CN117441897A

CN117441897A - Application of Pediococcus acidilactici RH2712 strain in immunoregulation

Info

Publication number: CN117441897A
Application number: CN202311004058.9A
Authority: CN
Inventors: 宋佳; 余萍; 林欣梅; 王婷婷; 彭永振; 矫艳平
Original assignee: Jiangxi Renren Health Microecological Technology Co ltd
Current assignee: Jiangxi Renren Health Microecological Technology Co ltd
Priority date: 2023-08-10
Filing date: 2023-08-10
Publication date: 2024-01-26

Abstract

The invention belongs to the technical field of microorganisms, and particularly relates to application of a Pediococcus acidilactici RH2712 strain in immunoregulation. The invention provides application of Pediococcus acidilactici RH2712 strain in immunoregulation, the Pediococcus acidilactici RH2712 strain has better safety, strong capability of resisting the reverse environment of the digestive tract, stronger immunoenhancement capability, and obvious immunoenhancement effect after the Pediococcus acidilactici RH2712 strain is treated at a high dose, the immune indexes of IL-4, IFN-gamma, TNF-alpha, igG and the like of an immunodepression mouse are restored to normal levels.

Description

Application of Pediococcus acidilactici RH2712 strain in immunoregulation

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to application of a Pediococcus acidilactici RH2712 strain in immunoregulation.

Background

Xinjiang milk lump is a crystalline fermented yoghourt, has unique flavor and rich nutritive value, can accelerate metabolism of human body, promote resistance of human body and provide vitamins and minerals, and is a traditional food with long history. As a dairy food, the milk lump not only has unique flavor, but also has higher nutritional value, and the nutritional content of raw milk is only 1/10 of that of the milk lump in general. The use of the milk lump not only can accelerate metabolism of human bodies, but also has rich calcium, is easy to absorb, and can effectively prevent cardiovascular diseases after long-term eating due to the low cholesterol content (Yang Shangjiao.2015. Screening of bacteriocin-producing lactobacillus in cheese in Xinjiang area, systematic development and bacteriostasis activity research [ D ] university of shihe.) of the milk lump. The fermentation process of the milk lump is simple, natural fermentation is carried out, the production mode mainly comprises manual production, and the milk product can be changed from liquid state to semisolid or solid state after the processes of natural fermentation, dehydration, natural drying and the like, so that the milk lump is finally obtained. The production principle of the milk lump is as follows: the milk is fermented under the action of lactobacillus to form lactic acid, then the pH value of the milk starts to be reduced, and after casein reaches corresponding equal point electricity, whey components in the cow milk can be discharged, and a lump coagulation phenomenon is shown, so that finally, a milk lump is formed.

Milk pimples contain a large amount of probiotics, wherein lactic acid bacteria separated from the milk pimple can decompose cow milk proteins into amino acids and polypeptides which are easier to be absorbed by human bodies, and the milk after fermentation of the lactic acid bacteria has a part of lactose components converted into lactic acid and the other part converted into whey, so that lactose intolerance response symptoms such as diarrhea and the like are not generated for lactase-deficient people (Yang Jixia.2013. Study of phenotype, genotype and probiotic characteristics of lactobacillus in yak cheese [ D ]. University of southwest). The Chinese patent application 202111588803.X discloses lactobacillus rhamnosus with mould inhibiting activity and application thereof, wherein the strain is separated from a natto pasture domestic yak milk pimple product in a Tibetan area with the elevation of 4500m, and has good growth performance and strong acid production capacity. The Chinese patent application 202210217216.8 discloses lactobacillus paracasei separated from Tibetan ali yak milk pimple and application thereof, and the lactobacillus paracasei AL1 Plateau LPA 1 strain (Lactobacillus paracasei AL plaeau LPA 1) is preserved in China Center for Type Culture Collection (CCTCC), the preservation number is CCTCC No. 20211312, and metabolic products generated by the strain can inhibit intestinal harmful bacteria.

No probiotic strains isolated from milk pimples are currently involved in immunomodulation.

Disclosure of Invention

Immunization is a rejection reaction of the body involving a variety of genes, proteins and cells. Immune dysregulation causes a number of diseases including allergies (allergies, immune complex type, delayed type immune diseases, cytotoxic type immune diseases), immunodeficiency, and impaired immune system.

Immunomodulation refers to the recognition and elimination of antigenic foreign matter by the body and maintains its physiological homeostasis and relatively stable physiological function. Immunomodulation involves interactions between immune cells and immune molecules in the immune system, as well as with other systems such as the neuroendocrine system, such that the immune response in its most appropriate form maintains the body at the most appropriate level.

Through a large number of experiments, the inventor separates a Pediococcus acidilactici RH2712 strain from traditional food milk pimples, and the strain can enhance the immune function of organisms, relieve immunosuppression and have a strong immune regulation effect.

In order to achieve the technical aim, the invention provides an application of the Pediococcus acidilactici RH2712 strain in immunoregulation.

The technical scheme provided by the invention is as follows:

in one aspect, the invention provides the use of the Pediococcus acidilactici RH2712 strain in immunomodulation.

In another aspect, the invention provides the use of Pediococcus acidilactici RH2712 strain for the preparation of a product for enhancing immune function or alleviating inhibition of immune function.

In some embodiments, the products include pharmaceuticals, nutraceuticals, and foods.

In some embodiments, the medicament comprises a therapeutically effective amount of Pediococcus acidilactici RH2712 strain and a pharmaceutically acceptable adjuvant.

In some specific embodiments, the adjuvant is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickening agents, emulsifiers, preservatives, stabilizers, hydration agents, emulsifying accelerators, buffers, absorbents, colorants, flavorants, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants.

