful bacteria in the intestinal tracts of the immunocompromised animals and improving the abundance of beneficial bacteria in the intestinal tracts of the immunocompromised animals.
The immune cells refer to macrophage RAW264.7;
preferably, the cytokine is selected from: cytokines TNF- α, IL-10 of macrophage RAW264.7, and/or cytokines IL-1β, TNF- α, IL-6, IL-17, IL-10 of immunocompromised animals;
preferably, the immunocompromised animal refers to cyclophosphamide-molded immunocompromised mice;
preferably, the immunoglobulin refers to sIgA;
preferably, the microbial community diversity index comprises: simpson index and PD_white_tree index;
preferably, the intestinal harmful bacteria are selected from: desulphurized vibrio, helicobacter;
preferably, the intestinal beneficial bacteria are selected from: bacteroides, prevotella, lachnospiraceae, NK4A136_group, unclassified Eubacterium, lactobacillus, and unclassified Muribaculaceae, candidatus _Saccharomonas.
An immunomodulatory product comprising: an active ingredient; the active ingredients include: lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a accession number of CCTCC NO: M2023768.
The immunoregulation product further comprises: auxiliary materials.
A product for inhibiting bacteria and/or regulating intestinal flora, comprising: an active ingredient; the active ingredients include: lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a accession number of CCTCC NO: M2023768.
The product for inhibiting bacteria and/or regulating intestinal flora further comprises: auxiliary materials.
An in vitro bacteriostasis method is characterized in that Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a preservation number of CCTCC NO: M2023768 is adopted for bacteriostasis.
The bacteriostasis refers to: inhibit one or more of Escherichia coli, staphylococcus aureus, salmonella and Streptococcus mitis.
The lactobacillus acidophilus LA15 provided by the invention has an effect on enhancing the content of cytokine TNF-alpha and IL-10 secreted by macrophage RAW 264.7.
Lactobacillus acidophilus strain LA15 with a preservation number (Lactobacillus acidophilus) is used for preparing an immunity enhancing product and application. Said is for enhancing immunoglobulin sIgA, cytokine IL-6, IL-1 beta, IL-17, TNF-alpha, IL-10 content
The application of lactobacillus acidophilus in regulating intestinal flora refers to increasing or decreasing the abundance of intestinal flora. The intestinal flora is as follows: improving Bacteroides, prevotella, maosporium, lactobacillus Candida_Saccharomona; [ Eubacterium ] _co-precursor groups; the content of beneficial bacteria such as Muribaculaceae; reducing the content of harmful bacteria such as Vibrio, helicobacter and the like.
The beneficial effects of the invention are as follows:
the invention screens a new strain from faeces samples, and the strain is lactobacillus acidophilus (Lactobacillus acidophilus) and named strain LA15 after molecular identification. A large number of experiments prove that the novel strain LA15 has higher gastric juice and bile salt solution tolerance capability, has stronger inhibition capability on escherichia coli, staphylococcus aureus, salmonella and streptococcus mitis, has sensitivity to various antibiotics and is safe to organisms. Meanwhile, the strain LA15 has higher adhesion capability to CaCO-2 cells, has remarkable improvement effect on proliferation rate of the macrophage RAW264.7, and can effectively regulate the content of pro-inflammatory factor TNF-alpha and anti-inflammatory factor IL-10 of the macrophage RAW 264.7.
The strain LA15 also has remarkable promotion effect on the body weight and organ index of the mice with low immunity, and has remarkable promotion effect on the level of the immunoglobulin and cytokines IL-1 beta, TNF-alpha, IL-6, IL-17 and IL-10 of the mice with low immunity. In addition, the strain LA15 can promote the abundance of beneficial bacteria such as Bacteroides, prevotella, lachnospiraceae NK4A136 group, unclassified Eubacterium, lactobacillus and unclassified Muribaculaceae, candidatus _Saccharomonas in the intestinal tract of the immunocompromised mice, and reduce the abundance of harmful bacteria such as Desulfovibrio and Helicobacter Helicobacter in the intestinal tract of the immunocompromised mice.
The lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 of the invention has the following preservation information:
preservation number: cctccc No. M2023768;
classification naming: lactobacillus acidophilus LA15;
preservation date: 2023, 5, 17;
preservation unit: china center for type culture Collection;
preservation address: chinese, wuhan, university of Wuhan.
