CN117159598B - Application of lactobacillus plantarum Lp18 in preparation of immunity-enhancing medicines or health-care foods and products - Google Patents
Application of lactobacillus plantarum Lp18 in preparation of immunity-enhancing medicines or health-care foods and products Download PDFInfo
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- CN117159598B CN117159598B CN202311446478.2A CN202311446478A CN117159598B CN 117159598 B CN117159598 B CN 117159598B CN 202311446478 A CN202311446478 A CN 202311446478A CN 117159598 B CN117159598 B CN 117159598B
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- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention relates to application of lactobacillus plantarum Lp18 in preparing a medicament or health-care food for enhancing immunity and a product thereof, belonging to the technical field of microorganisms. The invention provides a lactobacillus plantarum with a preservation number of CCTCC No. M20231524Lactiplantibacillus plantarum) The strain Lp18 is used for preparing medicines or health-care foods for enhancing immunity. Based on the strain Lp18, the invention also provides an immunity-enhancing medicament taking the strain Lp18 as an active ingredient and a health-care food with an immunity-enhancing function. The lactobacillus plantarum Lp18 provided by the invention can effectively relieve weight loss of an immunosuppressed mouse, improve organ indexes, repair intestinal tissue injury, regulate intestinal flora and has broad-spectrum antibacterial performance.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to application of lactobacillus plantarum Lp18 in preparation of immunity-enhancing medicines or health-care foods and products.
Background
The immune system has the ability to fight pathogenic microorganisms and prevent infectious diseases. When the immune system of the organism is damaged, the immune response is inhibited, resulting in the decrease of the organism's ability to defend, recognize and clear foreign antigens or pathogenic microorganisms, etc., and serious health hazards. The use of many drugs in clinic brings about certain side effects. Therefore, rational use of immunopotentiators for the prevention and treatment of immunosuppressive diseases is of great importance.
Probiotics are defined as "live microorganisms that, when ingested in sufficient quantity, are beneficial to the health of the host, are resistant to gastric acids, bile acids in the gastrointestinal tract, can colonize the intestinal tract, and enhance the host immune system. Probiotics can act on the signal pathway to affect body inflammation and immune response. Th cells exert an immunological effect by secreting a large number of cytokines, th1 cells secrete cytokines associated with cellular immunity (IL-2, TNF- α), th2 cells secrete cytokines associated with humoral immunity (IL-4, IL-6, IL-10). Probiotics are capable of modulating the secretion of Th1 and Th2 cytokines, maintaining cytokine secretion homeostasis, and thus modulating or assisting immune responses. The probiotics can promote secretion of secretory immunoglobulin A (SIgA), prevent pathogenic bacteria from invading intestinal tract, and regulate intestinal mucosa immune system.
Intestinal microorganisms have important influence on the immune system, intestinal micro-ecological disturbance reduces the integrity of intestinal mucosa barrier, increases pathogenic bacteria in intestinal tracts, reduces beneficial bacteria, causes the body immune system disturbance, inflammation or oxidative stress, and the like, and causes related diseases. The intestinal microorganisms can interact with host small intestinal epithelial cells and bacteria to maintain intestinal homeostasis and enhance host immune function. The probiotics change the flora composition of specific parts of the host, can inhibit the colonization of pathogenic bacteria in intestinal tracts, activate immune response to remove the pathogenic bacteria, and maintain the health of the intestinal mucosa protective layer of the host. In addition, some strains can inhibit the growth of harmful bacteria by producing bacteriocins. Probiotics can also provide energy to cells by fermenting carbohydrates to produce Short Chain Fatty Acids (SCFAs) to inhibit intestinal inflammation.
Lactobacillus plantarum is an important functional probiotic, and the strain has been reported to have various functions of relieving colonitis, resisting oxidization, reducing blood sugar and the like, and meanwhile, the immunoregulation effect of the strain is reported. However, the lactobacillus plantarum with the immunoregulation function reported at present has single function, and cannot realize multiple functions of repairing intestinal injury, promoting secretion of multiple cytokines, regulating balance of intestinal flora, broad-spectrum antibiosis and the like.
Therefore, there is still a need in the art to develop new strains of lactobacillus plantarum that have an immune enhancing effect and are rich in function and strong in antibacterial ability.
Disclosure of Invention
In order to solve the problems existing in the prior art: the lactobacillus plantarum Lp18 has single function, can not realize the problems of repairing intestinal injury, promoting secretion of various cytokines, regulating balance of intestinal flora, resisting bacteria in a broad spectrum and the like, and provides application and products of the lactobacillus plantarum Lp18 in preparation of immunity-enhancing medicines or health-care foods based on the requirements in the field.