In some embodiments, the pharmaceutical dosage form is selected from at least one of powders, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, powder mists, aerosols, suppositories, drops and drop pills.

In some embodiments, the strain is preserved in CGMCC with a preservation number of CGMCC No.25165.

In some specific embodiments, the 16S rDNA of the strain has a nucleotide sequence as shown in SEQ ID NO. 1.

In some embodiments, the application comprises the steps of:

(1) Inoculating the Pediococcus acidilactici RH2712 strain or a culture thereof into a basic culture medium for primary culture to obtain a primary culture;

(2) Transferring the primary culture into a basic culture medium for secondary culture to obtain a secondary culture;

(3) And inoculating the secondary culture into an optimized culture medium for tertiary culture to obtain a tertiary culture.

In some embodiments, the conditions for the primary, secondary, and tertiary culturing are 37 ℃ for 17 hours.

In some embodiments, the tertiary culture comprises a culture broth, a culture broth extract, a whole fungus extract, a fermentation broth extract, and/or a fungus powder.

In some specific embodiments, the tertiary culture may be a fermentation broth.

In some specific embodiments, the bacterial powder may be a lyophilized bacterial powder.

In some embodiments, the basal medium comprises a carbon source, a nitrogen source, a phosphorus source, trace elements, citric acid monohydrate, sodium acetate, and L-malic acid;

preferably, the carbon source is anhydrous glucose; and/or

The nitrogen source is yeast peptone and yeast extract; and/or

The phosphorus source is potassium dihydrogen phosphate; and/or

The microelements are magnesium sulfate and manganese sulfate.

In some specific embodiments, the carbon source has a mass to volume ratio of 15 to 25g/L; the mass volume ratio of the nitrogen source is 10-22g/L; the mass volume ratio of the phosphorus source is 1-5g/L; the mass volume ratio of the trace elements is 0.11-1.05g/L; the mass volume ratio of the citric acid monohydrate is 1-5g/L; the mass volume ratio of the sodium acetate is 2-8g/L; the mass volume ratio of the L-malic acid is 1-5g/L.

In some specific embodiments, the carbon source has a mass to volume ratio of 20g/L; the mass volume ratio of the nitrogen source is 15g/L; the mass volume ratio of the phosphorus source is 2g/L; the mass volume ratio of the trace elements is 0.51g/L; the mass volume ratio of the citric acid monohydrate is 2g/L; the mass volume ratio of the sodium acetate is 5g/L, and the mass volume ratio of the L-malic acid is 3g/L.

In some specific embodiments, the yeast peptone has a mass to volume ratio of 8-14g/L; the mass-volume ratio of the yeast extract is 2-8g/L; the mass volume ratio of the magnesium sulfate is 0.1-1.0g/L; the mass volume ratio of the manganese sulfate is 0.01-0.05g/L.

In some specific embodiments, the yeast peptone has a mass to volume ratio of 10g/L; the mass-volume ratio of the yeast extract is 5g/L; the mass volume ratio of the magnesium sulfate is 0.5g/L; the mass volume ratio of the manganese sulfate is 0.01g/L.

In some embodiments, the optimized medium comprises a carbon source, a nitrogen source, a phosphorus source, trace elements, citric acid monohydrate, sodium acetate, L-malic acid, tween 80, and calcium chloride;

preferably, the carbon source is anhydrous glucose; and/or

The nitrogen source is yeast peptone and yeast extract; and/or

The phosphorus source is potassium dihydrogen phosphate; and/or

The microelements are magnesium sulfate and manganese sulfate.

In some specific embodiments, the carbon source has a mass to volume ratio of 20 to 40g/L; the mass volume ratio of the nitrogen source is 28-54g/L; the mass volume ratio of the phosphorus source is 1-5g/L; the mass volume ratio of the trace elements is 0.31-1.05g/L; the mass volume ratio of the citric acid monohydrate is 1-5g/L; the mass volume ratio of the sodium acetate is 2-8g/L; the mass volume ratio of the L-malic acid is 1-5g/L; the mass volume ratio of the Tween 80 is 0.05-0.3g/L; the mass volume ratio of the calcium chloride is 0.1-1.0g/L.

In some specific embodiments, the nitrogen source has a mass to volume ratio of 40g/L; the mass volume ratio of the phosphorus source is 2g/L; the mass volume ratio of the trace elements is 0.51g/L; the mass volume ratio of the citric acid monohydrate is 2g/L; the mass volume ratio of the sodium acetate is 5g/L; the mass volume ratio of the L-malic acid is 3g/L; the mass volume ratio of the Tween 80 is 0.1g/L; the mass volume ratio of the calcium chloride is 0.5g/L.

In some embodiments, the yeast peptone has a mass to volume ratio of 8-14g/L; the mass-volume ratio of the yeast extract is 20-40g/L; the mass volume ratio of the magnesium sulfate is 0.3-1.0g/L; the mass volume ratio of the manganese sulfate is 0.01-0.05g/L.

In some embodiments, the yeast peptone has a mass to volume ratio of 10g/L; the mass-volume ratio of the yeast extract is 30g/L; the mass volume ratio of the magnesium sulfate is 0.5g/L; the mass volume ratio of the manganese sulfate is 0.01g/L.