Drawings
FIG. 1 effect of Lactobacillus acidophilus LA15 on proliferation rate of macrophage RAW 264.7.
FIG. 2 shows a graph of the modulation of the cytokine TNF- α, IL-10 by Lactobacillus acidophilus LA15 in macrophage RAW 264.7.
The labels on the abscissa of fig. 1-2 are listed below: CK refers to the control group and LA15 is lactobacillus acidophilus LA15 treated group.
FIG. 3 graph of the effect of Lactobacillus acidophilus LA15 on the body weight of immunocompromised mice.
FIG. 4 shows the effect of Lactobacillus acidophilus LA15 on immune organ index in immunocompromised mice.
FIG. 5 is a graph showing the results of measurement of immunoglobulin and cytokine contents in mouse intestinal tissues.
FIG. 6 is a graph of the Simpson index and the PD_whole_tree index in the alpha-diversity of intestinal microorganisms in mice.
FIG. 7 is a graph of a mouse intestinal flora level analysis.
In fig. 3 to 7, the marks on the abscissa of the respective diagrams are listed as follows:
NC is normal group mice, MC is model group mice, LA15 is probiotic group mice.
Detailed Description
The following describes the present invention in detail with reference to specific examples and experimental examples, but is not intended to limit the scope of the present invention.
Sources of biological materials
1. Experimental example 3 Escherichia coli, staphylococcus aureus, and Salmonella were purchased from the microorganism collection center of Guangdong province. Streptococcus mitis is Streptococcus mitis strain 5013 which has been registered with NCBI under the registration number MT512114. This strain 5013 is currently maintained by the applicant's laboratory and applicant promises to deliver to the public within 20 years from the date of application of the invention to verify the technical effect of the invention.
2. CaCO-2 cells used in Experimental example 5 are commercially available.
3. Macrophage RAW264.7 used in experimental example 6 is commercially available.
4. Experimental examples 7-9 Male Balb/c mice were used commercially.
Group 1 example, strain LA15 of the invention
The embodiment provides a lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with immunoregulation capability, which is characterized in that the preservation number is CCTCC NO: M2023768.
Any of culturing, propagating, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, transforming, selling, offering to sell a Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a preservation number of CCTCC NO: M2023768, and/or a Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a preservation number of CCTCC NO: M2023768 in combination with other probiotics, and/or an inhibiting effect of a Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a preservation number of CCTCC NO: M2023768 on pathogenic bacteria such as E.coli, staphylococcus aureus, salmonella, streptococcus mitis, etc., a bacteriostatic product, and/or an immunomodulating product, and/or a product for regulating intestinal flora, falls within the scope of the present invention.
Such other probiotics include, but are not limited to: lactobacillus plantarum, lactobacillus rhamnosus, lactobacillus delbrueckii subsp. Bulgaricus, lactobacillus delbrueckii subsp. Lactis, lactobacillus helveticus, lactobacillus casei, lactobacillus crispatus, lactobacillus fermentum, lactobacillus grignard, lactobacillus johnsonii, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus salivarius, saccharomyces cerevisiae, candida, wilm's yeast, pichia, saccharomyces, candida albicans, saccharomyces schwannoma, rhodotorula, schizosaccharomyces pombe, saccharomyces bauhini, bacillus thuringiensis, bacillus laterosporus, bacillus megaterium, bacillus mucilaginosus, bacillus azotembotrytis, clostridium butyricum, bifidobacterium adolescentis, bifidobacterium animalis, bifidobacterium bifidum, bifidobacterium starlike, bifidobacterium bifidum, bifidobacterium breve, bifidobacterium infantis (i.e.g., bifidobacterium longum subsp. Breve), bifidobacterium lactis (i.e.g., bifidobacterium lactis), bifidobacterium lactis, bifidobacterium parvom and bifidobacterium bifidum.
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 one lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with the preservation number of CCTCC NO: M2023768 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 strain LA15 of the invention
The embodiment of the group provides the application of lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with the preservation number of CCTCC NO: M2023768 in preparing antibacterial products and/or preparing immune regulation products and/or preparing in-vitro antibacterial products and/or preparing intestinal flora regulation products.
In specific embodiments, the bacteriostatic means inhibiting one or more than two of the following bacteria: coli, staphylococcus aureus, salmonella, streptococcus mitis;
preferably, the immunomodulation is selected from: increasing proliferation rate of immune cells, regulating cytokines, increasing body weight and organ index of immunocompromised animals, and regulating one or more of immunoglobulins;
preferably, the modulating intestinal flora is selected from: improving the microbial community diversity index of the intestinal tracts of the immunocompromised animals, reducing the abundance of harmful bacteria in the intestinal tracts of the immunocompromised animals and improving the abundance of beneficial bacteria in the intestinal tracts of the immunocompromised animals.