The technical scheme of the invention is as follows:
lactobacillus plantarum with preservation number of CCTCC No. M20231524Lactiplantibacillus plantarum) The strain Lp18 is used for preparing medicines or health-care foods for enhancing immunity.
The enhancing immunity comprises: repairing damaged intestinal tract, improving cytokine and immunoglobulin level, inhibiting weight loss, relieving spleen enlargement and thymus atrophy, and regulating intestinal flora.
The cytokines include: TNF- α, IL-6, IL-1β, IL-17;
preferably, the immunoglobulin refers to secreted immunoglobulin a.
The modulating the intestinal flora comprises: increasing ACE and Chao 1 index.
The modulating the intestinal flora further comprises: improving the Larix Gmelinidae-NK 4A 136-groupLachnospiraceae_ NK4A136_group) Unclassified helicobacter speciesunclassified_Oscillospiraceae) The genus of the tremellaOscillibacter) Ramulus et folium Gaultheriae Yunnanensis genusAlistipes) Genus Luo's bacteriumRoseburia) And Via the genus EubacteriumBlautia) Is a relative abundance of (c).
The relieving spleen enlargement and thymus atrophy refers to: lowering spleen index and raising thymus index.
An immunity enhancing medicament comprising: a pharmaceutically active ingredient; the pharmaceutically active ingredients include: lactobacillus plantarum with preservation number of CCTCC No. M20231524Lactiplantibacillus plantarum) Strain Lp18.
The medicine for enhancing immunity further comprises: pharmaceutically acceptable auxiliary materials.
A health food having an enhanced immune function, comprising: an active ingredient; the active ingredients include: lactobacillus plantarum with preservation number of CCTCC No. M20231524Lactiplantibacillus plantarum) Strain Lp18.
The health food with the function of enhancing immunity further comprises: edible auxiliary materials.
The beneficial effects of the invention are as follows:
the invention provides lactobacillus plantarum with the preservation number of CCTCC No. M20231524Lactiplantibacillus plantarum) The strain Lp18 is a new strain obtained by screening, and a large number of experiments prove that the strain not only has the immunity enhancing functions of improving various cytokines and immunoglobulins, inhibiting weight loss, relieving spleen enlargement, thymus atrophy and the like, also has effects in repairing intestinal injury caused by immunosuppression, and regulating intestinal flora disorder caused by immunosuppression, meanwhile, the invention also has broad-spectrum antibacterial capability, so the lactobacillus plantarum provided by the invention has the advantages of no toxicity, no toxicity and no toxicityLactiplantibacillus plantarum) The strain Lp18 can be used for preparing medicines for enhancing immunity or health-care foods with the function of enhancing immunity.
The lactobacillus plantarum Lp18 provided by the invention has a remarkable immune enhancement effect on immune suppression mice. The lactobacillus plantarum Lp18 adopted by the invention is separated from a farm yoghurt sample, is safe and non-pathogenic, and belongs to a microorganism species in a national recognized list which can be used for common foods. Animal experiments and mechanism studies prove that the composition has the effects of improving and relieving various adverse effects caused by immunosuppression. The food can be widely applied to the field of foods, the consumption possibility of consumers is increased, and the aim of enhancing immunity can be achieved through daily intake. Of course, the lactobacillus plantarum Lp18 can also be used for preparing immune enhancing medicaments. Meanwhile, the lactobacillus plantarum Lp18 provided by the invention has strong tolerance capability and wide bacteriostasis spectrum, can realize industrial production, and can realize remarkable immunity enhancement effect by adjusting cytokine level and intestinal flora.
The invention relates to lactobacillus plantarumLactiplantibacillus plantarum) The preservation information of strain Lp18 is as follows:
preservation number: CCTCC No. M20231524
Classification naming: lactobacillus plantarum Lp18
Latin name:Lactiplantibacillus plantarum Lp18
preservation date: 2023, 08 and 23
Preservation unit: china center for type culture Collection
Preservation address: chinese, wuhan, university of Wuhan.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum Lp18 of Experimental example 1 of the present invention.
FIG. 2 is a microscopic morphological image of Lactobacillus plantarum Lp18 of Experimental example 1 of the present invention.
FIG. 3 is a graph showing the results of measurement of the hemolytic activity of Lactobacillus plantarum Lp18 according to Experimental example 4 of the present invention.
FIG. 4 is a bar graph showing the weight change line graph and organ index of each group of mice in Experimental example 5 of the present invention.