Compared with the prior art, the Pediococcus acidilactici RH2712 strain provided by the invention has at least the following beneficial effects:

(1) The safety is good, the hemolysis is negative, and the medicine is sensitive to Ampicillin (AM), penicillin (PEN), imipenem (IP) and Chloramphenicol (CL);

(2) The digestive tract reverse environment resistance is strong, the growth can be continued after the culture is carried out for 4 hours under the condition of pH 3.0, the number of viable bacteria in the concentration of 1.5 percent of bile salt reaches 8.72, and the survival rate can still reach 76.20 percent after the simulated gastric juice is treated for 3 hours;

(3) Has stronger in-vitro immunity enhancement capability, and the extracellular polysaccharide content of the Pediococcus acidilactici RH2712 strain is 1070.12 mug/mL;

(4) Has stronger in vivo immunity enhancement capability, and after the treatment of the high-dose Pediococcus acidilactici RH2712 strain, the immune indexes (IL-4, IFN-gamma, TNF-alpha and IgG) of the immune suppression mice are restored to be equivalent to the normal level, and the immunity enhancement effect is obvious.

Preservation information:

biological material: RH2712

Classification naming: pediococcus acidilactici Pediococcus acidilacticii

Preservation number: CGMCC No.25165

Preservation date: 2022, 06, 23

Preservation unit: china general microbiological culture Collection center (China Committee for culture Collection of microorganisms)

Preservation address: no. 1 and No. 3 of the north cinquefoil of the morning sun area of beijing city.

Drawings

FIG. 1 is a morphology of candidate colonies on MRS medium.

FIG. 2 is a gram of candidate colonies.

FIG. 3 is a graph showing the results of phylogenetic tree of the RH2712 strain and the 16S rDNA sequences of related species. In the figure, the development tree nodes only show that the Bootstrap value is larger than 50%, and the superscript 'T' represents the model strain.

FIGS. 4 and 5 are scanning electron microscope images of RH2712 strain, wherein the scale of FIG. 4 is 10 μm and the scale of FIG. 5 is 2. Mu.m.

FIG. 6 is a graph showing the results of the RH2712 strain hemolysis experiment. Wherein 1 is a negative control bacterium: listeria enterica (Listeria innocua) CICC 10417;2 is positive control bacteria: staphylococcus aureus (Staphylococcus aureus) CCIC 10473;3 is a sample: pediococcus acidilactici RH2712 strain.

FIG. 7 is a glucose standard curve.

Detailed Description

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.

The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise. For example, reference to "a cell" includes a plurality of such cells, equivalents thereof known to those skilled in the art, and so forth.

The term "about" as used herein means a range of + -20% of the numerical values thereafter. In some embodiments, the term "about" means a range of ±10% of the numerical value following that. In some embodiments, the term "about" means a range of ±5% of the numerical value following that.

The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. All reagents or equipment were commercially available as conventional products without the manufacturer's attention. Numerous specific details are set forth in the following description in order to provide a better understanding of the invention. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.

The main reagents and materials related by the invention are as follows:

TABLE 1 Main reagents according to the invention

Table 2 the main instrumentation of the present invention

Isolation medium: 1% of yeast peptone, 0.3% of beef powder, 0.4% of yeast powder, 0.2% of potassium dihydrogen phosphate, 0.2% of citric acid, 0.5% of sodium acetate, 2% of glucose, 0.058% of magnesium sulfate, 0.025% of manganese sulfate, 0.06% of tween-80, 1% of calcium carbonate, 0.05g of neutral red (1% concentration of 5 mL), 1% (v/v) of tomato juice, 2% of agar powder and adjusting the pH to 5.5; sterilizing at 115 deg.C for 30min.

MRS medium: 1% of yeast peptone, 0.3% of beef powder, 0.4% of yeast extract, 0.2% of monopotassium phosphate, 0.2% of citric acid monohydrate, 0.5% of sodium acetate, 2% of anhydrous glucose, 0.058% of magnesium sulfate, 0.025% of manganese sulfate, 0.06% of tween 80 and 1% (v/v) of tomato juice, adjusting the pH value to 6.5, and sterilizing at 115 ℃ for 30min.

Enrichment medium: MRS medium at pH 5.0.

Basal medium: 8-14g/L of yeast peptone, 2-8g/L of yeast extract, 15-25g/L of anhydrous dextrose, 1-5g/L of citric acid monohydrate, 1-5g/L of L-malic acid, 2-8g/L of sodium acetate, 1-5g/L of monopotassium phosphate, 0.1-1.0g/L of magnesium sulfate, 0.01-0.05g/L of manganese sulfate and pH value of 6.6. Sterilizing at 115 deg.C for 30min.

Optimizing the culture medium: 8-14g/L of yeast peptone, 20-40g/L of yeast extract, 20-40g/L of anhydrous dextrose, 1-5g/L of citric acid monohydrate, 1-5g/L of L-malic acid, 2-8g/L of sodium acetate, 1-5g/L of monopotassium phosphate, 0.3-1.0g/L of magnesium sulfate, 0.01-0.05g/L of manganese sulfate, 0.05-0.3g/L of tween 80, 0.1-1.0g/L of calcium chloride and pH value of 6.6. Sterilizing at 115 deg.C for 30min.

Milk pimples were purchased from the inner Mongolian Hui Lun Bei Ere mol Gu Nashi Sanhe Hui nationality.

Example 1 screening and identification of strains

1.1 screening of strains

(1) Enrichment: taking a proper amount of milk lump sample in an enrichment medium, and enriching for 24 hours at 37 ℃;

(2) And (3) primary screening: and uniformly mixing the enriched samples, diluting the mixture into different gradients, and diluting and coating the mixture on a separation culture medium. Culturing at 37deg.C for 48 hr;

(3) Purifying: selecting single colony with typical characteristics of target strain (spherical, cell pairwise arrangement, no endophytic spore, gram positive strain), streaking, purifying and culturing in a separation culture medium, and repeating for 3 times until colony characteristics in a streak plate are consistent;

(4) And (5) microscopic examination: 2 individual colonies were picked from each purified plate for smear, gram stain, and observed under a microscope for consistency in color and bacterial shape to determine if the colonies in the plate were pure cultures. If the observed results are consistent, taking the obtained pure culture (plate colony) as a suspected strain, numbering the corresponding plate, and identifying; if the observed results under the mirror are inconsistent, the operation is continued.