In some embodiments, the immune cells refer to macrophage RAW264.7;
preferably, the cytokine is selected from: cytokines TNF- α, IL-10 of macrophage RAW264.7, and/or cytokines IL-1β, TNF- α, IL-6, IL-17, IL-10 of immunocompromised animals;
preferably, the immunocompromised animal refers to cyclophosphamide-molded immunocompromised mice;
preferably, the immunoglobulin refers to sIgA;
preferably, the microbial community diversity index comprises: simpson index and PD_white_tree index;
preferably, the intestinal harmful bacteria are selected from: desulphurized vibrio, helicobacter;
preferably, the intestinal beneficial bacteria are selected from: bacteroides, prevotella, lachnospiraceae, NK4A136_group, unclassified Eubacterium, lactobacillus, and unclassified Muribaculaceae, candidatus _Saccharomonas.
Group 3 examples, immunomodulatory products of the invention
The present set of embodiments provides an immunomodulatory product. All embodiments of this group share the following common features: the immunomodulating product comprises: an active ingredient; characterized in that the active ingredients comprise: lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a accession number of CCTCC NO: M2023768.
In a further embodiment, the immunoregulatory product 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 lactobacillus acidophilus (Lactobacillus rhamnosus) strain LA15 with cctccc NO: M2023768 into different dosage forms, such as powder, tablet, injection, oral liquid, suppository, gel, application, spray, lotion, granule, etc., according to different requirements in practical production and application, in combination with conventional technical means in the field of medicine preparation (for example, encyclopedia of formulation technology, pharmaceutical preparation technology, etc.).
In particular embodiments, the dosage form of the product is selected from: one or more of powder, tablet, liquid and capsule.
Group 4 example, bacteriostatic and/or intestinal flora-regulating products of the invention
The present group of embodiments provides a product for inhibiting bacteria and/or regulating intestinal flora. All embodiments of this group share the following common features: the product for inhibiting bacteria and/or regulating intestinal flora comprises: an active ingredient; the active ingredients include: lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a accession number of CCTCC NO: M2023768.
In a further embodiment, the product for inhibiting bacteria and/or regulating intestinal flora 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 technical means (for example, encyclopedia of preparation technology, pharmaceutical preparation technology and the like) in the field of preparation are combined for different demands in practical production application, and the technical staff can select and blend the medicinal auxiliary materials and prepare lactobacillus acidophilus (Lactobacillus rhamnosus) strain LA15 with CCTCC NO: M2023768 into different dosage forms, such as powder, tablets, injection, oral liquid and the like.
In particular embodiments, the antimicrobial agent is in a dosage form selected from the group consisting of: one or more of powder, tablet, liquid and capsule.
Group 5 example, in vitro method of inhibiting bacteria of the invention
The embodiment provides an in-vitro bacteriostasis method which is characterized in that Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a preservation number of CCTCC NO: M2023768 is adopted for bacteriostasis.
In a specific embodiment, the bacteriostasis means: inhibit one or more of Escherichia coli, staphylococcus aureus, salmonella and Streptococcus mitis.
The terms "above" and "below" as used herein include the present number.
"Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15", "Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15", "Lactobacillus acidophilus LA15", "LA15 strain", "strain LA15" herein all refer to: lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a accession number of CCTCC NO: M2023768.
Experimental example 1, isolation screening and identification of Lactobacillus acidophilus
(1) Isolation and selection of strains
Collecting a sample of infant feces, taking 0.5g to 4.5mL of sterile physiological saline, fully oscillating and dispersing the sample, taking 100 mu L of sample for gradient dilution, selecting a proper gradient to uniformly coat on an MRS agar plate, placing the plate under anaerobic conditions, culturing at 37 ℃ for 36-48 hours, selecting single bacterial colonies, carrying out gram staining, observing morphological characteristics of the bacterial colonies by microscopic examination, and primarily screening strains of suspected lactobacillus. The strain is repeatedly streaked and purified on MRS solid culture medium to obtain lactobacillus purified strain, and the purified strain is preserved in glycerol pipe at-80 ℃.