FIG. 5 is a graph of ileal tissue sections of groups of mice according to Experimental example 5 of the present invention.
FIG. 6 is a bar graph showing the serum cytokine and secreted immunoglobulin A levels of each group of mice of Experimental example 5 of the present invention.
FIG. 7 is a graph showing the results of analysis of the number of OTUs and alpha diversity of intestinal flora of mice of each group of experimental example 5 of the present invention.
FIG. 8 is a graph showing the results of analysis of intestinal flora levels of mice in each group according to experimental example 5 of the present invention.
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.
Biological material source
1. The E.coli, salmonella enteritidis, staphylococcus aureus, listeria monocytogenes, and Bacteroides fragilis used in Experimental example 3 of the present invention were all purchased from the Cantonese province microorganism collection. Streptococcus mitis is streptococcus mitis strain 5013 which is registered with NCBI and has the registration number of MT512114; klebsiella pneumoniae is Klebsiella pneumoniae strain 587, which has been registered with NCBI under the registration number MT573143; streptococcus pastoris 12758, which strain has been registered with NCBI under accession number MW445225.
The campylobacter salicifolius is a strain obtained by separating and screening adult feces, and the strain is identified as campylobacter salicifolius by morphological identification and molecular identification, has the same homology with ATCC14669 strain and has the same morphology as campylobacter salicifolius.
Strain 5013, strain 587, strain 12758 and the campylobacter salicifolius strain are currently held by the applicant's laboratory, who promises to issue to the public within 20 years from the date of application of the present invention for verification of the technical effects of the present invention.
2. BALB/c male mice used in Experimental example 5 of the present invention are commercially available.
Group 1 example, strain Lp18 of the invention
The embodiment provides Lactobacillus plantarum with the preservation number of CCTCC No. M20231524Lactiplantibacillus plantarum) The strain Lp18 is used for preparing medicines or health-care foods for enhancing immunity.
In a specific embodiment, the enhancing immunity comprises: repairing damaged intestinal tract, improving cytokine and immunoglobulin level, inhibiting weight loss, relieving spleen enlargement and thymus atrophy, and regulating intestinal flora.
In a further embodiment, the cytokine comprises: TNF- α, IL-6, IL-1β, IL-17;
preferably, the immunoglobulin refers to secreted immunoglobulin a.
In some embodiments, the modulating the intestinal flora comprises: increasing ACE and Chao 1 index.
In other embodiments, the modulating the intestinal flora further comprises: improving the Larix Gmelinidae-NK 4A 136-groupLachnospiraceae_NK4A136_group) Unclassified helicobacter speciesunclassified_ Oscillospiraceae) The genus of the tremellaOscillibacter) Ramulus et folium Gaultheriae Yunnanensis genusAlistipes) Genus Luo's bacteriumRoseburia) And Via the genus EubacteriumBlautia) Is a relative abundance of (c).
In a more specific embodiment, the alleviating splenomegaly and thymus atrophy refers to: lowering spleen index and raising thymus index.
Any one plant lactobacillus strain with CCTCC NO. M20231524 for culturing, expanding propagation, fermenting, enriching, producing, preparing, using, inoculating, amplifying, transforming, modifying, reforming, selling and offering for saleLactiplantibacillus plantarum) Behavior of strain Lp18 and/or Lactobacillus plantarum strain with preservation number of CCTCC NO: M20231524Lactiplantibacillus plantarum) Behavior of the strain Lp18 combined with other probiotics and/or using a lactobacillus plantarum strain with the preservation number of CCTCC NO: M20231524Lactiplantibacillus plantarum) The preparation of immunoregulatory and immunoenhancing products by strain LP18 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 creamer), bifidobacterium longum, bifidobacterium pseudocatenulatum, bifidobacterium thermophilum, and bifidobacterium acidophilus.
The person skilled in the art can select or adjust the pharmaceutical auxiliary materials conventionally according to the actual production requirement by combining 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), and further select a lactobacillus plantarum strain with the preservation number of CCTCC NO: M20231524Lactiplantibacillus plantarum) The strain Lp18 is produced in different dosage forms, under different storage conditions and with different shelf life, which is easy and can be easily done by a person skilled in the art without technical difficulties.
Group 2 examples, medicaments 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 medicine for enhancing immunity or regulating intestinal flora comprises the following components: a pharmaceutically active ingredient; the pharmaceutically active ingredients include: lactobacillus plantarum with preservation number of CCTCC No. M20231524 for enhancing immunityLactiplantibacillus plantarum) Strain Lp18.