The results showed that the single colony was about 1.5mm, white round protrusions, and high binding to the medium. Colonies were white on MRS neutral red plates, and the calcium-dissolving circles were apparent (FIG. 1). Gram positive, globular, sheeted, no spores (fig. 2).

1.2 identification of strains

(1) Physiological and biochemical characterization

The results of the physiological and biochemical tests of this example, which were completed by China center for type culture Collection of Industrial microorganisms, are shown in Table 3.

TABLE 3 physiological and biochemical characterization table

Wherein "+" represents positive and "w" represents weak positive; "-" represents negative.

(3) Identification of 16s rDNA sequence

The inventors selected 2 strains of bacteria that were positive for gram staining and negative for the contact enzyme test for inspection.

The 16s rDNA sequence is shown as SEQ ID NO. 1:

1 CGAACGAACT TCCGTTAATT GATTATGACG TGCTTGCACT GAATGAGATT

51 TTAACACGAA GTGAGTGGCG GACGGGTGAG TAACACGTGG GTAACCTGCC

101 CAGAAGCAGG GGATAACACC TGGAAACAGA TGCTAATACC GTATAACAGA

151 GAAAACCGCC TGGTTTTCTT TTAAAAGATG GCTCTGCTAT CACTTCTGGA

201 TGGACCCGCG GCGCATTAGC TAGTTGGTGA GGTAACGGCT CACCAAGGCG

251 ATGATGCGTA GCCGACCTGA GAGGGTAATC GGCCACATTG GGACTGAGAC

301 ACGGCCCAGA CTCCTACGGG AGGCAGCAGT AGGGAATCTT CCACAATGGA

351 CGCAAGTCTG ATGGAGCAAC GCCGCGTGAG TGAAGAAGGG TTTCGGCTCG

401 TAAAGCTCTG TTGTTAAAGA AGAACGTGGG TGAGAGTAAC TGTTCACCCA

451 GTGACGGTAT TTAACCAGAA AGCCACGGCT AACTACGTGC CAGCAGCCGC

501 GGTAATACGT AGGTGGCAAG CGTTATCCGG ATTTATTGGG CGTAAAGCGA

551 GCGCAGGCGG TCTTTTAAGT CTAATGTGAA AGCCTTCGGC TCAACCGAAG

601 AAGTGCATTG GAAACTGGGA GACTTGAGTG CAGAAGAGGA CAGTGGAACT

651 CCATGTGTAG CGGTGAAATG CGTAGATATA TGGAAGAACA CCAGTGGCGA

701 AGGCGGCTGT CTGGTCTGTA ACTGACGCTG AGGCTCGAAA GCATGGGTAG

751 CGAACAGGAT TAGATACCCT GGTAGTCCAT GCCGTAAACG ATGATTACTA

801 AGTGTTGGAG GGTTTCCGCC CTTCAGTGCT GCAGCTAACG CATTAAGTAA

851 TCCGCCTGGG GAGTACGACC GCAAGGTTGA AACTCAAAAG AATTGACGGG

901 GGCCCGCACA AGCGGTGGAG CATGTGGTTT AATTCGAAGC TACGCGAAGA

951 ACCTTACCAG GTCTTGACAT CTTCTGCCAA CCTAAGAGAT TAGGCGTTCC

1001 CTTCGGGGAC AGAATGACAG GTGGTGCATG GTTGTCGTCA GCTCGTGTCG

1051 TGAGATGTTG GGTTAAGTCC CGCAACGAGC GCAACCCTTA TTACTAGTTG

1101 CCAGCATTCA GTTGGGCACT CTAGTGAGAC TGCCGGTGAC AAACCGGAGG

1151 AAGGTGGGGA CGACGTCAAA TCATCATGCC CCTTATGACC TGGGCTACAC

1201 ACGTGCTACA ATGGATGGTA CAACGAGTCG CGAAACCGCG AGGTTTAGCT

1251 AATCTCTTAA AACCATTCTC AGTTCGGACT GTAGGCTGCA ACTCGCCTAC

1301 ACGAAGTCGG AATCGCTAGT AATCGCGGAT CAGCATGCCG CGGTGAATAC

1351 GTTCCCGGGC CTTGTACACA CCGCCCGTCA CACCATGAGA GTTTGTAACA

1401 CCCAAAGCCG GTGGGGTAAC CTTTTAG

as shown in Table 4, the sequence alignment showed 99.79% homology of the gene sequence of the strain RH2712 with Pediococcus acidilactici (Pediococcus acidilactici).

Adopting MEGA software, constructing phylogenetic tree by adopting an adjacent connection method, and carrying out similarity calculation 1000 times, wherein the result is shown in the table. Phylogenetic analysis showed that the strain RH2712 was on the phylogenetic tree in one branch with Pediococcus acidilactici DSM 20284T (FIG. 3).