MRS culture medium formula: 10g of peptone, 5g of beef extract powder, 5g of yeast powder, 20g of glucose, 5g of anhydrous sodium acetate, 2g of diammonium hydrogen citrate, 1mL of Tween 80 and K 2 HPO 4 2g、MgSO 4 0.2g、MnSO 4 0.05g of 1000ml of distilled water, pH6.5, and 121℃for 20min, 1.5g of agar powder was added to the solid MRS medium.
(2) Molecular biological identification
Culturing the screened target strain, collecting thalli, extracting genome DNA, detecting PCR amplification products by agarose gel electrophoresis by adopting an upstream primer 27, a F (AGTTTGATCMTGGCTCAG) and a downstream primer 1492, R (GGTTACCTTGTTACG ACTT), and sequencing the PCR amplification products. Wherein the PCR reaction system comprises: 10 Xbuffer 10. Mu.L, 10mM dNTP 2. Mu.L, 1. Mu.L each of upper and lower primers, 2. Mu.L of DNA template, 0.5. Mu.L of Taq enzyme, ddH 2 O34.5. Mu.L. 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. Detecting PCR product by gel electrophoresis, and delivering to Wohan Jin Kairui biological engineeringSequencing by Programming Limited. The identified gene sequences were aligned in NCBI database using BLAST tool, and based on molecular biology identification, the strain was identified as Lactobacillus acidophilus, designated Lactobacillus acidophilus LA15, and deposited as follows:
preservation number: cctccc No. M2023768;
classification naming: lactobacillus acidophilus LA15;
preservation date: 2023, 5, 17;
preservation unit: china center for type culture Collection;
preservation address: chinese, wuhan, university of Wuhan.
Experimental example 2 gastrointestinal tract tolerance experiment
Artificial gastric juice: PBS solution is prepared, 0.3% pepsin is added, the pH value is regulated to 2.5 by 1mol/LHCL, and then the solution is fully dissolved and filtered and sterilized by a microporous filter membrane with the thickness of 0.22 mu m for later use.
Bile salt solution: in MRS culture medium, 0.3% of ox gall salt and 0.2% of sodium thioglycolate are added, and after being fully dissolved, the mixture is filtered and sterilized by a 0.22 mu m microporous filter membrane for standby.
Activating the strain to be detected for 2 generations and then adjusting the concentration of bacterial liquid to 10 8 CFU/mL. And (3) centrifuging 1mL of bacterial suspension, collecting thalli, respectively inoculating 1mL of prepared artificial gastric juice and bile salt solution, uniformly mixing, digesting at 37 ℃, simultaneously respectively taking the digests of 0h and 3h to detect the number of viable bacteria, and calculating the survival rate, wherein the result is shown in Table 1. Wherein, the survival rate (%) =Nt/N0×100%, where N0 represents the viable count of the strain 0h, lgCFU/mL, nt represents the viable count of the strain 3h, lgCFU/mL
TABLE 1 Lactobacillus acidophilus tolerance to the gastrointestinal tract
TABLE 1 survival rate of Lactobacillus acidophilus LA15 against artificial gastric juice and bile salt solution
| Strain | Artificial gastric juice (%) | Bile salts (%) |
| Lactobacillus acidophilus LA15 | 98.90±0.79 | 95.68±0.67 |
The experimental result shows that the survival rate of lactobacillus acidophilus LA15 is 98.9 percent after gastric juice is digested for 3 hours, and the survival rate of lactobacillus acidophilus LA15 is 95.68 percent after bile salt is digested for 3 hours, which shows that the lactobacillus acidophilus LA15 has stronger gastric acid and bile resistance.
Experimental example 3 inhibition ability of Lactobacillus acidophilus against four pathogenic bacteria
E.coli, staphylococcus aureus and salmonella are respectively inoculated into a liquid LB culture medium for 24 hours at 37 ℃, and streptococcus mitis is inoculated into BHI and 5% sheep blood solid culture medium for 16 hours at 37 ℃ for bacteriostasis experiments. An oxford cup method is adopted, the oxford cup is put in a sterile plate, escherichia coli, staphylococcus aureus and salmonella are mixed into an MRS culture medium according to a certain proportion, and the number of viable bacteria of pathogenic bacteria of a system is 10 6 Order of magnitude, mixing the streptococcus mitis into an improved MRS culture medium according to a certain proportion, wherein the number of living pathogenic bacteria of the system is 10 6 Order of magnitude, after taking out oxford cup, 200. Mu.L of Lactobacillus acidophilus solution (10 8 CFU), the diameter of the inhibition zone was measured after incubation at 37 ℃ for 24h.