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, for different demands in practical production and application, the common technical means in the field of medicine preparation (for example, encyclopedia of preparation technology, medicine preparation technology, microbial agent technical research and application, and the like) are combined, and the pharmaceutically acceptable auxiliary materials can be selected and formulated by the person skilled in the art, and the plant lactobacillus with the preservation number of CCTCC NO: M20231524 is preparedLactiplantibacillus plantarum) The strain Lp18 can be made into various dosage forms, such as powder, tablet, 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 3 example, health food 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 functions of enhancing immunity or regulating intestinal flora comprises the following components: an active ingredient; the active ingredients include: lactobacillus plantarum with preservation number of CCTCC No. M20231524 for enhancing immunityLactiplantibacillus plantarum) Strain Lp18.
In a further embodiment, the health food with immunity enhancing function further comprises: edible 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 combine the conventional technical means (such as food technology, new food processing technology, food technology experimental course, etc.) in the field of medicine preparation according to different requirements in practical production application, and the plant lactobacillus with the preservation number of CCTCC NO: M20231524Lactiplantibacillus plantarum) The strain Lp18 can be made into various dosage forms, such as powder, tablet, 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, 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: lactobacillus plantarum with preservation number of CCTCC No. M20231524 for enhancing immunityLactiplantibacillus plantarum) Strain Lp18.
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, for different demands in practical production application, the common technical means in the field of medicine preparation (for example, encyclopedia of preparation technology, pharmaceutical preparation technology, microbial agent technical research and application, and the like) are combined, and the man skilled in the art can select and blend the auxiliary materials and store Lactobacillus plantarum with the preservation number of CCTCC NO: M20231524Lactiplantibacillus plantarum) The strain LP18 can be made into various dosage forms, such as powder, tablet, injection, oral liquid, suppository, gel, patch, spray, lotion, granule, etc.
In particular embodiments, the dosage form of the product is selected from: one or more of powder, tablet, liquid and capsule.
In a specific embodiment, the bacteriostasis spectrum of the bacteriostat includes: coli, salmonella enteritidis, staphylococcus aureus, listeria monocytogenes, klebsiella pneumoniae, streptococcus mitis, streptococcus pastoris, campylobacter salicifolius.
Experimental example 1, isolation screening and identification of Lactobacillus plantarum
(1) Isolation and selection of strains
The lactobacillus plantarum is separated from a farm yoghurt sample. Samples were diluted under sterile conditions with a 0.85% physiological saline gradient, and the appropriate gradient was selected and spread on LBS agar plates and incubated at 37 ℃ for 48-72 hours. And visually observing colony morphology, picking suspected single colonies, and performing preliminary screening, purification and culture after microscopic observation. The purified strain was cultured at 37℃for 8-12 hours using MRS liquid, and after centrifugation to remove the supernatant, it was resuspended in sterile 30% glycerol aqueous solution and stored at-80 ℃.
LBS media formulation (g/L): yeast extract powder 5.0 g, tryptone 10.0 g, monopotassium phosphate 6.0 g, ferrous sulfate 0.034 g, magnesium sulfate 0.575 g, glucose 20.0 g, sodium acetate 25.0 g, ammonium citrate 2.0 g, manganese sulfate 0.12 g and pH 5.5, tween-80 mL and glacial acetic acid 1.3 mL are added, heated and stirred to dissolve in distilled water of 1000 mL, and the mixture is autoclaved at 121 ℃ for 15 min. Agar 15.0. 15.0 g was added to the solid medium.
MRS Medium preparation method referring to GB4789.35-2016, 1.5% agar was added to a solid medium.
(2) Morphological characterization and 16S rDNA identification
The colony and the bacterial form of the lactobacillus plantarum are shown in fig. 1 and 2 respectively. The lactobacillus plantarum grows well on an MRS solid plate, and single bacterial colony is milky white, round convex, medium in size, neat in edge and smooth in surface. The form of the microscopic thallus is rod-shaped, and the thallus is arranged singly, in pairs or in chain, so that the microscopic thallus accords with the staining characteristics of gram-positive bacteria.