TABLE 4 sequence alignment results

Strain species	Similarity degree
		Pediococcus acidilactici DSM 20284 ^T (GL397069)	99.79％
Pediococcus pentosaceus DSM 20336 ^T (JQBF01000022)	98.46％
		Pediococcus stilesii LMG 23082 ^T (AJ973157)	97.83％
Pediococcus claussenii ATCC BAA-344 ^T (CP003137)	96.78％
		Pediococcus argentinicus DSM 23026 ^T (JQCQ01000064)	96.64％
Pediococcus parvulus JCM 5889 ^T (D88528)	95.38％
		Pediococcus ethanolidurans DSM 22301 ^T (JQBY01000053)	95.24％
Pediococcus cellicola DSM 17757 ^T (JQBR01000021)	95.10％
		Pediococcus inopinatus DSM 20285 ^T (JQBC01000099)	94.89％
Pediococcus damnosus JCM 5886 ^T (D87678)	94.54％

The scanning electron microscope results showed that Pediococcus acidilactici RH2712 strain was ellipsoidal, (0.6-0.9 μm) x (0.7-1.0 μm), and arranged singly or in pairs (FIGS. 4 and 5).

The identification result shows that the RH2712 strain is Pediococcus acidilactici, and the strain is preserved in the China general microbiological culture Collection center (CGMCC) No.25165.

Example 2 safety evaluation

(1) Evaluation of hemolysis

The results showed that RH2712 strain was negative for hemolysis (FIG. 6).

(2) Evaluation of drug sensitivity

Drug susceptibility experiments are shown in table 5. The results showed that RH2712 strain was sensitive to Ampicillin (AM), penicillin (PEN), imipenem (IP), chloramphenicol (CL).

TABLE 5 evaluation results of drug sensitivity

EXAMPLE 3 Pediococcus acidilactici RH2712 strain cultivation method

The formulation of the basal medium used in this example was: 10g/L of yeast peptone, 5g/L of yeast extract, 20g/L of anhydrous dextrose, 2g/L of citric acid monohydrate, 3g/L of L-malic acid, 5g/L of sodium acetate, 2g/L of potassium dihydrogen phosphate, 0.5g/L of magnesium sulfate, 0.01g/L of manganese sulfate and pH value of 6.6. Sterilizing at 115 deg.C for 30min.

The formulation of the optimized medium used in this example was: 10g/L of yeast peptone, 30g/L of yeast extract, 30g/L of anhydrous glucose, 2g/L of citric acid monohydrate, 3g/L of L-malic acid, 5g/L of sodium acetate, 2g/L of monopotassium phosphate, 0.5g/L of magnesium sulfate, 0.01g/L of manganese sulfate, 0.1g/L of Tween 80, 0.5g/L of calcium chloride and pH value of 6.6. Sterilizing at 115 deg.C for 30min.

Primary culture: the thallus recovered in the freezing tube is transferred into 10mL of basic culture medium according to the inoculation amount of 10 percent, and the bacterial suspension is obtained by static culture for 17 hours at 37 ℃.

Secondary culture: the bacterial suspension is transferred into 100mL of basic culture medium, the liquid loading amount is 40 percent, and the bacterial suspension is subjected to stationary culture for 17 hours at 37 ℃. And (3) three-stage culture: the bacterial suspension is inoculated into 200mL of optimized culture medium with 3% of inoculation amount, the liquid loading amount is 40%, and the bacterial suspension is subjected to stationary culture at 37 ℃ for 17 hours.

Yield after completion of culture: 1.203% and viable count of 6.30X10 ⁹ CFU/mL。

EXAMPLE 4 Pediococcus acidilactici RH2712 strain cultivation method

The formulation of the basal medium used in this example was: 14g/L yeast peptone, 8g/L yeast extract, 25g/L anhydrous dextrose, 5g/L citric acid monohydrate, 5g/L malic acid, 8g/L sodium acetate, 5g/L potassium dihydrogen phosphate, 1.0g/L magnesium sulfate, 0.05g/L manganese sulfate and pH 6.6. Sterilizing at 115 deg.C for 30min.

The formulation of the optimized medium used in this example was: 14g/L yeast peptone, 40g/L yeast extract, 40g/L anhydrous dextrose, 5g/L citric acid monohydrate, 5g/L malic acid, 8g/L sodium acetate, 5g/L potassium dihydrogen phosphate, 1.0g/L magnesium sulfate, 0.05g/L manganese sulfate, 0.3g/L tween 80, 1.0g/L calcium chloride and pH 6.6. Sterilizing at 115 deg.C for 30min.

Yield after completion of culture: 1.190% and viable count of 5.70X10% ⁹ CFU/mL。

EXAMPLE 5 gastrointestinal adverse environmental resistance of Pediococcus acidilactici RH2712 Strain

After probiotics enter the stomach, gastric acid can lead to activity reduction and vitality reduction of the probiotics, bile salts can lead to vitality reduction of the probiotics after entering the small intestine, and residual bile salts can also influence the field planting and the immunity vitality of the probiotics in the large intestine after entering the large intestine. Gastric acid pH of a person in a fasting state is about 0.9.1.5, gastric juice is diluted after eating, pH is about 3.5, and common probiotics are difficult to survive. Thus, probiotics are often affected by the reverse environment of the digestive tract in their functioning.

5.1 acid resistant survival assay

Bacterial strain preparation:

1) Primary culture: taking out the strain freezing tube preserved at-80 ℃, thawing at room temperature, mixing uniformly, streaking 1-cycle bacteria liquid on an MRS solid flat plate, and standing and culturing at 37 ℃ for 48 hours;

2) Secondary culture: selecting single colony to 5mL MRS liquid culture medium, and culturing at 37 ℃ for 20h;

3) And (3) three-stage culture: 5mL of the bacterial liquid is inoculated into 100mL of MRS liquid culture medium and cultured for 17h at 37 ℃.