TABLE 2 diameter of zone of inhibition of Lactobacillus acidophilus against pathogenic bacteria
| Strain | Coli bacterium | Staphylococcus aureus | Salmonella bacteria | Streptococcus mitis |
| Lactobacillus acidophilus | 43±0.5 | 22±1.0 | 45±0.5 | 24±0.5 |
As shown in the table above, lactobacillus acidophilus can effectively inhibit intestinal pathogens, especially escherichia coli, salmonella, and antibacterial rings with diameters of 43mm and 45mm respectively, and has strong inhibition capability.
Experimental example 4 Lactobacillus acidophilus antibiotic susceptibility test
The test uses K-B (drug sensitive paper agar diffusion method) to measure antibiotic sensitivity. Activating the strain to be tested, preparing a bacterial suspension and adjusting the concentration to be 10 8 CFU/mL is taken, 200 mu L of bacterial suspension is added to the surface of an MRS solid flat plate, bacterial liquid is uniformly coated on the flat plate by using a sterile cotton swab, antibiotic drug sensitive paper sheets are attached to the MRS solid culture medium by using sterile forceps, marking is carried out, and after culturing for 24 hours at 37 ℃, the diameter of a bacteriostasis ring is measured by using a vernier caliper. Table 3 shows the sensitivity results of Lactobacillus acidophilus LA15 to common antibiotics, and the experimental results show that: lactobacillus acidophilus LA15 showed sensitivity to all 13 antibiotics, indicating that the strain is safe and useful in biological products or dietary supplements.
TABLE 3 sensitivity of Lactobacillus acidophilus to different antibiotics
| Sequence number | Antibiotics | Diameter of inhibition zone (mm) | Sensitivity to |
| 1 | Tetracycline | 37 | S |
| 2 | Erythromycin | 29 | S |
| 3 | Amoxicillin | 34 | S |
| 4 | Penicillin | 40 | S |
| 5 | Ampicillin (Amoxicillin) | 33 | S |
| 6 | Vancomycin | 26 | S |
| 7 | Oxacillin (oxacillin) | 24 | S |
| 8 | Azithromycin | 22 | S |
| 9 | Rifampicin | 25 | S |
| 10 | Cefaclor (Cefaclor) | 31 | S |
| 11 | Rifampicin | 25 | S |
| 12 | Ceftriaxone | 31 | S |
| 13 | Cefixime | 27 | S |
Experimental example 5 measurement of adhesion of Lactobacillus acidophilus to CaCO-2 cells
Culturing strain in MRS culture medium for 24 hr, centrifuging, collecting thallus, washing with PBS for 3 times, suspending in DMEM culture solution without double antibody, and regulating the concentration of bacterial suspension to 10 8 CFU/mL. Regulating CaCO-2 cell suspension with good cell state to 10 5 cell/mL, inoculating 1mL to 12-hole cell culture plates, incubating in an incubator with a concentration of 5% CO2 until the cells grow to a monolayer, washing twice with sterile PBS, and performing cell counting by a blood cell counting plate; adding 1mL of lactobacillus acidophilus bacterial suspension, incubating for 2 hours at 37 ℃ in a 5% CO2 incubator, washing cells for 3 times by using sterile PBS, removing non-adhered bacterial suspension, adding 0.2mL of pancreatin-EDTA buffer solution into each hole to digest the cells for 5 minutes, adding 0.8mL of PBS after digestion, blowing evenly, and taking bacterial liquid for dilution and live bacterial counting. 3 replicates were run for each experiment and the adhesion of the strain to the cells was examined by adhesion experiments and the results are shown in Table 4.
TABLE 4 determination of adhesion ability of Lactobacillus acidophilus to CaCO-2 cells
| Strain | Adhesion capability (CFU/cell) |
| Lactobacillus acidophilus LA15 | 3.54±0.18 |
As can be seen from table 4 above: the lactobacillus acidophilus LA15 cells have higher adhesion capability, and the adhesion capability to CaCO-2 cells is 3.54CFU/cell. This shows that lactobacillus acidophilus LA15 can adhere to intestinal epithelial cells and can be well planted in intestinal tracts, so that the probiotic performance is exerted and the microecological balance of the organism is regulated.