The screened target strain is subjected to liquid amplification, thalli are collected, genome DNA is extracted, and PCR amplification reaction is carried out by adopting universal primers 27F and 1492R described in Chinese patent No. ZL 202210478937.4. The PCR amplification procedure was: 94. pre-denaturation at 5 min at 94℃for 30 s, annealing at 55℃for 30 s, elongation at 72℃for 90 s for 35 cycles, and elongation at 72℃for 10 min. And detecting the content and purity of the PCR amplified product, sequencing the PCR amplified product after the PCR amplified product is qualified by a Wohan Jin Kairui bioengineering limited company, performing homology comparison by using a BLAST tool in an NCBI database according to the sequencing result, identifying the obtained strain as lactobacillus plantarum, determining the strain as lactobacillus plantarum according to the molecular biological identification result, naming the strain as lactobacillus plantarum Lp18, and carrying out preservation.
The preservation information of the lactobacillus plantarum Lp18 is as follows:
preservation number: CCTCC No. M20231524
Classification naming: lactobacillus plantarum Lp18
Latin name:Lactiplantibacillus plantarum Lp18
preservation date: 2023, 08 and 23
Preservation unit: china center for type culture Collection
Preservation address: chinese, wuhan, university of Wuhan.
Experimental example 2 Lactobacillus plantarum Lp18 tolerance experiment
Artificial gastric juice: PBS solution is prepared, 0.3% pepsin is added, the pH is regulated to 2.5 by 1 mol/l HCl, and after the PBS solution is fully dissolved, the PBS solution is filtered and sterilized by a microporous filter membrane with the thickness of 0.22 mu m for later use.
Bile salt solution: sodium thioglycolate (0.2% (w/v)) is added into MRS liquid culture medium, 0.3% of ox gall salt is added, and after the ox gall salt is fully dissolved, the ox gall salt is filtered and sterilized by a microporous filter membrane with the thickness of 0.22 mu m for standby.
Lactobacillus plantarum is activated and cultured for two generations in MRS liquid culture medium. Centrifuging the activated strain liquid, discarding the supernatant, collecting thallus, and adjusting the concentration of the bacterial liquid to 10 8 CFU/mL. Centrifuging 1 mL bacteria liquid, washing twice with PBS, collecting bacteria after centrifugation, respectively adding into 1 mL simulated artificial gastric juice and simulated artificial intestinal juice, then placing at 37 ℃ for incubation, respectively taking digestion solutions of 0 h and 3 h to detect viable bacteria, and calculating survival rate. Strain survival (%) =nt/n0×100 where N0 represents the number of viable bacteria (log CFU/mL) of strain 0 h and Nt represents the number of viable bacteria (log CFU/mL) of strain 3 h.
The tolerability results of lactobacillus plantarum Lp18 in gastric juice and bile salts are shown in table 1. As shown in Table 1, the survival rate of the strain after being treated in gastric juice and bile salt for 3 h is 99.08% and 97.30%, respectively, and the survival rate is high, which indicates that the strain has strong gastric juice bile salt tolerance.
Experimental example 3 Lactobacillus plantarum Lp18 antagonistic to pathogenic bacteria experiment
1. Preparing lactobacillus plantarum Lp18 bacterial liquid: lactobacillus plantarum Lp18 is inoculated into a liquid MRS culture medium according to a certain proportion, and is cultured overnight at 37 ℃ to obtain antagonistic bacteria liquid. Preparing an index bacterial liquid: activating Escherichia coli, salmonella enteritidis and Staphylococcus aureus strain on LB solid plate medium, culturing at 37deg.C for 24 h, and collecting thallus Porphyrae to normal saline to obtain bacterial suspension 10 8 CFU/ml; listeria monocytogenes was cultured overnight in BHI anaerobic tubes at 37deg.C, and the concentration of the bacterial suspension was adjusted to 10 8 CFU/ml; anaerobic culturing Bacteroides fragilis in TSA+5% sheep blood agar plate at 37deg.C for 24 hr, and collecting thallus Porphyrae until physiological saline is adjustedThe concentration of the bacterial suspension is 10 8 CFU/ml; klebsiella pneumoniae, streptococcus mitis and Streptococcus pasteurii are cultured in BHI and 5% sheep blood solid culture medium at 37deg.C for 16 h, and the concentration of bacterial suspension is adjusted to 10 8 CFU/mL。
2. And (3) bacteriostasis circle experiment: cooling the culture medium corresponding to each pathogenic bacteria to about 55deg.C, mixing with the strain suspension of the indicator strain at a certain ratio, and adjusting the concentration of the indicator bacteria to 10 6 CFU/ml, then rapidly pour the mixture into a plate pre-placed on oxford cup, take out oxford cup after the culture medium has cooled and solidified, add 200. Mu.l of antagonistic bacteria solution (10 8 CFU/ml), 200 μl of sterile MRS liquid medium was used as a blank control, left to stand for several minutes, placed in a 37 ℃ incubator, incubated for 24 h, observed, and the diameter of the zone of inhibition was measured with vernier calipers, three replicates per bacterium, and the results are shown in table 2.