Bacterial solutions of three passages were inoculated into MRS medium at pH 2.0 and pH 3.0, respectively, at an inoculum size of 10%. Stationary culture at 37deg.C, sampling at 0h, 1h, 2h and 4h, and 10-fold incremental gradient dilution with sterilized normal saline (to 10 ^-7 Dilution), 10 ^-4 -10 ^-7 1mL of bacterial liquid with dilution is subjected to mixed bacterial counting operation, each dilution is repeated for 2 times, and the bacterial liquid is subjected to stationary culture at 37 ℃ for 36-48 hours and then counted. 1h, 2h and 4h of sampling and counting the number of viable bacteria (expressed by N'); the number of viable bacteria (N ₀ Representation) is calculated according to the following formula: acid-resistant survival (%) = lg cfu N'/lg cfu N of the test strain ₀ ×100％。

The result shows that after 2 hours of the RH2712 strain under the condition of pH 3.0, the number of viable bacteria is reduced compared with that of a 0-hour control, the number of viable bacteria reaches 8.20, after 4 hours, the number of viable bacteria is increased, and the number of viable bacteria reaches 8.25; the survival rate of the strain reaches 36.57% after 2 hours under the condition of pH 2.0; pH 3.0 there was a significant decrease in pH 2.0 compared to pH 2.0 (Table 6). The RH2712 strain can grow normally at pH 3.0, and has good acid resistance, and can survive in the normal reverse environment of the digestive tract.

TABLE 6 acid resistant survival assay for RH2712 Strain

5.2 determination of bile salt survival

Inoculating the bacterial solutions of the three passages respectively into MRS liquid culture medium containing 0.3%, 0.5%, 1.0% and 1.5% of ox gall salt concentration according to 10% inoculum size, standing at 37deg.C for culturing, sampling at 0h, 1h, 2h and 4h, respectively performing 10-fold incremental gradient dilution with sterilized normal saline (to 10% ^-7 Dilution), 10 ^-4 -10 ^-7 1mL of bacterial liquid with dilution is subjected to mixed bacterial counting operation, each dilution is repeated for 2 times, and the bacterial liquid is subjected to stationary culture at 37 ℃ for 36-48 hours and then counted. 1h, 2h and 4h of sampling and counting the number of viable bacteria (expressed by N'); the number of viable bacteria (N ₀ Representation) is calculated according to the following formula: bile salt-tolerant survival (%) = lg cfu N'/lg cfu N of the test strain ₀ ×100％。

The results showed that the RH2712 strain was well tolerated by bile salts. Under the condition of different bile salt concentrations, the viable bacteria log values all show a trend of descending and then ascending along with the extension of the culture time of the bacterial liquid. After the bacterial liquid is cultured for 4 hours under the condition of different bile salt concentrations, the number of viable bacteria is reduced along with the increase of the bile salt concentration, when the bile salt concentration is 0.3%, the number of viable bacteria reaches 9.20, and when the bile salt concentration is 1.5%, the number of viable bacteria still reaches 8.72 (Table 7).

TABLE 7 determination of bile salt resistance survival of RH2712 Strain

5.3 gastric juice simulation experiment

Shaking the bacterial liquid for three times, taking 10mL of bacterial suspension, centrifuging (5000 Xg, 10min,4 ℃) to obtain bacterial mud, flushing with PBS buffer solution for 2 times, re-suspending the obtained bacterial mud in 10mL of simulated gastric fluid, digesting for 3h at 37 ℃, sampling at 0h and 3h respectively, and performing primary series with sterilized physiological salineColumn 10-fold increasing gradient dilution (to 10 ^-7 Dilution), 10 ^-4 -10 ^-7 1mL of bacterial liquid with dilution is subjected to mixed bacterial counting operation, each dilution is repeated for 2 times, and the bacterial liquid is subjected to stationary culture at 37 ℃ for 36-48 hours and then counted. The number of viable bacteria (denoted by N) of the test strain in the third-generation culture solution after the third generation of activation; the viable count (denoted by N ") measured by sampling and counting for 0h and 3h was calculated according to the following formula: the test strain simulated gastric juice test survival (%) =lg cfu N "/lg cfu n×100%.

The results showed that the RH2712 strain had a viable bacterial count of 9.37 before simulated gastric fluid treatment (OR, bacterial fluid after 3 generations of activation); after 3h treatment, the survival rate still reached 76.20% (Table 8).

Table 8 results of simulated gastric fluid experiments

Example 6 in vitro enhanced immunity function assay of Pediococcus acidilactici RH2712 Strain

6.1 preparation of Strain

The preparation method of the strain of this example is the same as that of example 5.

6.2 preparation of samples to be tested

Boiling the cultured bacterial liquid at 100deg.C for 10min to inactivate enzymes degrading polysaccharide; after cooling, 17% (v/v), 85% trichloroacetic acid was added as a volume ratio of bacterial liquid to trichloroacetic acid. Then centrifuging at 4deg.C and 8000rpm for 20min, removing cell and protein precipitate, and collecting supernatant. Adding three times volume of absolute ethanol into the supernatant, centrifuging at 4deg.C and 10000rpm for 25min, and precipitating to obtain total extracellular polysaccharide.

6.3 drawing a Standard Curve

Accurately weighing 10mg of anhydrous glucose, dissolving in a 100mL volumetric flask, adding a proper amount of distilled water to a constant volume to prepare 100 mug/mL glucose standard solution, taking 6 test tubes, respectively adding 0, 0.2, 0.4, 0.6, 0.8 and 1.0mL glucose standard solution, adding distilled water to 1.0mL, adding 1.0mL of 5% phenol solution into each test tube, shaking, rapidly adding 5.0mL concentrated sulfuric acid, standing at room temperature for 30min, and measuring OD by using an enzyme-labeling instrument ₄₉₀ Values, OD, on the abscissa of the mass concentration of glucose standard solution ₄₉₀ Values are plotted as ordinate against a standard curve (fig. 7).