Experimental example 6 Regulation of immune cells by Lactobacillus acidophilus
(1) Effect of Lactobacillus acidophilus on proliferation Raw264.7 of macrophages
Cell concentration was adjusted to 1 x 10 in 96-well plates 4 cell/mL, 100. Mu.L/well was inoculated in 96-well plates and cultured for 24h until cells grew on the wall. Control group (CK): adding 100 mu L of cell culture solution into the plate hole without any other treatment, and culturing in a 5% CO2 incubator at 37 ℃ for 24 hours; probiotic group, lactobacillus acidophilus LA15 treatment group (LA 15): mu.L of the bacterial suspension (resuspended in DMEM) was added to the well plate and incubated at a ratio of 100:1 for 24h. After the cell culture was completed, 10. Mu.L of CCK-8 reagent was added, incubated at 37℃for 1 hour in the absence of light, then the supernatant in the well plate was aspirated, washed 2 times with PBS, and the absorbance (OD value) of each well at 450nm was measured with an ELISA reader.
Cell viability calculation formula: cv=od 1 /OD 2 ×100％。
In the formula, CV-cell viability (%); OD (optical density) 1 -OD values at 450nm wavelength for each treatment group; OD (optical density) 2 OD values of the blank at a wavelength of 450 nm.
As can be seen from fig. 1: compared with the control group (CK), the probiotic group, namely lactobacillus acidophilus LA15 treatment group (LA 15), remarkably improves the proliferation rate of macrophages, the proliferation rate reaches 158.4%, and the lactobacillus acidophilus LA15 can enhance the immune function of the organism by inducing the activity enhancement of the macrophages.
(2) Cytokine modulating effect of lactobacillus acidophilus on macrophage RAW264.7
The cell concentration was adjusted to 1X 10 6 cell/mL,0.5 mL/well was inoculated in a 24-well plate and cultured for 24 hours (adherent growth). Control group (CK): adding only 0.5mL of cell culture solution into the plate hole, and culturing in a 5% CO2 incubator at 37 ℃ for 24 hours without any other treatment; probiotic group: 0.5mL of bacterial suspension (serum-free resuspended in DMEM) was added to the well plate 1X 10 8 CFU/mL, according to 100:1 for 24 hours. After the cell culture is completed, the supernatant is taken out, and then centrifuged at 1000rpm for 10min, and the supernatant is taken out, and the content of the pro-inflammatory factor TNF-alpha and the anti-inflammatory factor IL-10 is measured by ELISA kit.
The cytokine TNF- α, IL-10, is an immune effector molecule that is produced during the innate immune response phase. The experimental results of the invention show that: as shown in FIG. 2, the probiotic group, lactobacillus acidophilus LA15 treated group (LA 15), was able to significantly increase the levels of TNF-. Alpha.and IL-10, respectively 765.78pg/mL and 341.18pg/mL, compared to the control group (CK). This suggests that lactobacillus acidophilus LA15 is able to stimulate macrophages to secrete pro-and anti-inflammatory factors, playing an important role in activating innate immunity.
Experimental example 7 influence of Lactobacillus acidophilus on body weight and immune organ index of immunocompromised mice
Male Balb/c mice (6-8 weeks; 20.+ -.2 g) were housed in Hubei province disease prevention control center animal house, laboratory animals use license number: heart (good fortune) 202210219 was evaluated. Animal experiment is maintained at 20-22 deg.C and humidity of 40-60%, and the circulation is 12h illumination/12 h darkness. Mice were free to be autoclaved on standard laboratory diet and water. After one week of acclimation, mice were divided into 3 groups (n=12): normal group (NC group), model group (MC group), probiotic group (LA 15,1×10) 9 CFU/d). The normal group and the model group are subjected to gastric lavage by sterile normal saline, and the probiotic group is subjected to gastric lavage by lactobacillus acidophilus LA15,1 time a day for 14 continuous days, wherein CTX 80mg/kg/d is intraperitoneally injected by the other mice except the normal group of mice on days 7,8 and 9. After the experiment is finished, dislocation is killed, thymus is taken, spleen is weighed, and spleen index and thymus index are calculated.