Lactobacillus plantarum Lp18 has inhibitory activity against E.coli, salmonella enteritidis, staphylococcus aureus, listeria monocytogenes, bacteroides fragilis, klebsiella pneumoniae, streptococcus mitis, streptococcus Pasteurensis, and Campylobacter salicifolius. The lactobacillus plantarum Lp18 is shown to have the capability of inhibiting the growth of various pathogenic bacteria.
3. Co-culture bacteriostasis experiment
Preparation of lactobacillus plantarum: lactobacillus plantarum Lp18 is inoculated in MRS liquid culture medium, and is cultured at a constant temperature of 37 ℃ and standing overnight.
Preparation of pathogenic bacteria: the pathogenic strain bacteroides fragilis is inoculated on a TSA+5% sheep blood agar plate for anaerobic culture for 24 hours at 37 ℃, then bacteria are washed by sterile PBS, and the concentration of bacteria liquid is regulated for subsequent experiments. The listeria monocytogenes is inoculated in a BHI anaerobic tube liquid culture medium for anaerobic overnight culture at 37 ℃ to prepare pathogenic bacteria liquid.
Co-culture experiments: lactobacillus plantarum Lp18 and pathogenic bacteria are respectively mixed according to 10 percent 8 CFU/mL and 10 7 CFU/mL was inoculated into 7 mL of BHI anaerobic tube liquid medium, and cultured by standing for 16 hours using PALCAM agar medium and BBE agar medium, respectivelyThe number of live Listeria monocytogenes and Bacteroides fragilis was measured by base counting, and the results are shown in Table 3.
From Table 3, lactobacillus plantarum Lp18 can well inhibit the growth of Listeria monocytogenes and Bacteroides fragilis, has strong antibacterial ability to the Listeria monocytogenes and Bacteroides fragilis, and has an inhibition rate of more than 99%.
Experimental example 4 detection of the haemolytic Activity of Lactobacillus plantarum Lp18
After the strain to be tested is activated by MRS liquid culture medium, the supernatant is removed by centrifugation, and bacterial mud is reserved for standby. The bacterial mud is streaked and inoculated on Columbia blood agar medium containing 5% sheep blood, after culturing for 36-48 hours at 37 ℃, the presence or absence of hemolytic circles around colonies on the flat plate is observed, and streptococcus pyogenes is used as a quality control strain. The results of the hemolytic activity of the strain are shown in FIG. 3. As can be seen from fig. 3: the streptococcus pyogenes has obvious transparent hemolytic ring, is beta hemolysis, and has no obvious hemolysis phenomenon around the strain to be tested and the LGG, thus indicating no hemolysis capability.
Example 5 application of Lactobacillus plantarum Lp18 in alleviating immunosuppressed mice
Establishment of a mouse immunocompromised model:
BALB/c male mice (18-20 g) without specific pathogen were purchased from Beijing Bei Fu Biotechnology Co., ltd, and were kept in Hubei province food and drug safety evaluation center animal room, kept at 22.+ -. 2 ℃ and humidity 50.+ -. 5%, and were light and dark cycled for 12 hours, allowing free drinking and eating. Mice were first adaptively bred for one week prior to the experiment, and then randomly divided into three groups, respectively: normal control group (NC), immunosuppression group (MC), lactobacillus plantarum group (Lp 18). During the whole experiment, NC group and MC group mice were perfused with 0.2 mL sterile physiological saline every day, and Lp18 group mice were perfused with equal amounts of Lactobacillus plantarum bacterial suspension (viable count 5×10) 9 CFU/mL). On experiment days 8-10, continuous injection of cyclophosphamide (CTX, 80 mg/kg/d) for 3 days was performed on MC and Lp18 mice to establish an immunosuppressive mouse model, and an equal volume of sterile physiological saline was injected into NC. Mice were sacrificed on day 15 and samples were collected for post-treatmentAnd (5) continuing analysis. The detailed animal experiments are shown in table 4.