6.4 extracellular polysaccharide content determination

A large number of pharmacological and clinical researches show that a plurality of polysaccharide compounds have the function of regulating immunity, can activate immune receptors and improve the immune function of organisms. Vinderrola et al report that EPS (exopolysaccharide) produced by Lactobacillus equi induces response of digestive tract mucosa by cytokine release into circulating blood, regulates protective immunity, maintains stability of environment in intestinal tract, increases leA level of large intestine and small intestine, and affects immunity of body tissue (Vinderrola G et al effects of the oral administration of the exopolysaccharide produced by Lactobacillus kefiranofaciens on the gut mucosal immunity. Cytokine.2006;36 (5-6): 254-260).

Adding equal amount of distilled water into the prepared precipitate for resuspension (equal amount of bacterial liquid), and measuring the EPS content of lactobacillus by using a phenol-sulfuric acid method, wherein the reaction system is as follows: the ratio of the sample to 5% phenol and concentrated sulfuric acid is 1:1:5, shaking uniformly, standing for 30min at room temperature, and measuring OD by using an enzyme-labeled instrument ₄₉₀ Values.

The result shows that the exopolysaccharide content in the fermentation supernatant of the Pediococcus acidilactici RH2712 strain is 1070.12 mug/mL, and the Pediococcus acidilactici RH2712 strain has a very strong immunity enhancing function.

Example 7 determination of the in vivo immunity enhancing function of Pediococcus acidilactici RH2712 Strain

7.1 establishing a mouse immunosuppression model

The mice of this example were Specific Pathogen Free (SPF) grade male ICR mice (18-22 g) purchased from Liaoning long biotechnology Co. The test mice were fed with water freely under laboratory conditions for 1 week. The mice to be tested were randomly divided into 5 groups of 10 animals each, which were respectively a solvent control group (pure water), a model group, a low dose group, a medium dose group and a high dose group, and the experimental animals of the other groups except the solvent control group were first intraperitoneally injected with cyclophosphamide 80 mg/(kg·d) daily after the start of the experiment for 3 days continuously to establish an immunosuppression model.

In practiceAfter the test starts, 4 days, respectively dissolving and irrigating the bacterial powder every day, and then administering various samples with different dosages, wherein the gastric lavage volume is 10mL/kg from 1 time every day to 30 days, and the solvent control group and the model group are irrigated with equal volumes of pure water. Wherein, the solvent control group (pure water), the model group, the 1.0 g/(kg.bw) low dose group, the 2.0 g/(kg.bw) medium dose group and the 4.0 g/(kg.bw) high dose group, wherein, g is the mass unit of the bacterial powder. The number of the viable bacteria of the lyophilized powder of the Pediococcus acidilactici RH2712 strain is 5.0x10 ⁹ CFU/g meter.

7.2 measurement of organ index

The mice were sacrificed immediately after 24h of last dose on day 40, after blood was taken from the eyeballs, thymus, spleen, liver, kidney and heart were dissected and isolated, and the viscera surface blood was blotted with filter paper and weighed separately. The organ index is calculated according to the quality of each organ, and the calculation formula is as follows:

the results of the organ index change are shown in Table 9.

TABLE 9 Effect of Pediococcus acidilactici RH2712 strain treatment on immunosuppressed mouse organ indexes

Note that: * P <0.05 compared to model group.

The results show that the organ indexes of the mice in the model group are reduced compared with the normal control group; after the Pediococcus acidilactici RH2712 strain is treated, compared with a model group of mice, thymus indexes of mice in a medium-dose group and a high-dose group are obviously improved, spleen indexes of mice in different dose groups are obviously improved, and kidney indexes of mice in the high-dose group are obviously improved. The results indicated that the Pediococcus acidilactici RH2712 strain had a dose-dependent therapeutic effect on the immune organ index of immunosuppressed mice, with the most significant improvement in thymus index and kidney index of immunosuppressed mice, returning to substantially normal levels.

7.3 determination of immune index

The mice of each experimental group were collected with eyeball blood, placed at 37℃for 1h,4℃for 1h, centrifuged at 2800r/min for 10min, and serum was prepared and stored at-20 ℃. The activities of IL-4, IL-10, IFN-gamma, TNF-alpha and IgG in serum were measured using enzyme-linked immunosorbent assay (ELISA) kit, and the measurement results are shown in Table 10.

TABLE 10 influence of Pediococcus acidilactici RH2712 strain treatment on immune indicators of immunosuppressive mice

Note that: * P <0.05 compared to model group; * Comparing p <0.01 to the model group; where pg/ng is the mass of each immune factor and mL is the volume of mouse serum.

Compared with the mice in the solvent control group, the serum IL-4, IFN-gamma, TNF-alpha and IgG mass concentration of the mice in the model group are obviously reduced, and the IL-10 content is obviously increased. Indicating that the model of cyclophosphamide induced immunosuppression was successfully modeled.