As shown in fig. 3, after 3 days of continuous intraperitoneal injection of cyclophosphamide, both other groups of mice were significantly reduced compared to the normal group (NC). At day 10, the probiotic group of lactobacillus gastrolavage LA15 (LA 15) slowly increased in body weight while the mice in model group (MC) continued to decrease in body weight. On the last day of the experiment, the body weight of the mice in the MC group was significantly reduced compared to the NC group; the body weight of the probiotic group (LA 15) mice was significantly increased (P < 0.05) compared to the MC group. Thymus and spleen are important immune organs of the body and are also the sites for immune cell growth and proliferation. Analysis of immune organ indexes shows that compared with a normal group (NC), spleen indexes and thymus indexes (figure 4) of a model group (MC) are obviously reduced, and compared with the model group (MC), lactobacillus acidophilus LA15 of a probiotic group (LA 15) can improve the spleen indexes and thymus indexes of mice, so that immune organ index reduction caused by cyclophosphamide injection is obviously improved, and the influence of immunosuppression on important immune organ development is resisted.
Experimental example 8 influence of Lactobacillus acidophilus on immunoglobulins and cytokines in immunocompromised mice
Small intestine tissue homogenate supernatants were collected and assayed for sIgA levels, and for cytokines IL-1β, TNF- α, IL-6, IL-17 and IL-10 using ELISA kits. The results are expressed as cytokine concentrations in the supernatant of tissue homogenates per ml of small intestine using standard cytokines provided in the kit.
sIgA is a major immunoglobulin that can enhance humoral immunity. As shown in fig. 5, it can be seen that the sIgA content in the small intestine tissue of the mice of the MC group was significantly reduced after injection of cyclophosphamide, and the sIgA content of the mice of the probiotic group (LA 15) was significantly increased, compared to the NC group. The results indicate that LA15 can enhance body humoral immunity by stimulating the production of sIgA. The result of cytokine detection in small intestine tissue shows that compared with NC group, the content of IL-1 beta, TNF-alpha, IL-6 and IL-17 pro-inflammatory factors in MC group small intestine is obviously reduced (P < 0.05), and the content of anti-inflammatory factor IL-10 is obviously reduced; compared with MC group, after the stomach is irrigated with lactobacillus acidophilus, the content of four pro-inflammatory factors is obviously increased (P < 0.05), the content of anti-inflammatory factors IL-10 is obviously increased, and the result shows that lactobacillus acidophilus LA15 can obviously improve the immunity of the mice with low immunity caused by cyclophosphamide.
Experimental example 9 influence of Lactobacillus acidophilus on intestinal flora of immunocompromised mice
After the experiment, the mice were sacrificed by cervical dislocation, the abdomen was sterilized and dissected, and the cecal contents of each group of mice were taken. Microbial genomic DNA was extracted from a mouse fecal sample and quantified by uv spectrophotometry. The 16s rdna 3-V4 hypervariable regions of all bacteria in the sample were sequenced on a Mi Seq Illumina sequencing platform to determine the characteristics of the intestinal microbiota.
The α -diversity indicates the species microbial community diversity, as shown in fig. 6, and according to the trends of the differences between the simpson index and the pd_whole_tree index groups, the injection of cyclophosphamide resulted in a significant decrease (P < 0.05) in the α -diversity index of the intestinal flora of mice, while the probiotic groups were all elevated, especially the pd_whole_tree index, and the probiotic groups were significantly elevated (P < 0.01) compared to the model group. The lactobacillus acidophilus LA15 intervention can improve the alpha diversity index of intestinal flora of mice with low immunity and can increase the diversity of microorganisms in the intestinal tract of the mice.
Intestinal flora is an important factor in determining the health of the body. The present invention analyzes the intestinal microbiota of mice at the genus level (fig. 7), and finds that the composition of the microorganisms in different groups is different and the relative abundance content of the bacterial taxa is different. In the MC group, the relative abundance of devulcanizvibrio, helicobacter, and probiotic (LA 15) mice was increased, while the relative abundance was decreased after the intervention of lactobacillus acidophilus LA15, compared to the NC group; the relative abundance of Bacteroides, prevotella, lachnospiraceae, NK4A136_group, unclassified Eubacterium, lactobacillus, unclassified Murilbaceae, candida_Saccharomonas was reduced compared to the NC group, whereas in the probiotic group (LA 15) mice, there was an increasing trend. Wherein the genus Bacteroides, the genus Prevotella, the genus Murilbaculoides, are capable of producing Short Chain Fatty Acids (SCFAs), the SCFAs induce the production of pro-inflammatory cytokines alone or in combination with toll-like receptor ligands, [ Eubacterium ] _co-pro-stanols_group can lower cholesterol, prevent cardiovascular disease, murilicuiculatae is positively associated with immune responses such as natural killer cells and NF-. Kappa.B signaling, and Murilicuiculatae family members can interact with innate or adaptive immune responses via IgA. According to document Oral Microbiota IsAssociatedWith Immune Recoveryin
HumanImmunodecifiencyVirus-Infected Individuals reports that Candida_Saccharomonas can play an important role in the immune recovery of immunodeficient patients.