(1) Mouse body weight and organ index changes
Mice were weighed and recorded daily during the experiment and observed for changes in body weight. Mice were sacrificed after the end of the experiment, spleens and thymus were collected, weighed, and immune organ indexes were calculated. Spleen/thymus index (mg/g) =spleen/thymus mass (mg)/body weight (g). After three days of continuous CTX injection, the weights of the mice in the MC group and the mice in the LP group are reduced, wherein the weight of the mice in the MC group is seriously reduced, the weights of the mice in the MC group and the mice in the LP18 group are still continuously reduced after the administration is stopped, and the weight of the mice in the LP18 group is obviously higher than that of the mice in the MC group at the 14 th dayp<0.01, panel a in fig. 4). Wherein, the weight of the MC group mice is reduced by 21.3% compared with the weight of the LP group mice, the weight of the LP group mice is reduced by 5.4% compared with the weight of the LP group mice, and the LP18 remarkably inhibits the weight reduction of the mice. As can be seen from the graphs B-C in FIG. 4, CTX treatment significantly improved the spleen index of mice compared to NC groupp<0.001 Reducing thymus indexp<0.0001). After the dry prognosis of the lactobacillus plantarum Lp18, the spleen index of the mice is obviously lower than that of the MC groupp<0.05 74.5% of MC group, the thymus index is obviously higher than that of MC groupp<0.001 4.2 times that of the MC group. The lactobacillus plantarum Lp18 can regulate immune response, delay weight loss of mice and effectively relieve splenomegaly and thymus atrophy.
(2) Ileum tissue pathology observation of mice
After the mice were sacrificed, the ileal tissues of the mice were collected 0.5 cm, soaked and fixed with 10% formaldehyde solution, then hematoxylin-eosin (HE) stained, and finally the intestinal structures were observed using an optical microscope. As shown in FIG. 5, the NC group ileum tissue is complete, the morphological structure is normal, the epithelial villi are arranged in a slender manner, and no obvious lesions are seen. The MC group ileum mucous membrane is seriously damaged, the intestinal myolayer and the intestinal gland are deleted, the intestinal villi is destroyed and seriously reduced, the intestinal tissue structure is incomplete, and inflammatory cell infiltration is visible. Compared with the MC group, the Lp18 group mice have recovered intestinal damage, and the intestinal morphology and structure are complete, which indicates that the lactobacillus plantarum Lp18 can strengthen the intestinal mucosa barrier and reduce the ileum tissue damage.
(3) Mouse serum cytokine and immunoglobulin A content determination
After the experiment, the orbital blood of the mice is collected, the blood is kept stand at room temperature for 30 min, and centrifuged (3000 r/min,10 min), and the upper serum is collected, and the contents of the peripheral blood cytokines tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-1 beta (IL-1 beta), interleukin-17 (IL-17) and secretory immunoglobulin A (SIgA) of each group of mice are respectively measured according to the detection method of the specification of ELISA kit manufacturers. As shown in FIG. 6, the TNF-. Alpha.and IL-17 contents in the MC group were extremely significantly reduced as compared with the control groupp<0.01 IL-6 and IL-1 beta content is obviously reducedp<0.05). The dry prognosis of the plant lactobacillus Lp18 obviously improves the levels of TNF-alpha, IL-6, IL-1 beta, IL-17 and SIgA in the serum of micep<0.05，p<0.001). Wherein the SIgA content in serum is 1.7 times of that of the model group, and is obviously higher than that of the model group. The lactobacillus plantarum Lp18 can promote secretion of organism cytokines and immunity of organism intestinal mucosa, maintain intestinal homeostasis, further improve organism immunity and maintain organism health.
(4) Lactobacillus plantarum for regulating immune suppression of intestinal flora of mice
After the end of the experiment, the mice were sacrificed, dissected, their cecal content was collected, and the cecal content of the mice was subjected to 16S sequencing and analysis. As shown in panel a of fig. 7, the number of OTUs shared by MC group and NC group was 366, and the number of OTUs shared by Lp group and NC group was 511, indicating that lactobacillus plantarum Lp18 intervention was able to increase the common flora in the intestinal tracts of immunosuppressive mice and normal feeding mice. Analysis of alpha-diversity of mice intestinal flora showed (panel B of fig. 7) that the ACE and Chao 1 index of the intestinal flora of mice in the model group was significantly reduced compared to the control group [ (]p<0.001 Indicating that CTX induces a disturbance in the intestinal flora of mice, resulting in a decrease in intestinal flora diversity. After feeding the lactobacillus plantarum, the indexes of the intestinal flora ACE and Chao 1 of the mice are obviously higher than those of a model groupp<0.05 Indicating that Lp18 can regulate intestinal flora and increaseThe richness of the intestinal flora of the immunized mice is added.
Analysis of changes in relative abundance of levels of genus in cecal content of mice between groups showed that the model group was composed of the group consisting of chaetoceraceae NK4a136 @Lachnospiraceae_NK4A136_group) Unclassified helicobacter speciesunclassified_Oscillospiraceae) The genus of the tremellaOscillibacter) Ramulus et folium Gaultheriae Yunnanensis genusAlistipes) Genus Luo's bacteriumRoseburia) And Via the genus EubacteriumBlautia) The relative abundance is obviously reduced compared with the control groupp<0.05，p<0.01 Genus helicobacterHelicobacter) And unclassified genus UbbelobactercoprostanoligenesGroup%unclassified_[Eubacterium]_coprostanoligenes_group) The relative abundance is obviously increased compared with the control groupp<0.05，p<0.01 Lactobacillus plantarum Lp18 treatment was effective in ameliorating this phenomenon (FIG. 8). Compared with MC group, lactobacillus plantarum Lp18 group mice have significantly increased relative abundance of Protocolzfeldae-NK 4A 136-group, unclassified Oscillaceae, oscillating genus, odoramella, rauzopsis and Eubacterium mucilaginosum in the intestinal tractp<0.05，p<0.01, panels A-F of FIG. 8), particularly of the genus Odormitoria, genus Ralstonia and genus Eubacterium viscosum, in relative abundance at or above the control level, while the genus helicobacter and unclassified genus EubacteriumcoprostanoligenesThe relative abundance of the group is obviously reducedp<0.05，p<0.01, G-H of fig. 8). The group consisting of the chaetoceros NK4a136, unclassified spirochete family of the family of tremella, the genus Oscillating and the genus Odormitopsis can ferment carbohydrates, SCFAs are produced, which provide host cells with the ability to function as anti-inflammatory agents; literature (use of Chinese characters)BlautiaA new functional genus with potential probiotic properties it is reported that the genus Eubacterium mucilaginosum can produce bacteriocins in addition to regulating the production of SCFAs, thereby preventing colonization of the intestinal tract by pathogens. The result shows that the lactobacillus plantarum Lp18 can obviously increase the abundance of beneficial bacteria, reduce the abundance of harmful bacteria, promote the stable state recovery of intestinal flora, further enhance the barrier function of intestinal tracts and improve the immunity of hosts.
Claims (6)
1. Lactobacillus plantarum with preservation number of CCTCC No. M20231524Lactiplantibacillus plantarum) The strain Lp18 is used for preparing medicines or health-care foods for enhancing immunity.
2. The Lactobacillus plantarum with the preservation number of CCTCC No. M20231524 according to claim 1Lactiplantibacillus plantarum) Use of strain Lp18 in the preparation of an immunity enhancing medicament or health food, wherein the immunity enhancing comprises: repairing damaged intestinal tract, improving cytokine and immunoglobulin level, inhibiting weight loss, relieving spleen enlargement and thymus atrophy, and regulating intestinal flora.
3. The Lactobacillus plantarum with the preservation number of CCTCC No. M20231524 according to claim 2Lactiplantibacillus plantarum) Use of strain Lp18 in the preparation of an immunity enhancing medicament or health food, wherein the cytokine comprises: TNF- α, IL-6, IL-1β, IL-17;
and/or, the immunoglobulin refers to secreted immunoglobulin a.
4. The Lactobacillus plantarum with the preservation number of CCTCC No. M20231524 according to claim 2Lactiplantibacillus plantarum) Use of strain Lp18 for the preparation of an immunity enhancing medicament or health food, characterized in that the modulation of intestinal flora comprises: increasing ACE and Chao 1 index.
5. The Lactobacillus plantarum strain with the preservation number of CCTCC No. M20231524 according to claim 4Lactiplantibacillus plantarum) Use of strain Lp18 for the preparation of an immunity enhancing medicament or health food, characterized in that the modulating the intestinal flora further comprises: improving the Larix Gmelinidae-NK 4A 136-groupLachnospiraceae_ NK4A136_group) Unclassified helicobacter speciesunclassified_Oscillospiraceae) Xylobacter gram (Xylobacter)Belongs to%Oscillibacter) Ramulus et folium Gaultheriae Yunnanensis genusAlistipes) Genus Luo's bacteriumRoseburia) And Via the genus EubacteriumBlautia) Is a relative abundance of (c).
6. The Lactobacillus plantarum with the preservation number of CCTCC No. M20231524 according to claim 2Lactiplantibacillus plantarum) The application of the strain Lp18 in preparing medicaments or health-care foods for enhancing immunity is characterized in that the effects of relieving splenomegaly and thymus atrophy are as follows: lowering spleen index and raising thymus index.
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