The above results indicate that: cyclophosphamide can reduce the diversity of intestinal microorganisms, enrich harmful flora, reduce the abundance of beneficial bacteria and further cause the disturbance of intestinal flora and the dysbiosis of intestinal tracts; and lactobacillus acidophilus LA15 regulates intestinal microorganism ecology by improving the diversity of intestinal microorganism communities and increasing the abundance of intestinal beneficial bacteria, and lightens the immunosuppression effect of cyclophosphamide on organisms.
Claims (10)
1. Lactobacillus acidophilus strain LA15 with immunoregulatory capability, which is characterized by having a preservation number of CCTCCNO: M2023768.
2. The Lactobacillus acidophilus (Lactobacillus acidophilus) strain LA15 with a preservation number of CCTCCNO: M2023768 is applied to the preparation of antibacterial products and/or the preparation of immune regulation products and/or in vitro antibacterial products and/or the preparation of products for regulating intestinal flora.
3. Use of lactobacillus acidophilus strain LA15 with a preservation number of cctcno: M2023768 according to claim 2 for the preparation of a bacteriostatic product and/or for the preparation of an immunomodulating product and/or for the preparation of a product for the regulation of intestinal flora in vitro, wherein bacteriostatic means inhibiting one or more than two of the following bacteria: coli, staphylococcus aureus, salmonella, streptococcus mitis;
and/or, the immunomodulation is selected from: increasing proliferation rate of immune cells, regulating cytokines, increasing body weight and organ index of immunocompromised animals, and regulating one or more of immunoglobulins;
and/or, the modulating intestinal flora is selected from: improving the microbial community diversity index of the intestinal tracts of the immunocompromised animals, reducing the abundance of harmful bacteria in the intestinal tracts of the immunocompromised animals and improving the abundance of beneficial bacteria in the intestinal tracts of the immunocompromised animals.
4. Use of lactobacillus acidophilus strain LA15 with a preservation number of cctcno: M2023768 according to claim 3 for the preparation of a bacteriostatic product and/or for the preparation of an immunomodulating product and/or for the preparation of an in vitro bacteriostatic and/or intestinal flora regulating product, wherein said immune cells are macrophages RAW264.7;
and/or, the cytokine is selected from: cytokines TNF- α, IL-10 of macrophage RAW264.7, and/or cytokines IL-1β, TNF- α, IL-6, IL-17, IL-10 of immunocompromised animals;
and/or, said immunocompromised animal refers to cyclophosphamide-molded immunocompromised mice;
and/or, immunoglobulin refers to sIgA;
and/or, the microbial community diversity index comprises: simpson index and PD_white_tree index;
and/or the intestinal harmful bacteria are selected from: desulphurized vibrio, helicobacter;
and/or the intestinal beneficial bacteria are selected from: bacteroides, prevotella, lachnospiraceae, NK4A136_group, unclassified Eubacterium, lactobacillus, and unclassified Muribaculaceae, candidatus _Saccharomonas.
5. An immunomodulatory product comprising: an active ingredient; characterized in that the active ingredients comprise: lactobacillus acidophilus strain LA15 with a accession number of CCTCC NO: M2023768.
6. An immunomodulatory product according to claim 5, further comprising: auxiliary materials.
7. A product for inhibiting bacteria and/or regulating intestinal flora, comprising: an active ingredient; characterized in that the active ingredients comprise: lactobacillus acidophilus strain LA15 with a accession number of CCTCC NO: M2023768.
8. A product for inhibiting bacteria and/or regulating intestinal flora according to claim 7, further comprising: auxiliary materials.
9. An in vitro bacteriostasis method is characterized in that Lactobacillus acidophilus strain LA15 with a preservation number of CCTCCNO: M2023768 is adopted for bacteriostasis.
10. The in vitro method of claim 9, wherein the bacteriostasis means: inhibit one or more of Escherichia coli, staphylococcus aureus, salmonella and Streptococcus mitis.
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