After the Pediococcus acidilactici RH2712 is applied to the immunosuppressed mice, the result shows that compared with the mice in a model group, the serum IL-4 content of the mice in a medium-high dose group is obviously improved (p is less than 0.05), and the mice are equivalent to the mice in a control group, and can reach 43.29pg/mL and 44.15pg/mL respectively; serum IL-10 mass concentration of mice in medium and high dose groups is significantly reduced (p < 0.05), wherein the IL-10 mass concentration of mice in the high dose group can be restored to 75.09ng/mL, which is close to the normal level; the IFN-gamma mass concentration of the mice in the low, medium and high dose groups is obviously improved, wherein the IFN-gamma mass concentration of the mice in the high dose groups can be recovered to 210.47pg/mL; the TNF-alpha mass concentration of the mice in the low, medium and high dose groups is obviously improved, wherein the difference between the low and medium dose groups is obvious (p < 0.05), the difference between the high dose groups is extremely obvious (p < 0.01), the TNF-alpha content is restored to the level approximately equivalent to that of the solvent control group, and the mass concentration can reach 244.13pg/mL; the IgG mass concentrations of the low, medium and high dose group mice were recovered to different extents, with the medium and high dose groups differing significantly (p < 0.05). The results show that the Pediococcus acidilactici RH2712 strain has a good recovery function on the immunosuppression of organisms and has the capability of enhancing the immunity.

Comparative example

In view of the temporary inability to obtain pediococcus acidilactici CCFM6432 from the guangdong collection in chinese patent application CN110079485A, this comparative example uses the same experimental procedure in CN110079485A to indirectly effect comparison with pediococcus acidilactici CCFM 6432.

32 male C57BL/6 mice of 6 weeks old were purchased from Liaoning long biotechnology Co., ltd, and after one week of adaptation to the environment, were randomly divided into four groups according to body weight: normal control (free diet, gavage control solvent), model, drug intervention, pediococcus acidilactici RH2712, each containing 8 mice. Model, drug intervention and Pediococcus acidilactici RH2712 groups were chronically unpredictable Wen Heying stimulated while drinking water was free. Depression model group lavage control solvent (pure water), drug intervention group lavage 10mg/kg fluoxetine, pediococcus acidilactici RH2712 group lavage 10 ⁹ CFU/mL bacterial liquid. The test cycle proceeds to 6 weeks, at which time daily chronic unpredictable stress and drug and probiotic intervention is stopped.

Chronic unpredictable stress depression mouse model (model group): 1-2 kinds of stimulation are randomly adopted every day, and the time of the stimulation every day is randomly determined, so that the circadian rhythm is avoided. Each method was no more than four times for five weeks. Stimulus factors include: (1) fasted for 24 hours; (2) water forbidden and empty bottle stimulated for 24 hours; (3) clamping the tail for 3min; (4) wet padding for 24 hours; (5) braking for 1-2h; (6) 45 DEG inclined cage boxes for 24h; (7) continuously illuminating for 24 hours; (8) no padding 24h; (9) forced swimming for 15 minutes; and (10) carrying out solitary culture for 24h.

Mice were euthanized on the sixth weekend, fresh mouse spleens (about 50 mg) were taken, 5mL of pre-chilled PBS buffer was added, sheared, ground, and sun-filtered with 200 mesh nylon to give a tissue cell suspension. 2mL of the erythrocyte lysate was added to lyse for 4min, the lysate was removed by centrifugation at room temperature (300 Xg, 5 min), and the resulting cell pellet was washed twice with pre-chilled PBS and resuspended in a defined amount of PBS to give a lymphocyte suspension. Antibody labeling of Regulatory T cells (Tregs) was performed according to the instructions of eBioscience Mouse Regulatory T Cell Staining Kit (Invitrogen Corporation, carlsbad, CA, USA). Tregs (cd4+cd25+foxp3+) were detected using a flow cytometer.

The experimental results are shown in table 11, chronic stress can lead to a significant reduction in the proportion of Tregs in the spleen of mice, i.e. the immune competence of mice is impaired; both fluoxetine and Pediococcus acidilactici RH2712 groups significantly improved the immune status of depressed mice. And the Pediococcus acidilactici RH2712 group has more recovery than the Tregs proportion (about 2.77% of the data recorded by CN 110079485A) in Pediococcus acidilactici CCFM6432, and has better immunity improving capability.

Table 11 results of comparative tests

Grouping	Tregs/total cells (%)
		Normal control group	3.02±0.1 ^**
Model group	2.05±0.3
		Pharmaceutical intervention group	2.75±0.1 ^*
Pediococcus acidilactici RH2712 group	2.92±0.2 ^*

Note that: * P <0.05 compared to model group; * Comparing p <0.01 to the model group;

finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. Use of Pediococcus acidilactici RH2712 strain in immunomodulation.

2. Application of Pediococcus acidilactici RH2712 strain in preparing products for enhancing immune function or relieving immune function inhibition;

preferably, the products include pharmaceuticals, nutraceuticals and foods.

3. The use according to claim 2, wherein the medicament comprises a therapeutically effective amount of Pediococcus acidilactici RH2712 strain and pharmaceutically acceptable excipients;

preferably, the dosage form of the medicament is at least one selected from powder, tablets, granules, capsules, solutions, emulsions, suspensions, injections, sprays, powder mists, aerosols, suppositories, drops and drop pills;

preferably, the auxiliary material is selected from at least one of solvents, diluents, disintegrants, precipitation inhibitors, surfactants, glidants, binders, lubricants, dispersants, suspending agents, isotonic agents, thickeners, emulsifiers, preservatives, stabilizers, hydration agents, emulsification accelerators, buffers, absorbents, colorants, flavoring agents, sweeteners, ion exchangers, mold release agents, coating agents, flavoring agents, and antioxidants.

4. The use according to claim 1 or 2, wherein the strain is preserved in CGMCC with the preservation number of CGMCC No.25165.

5. The use according to claim 4, wherein the nucleotide sequence of the 16S rDNA of the strain is shown in SEQ ID NO. 1.

6. The use according to claim 5, characterized by the steps of:

(3) Inoculating the secondary culture into an optimized culture medium for tertiary culture to obtain a tertiary culture;

preferably, the conditions of the primary culture, the secondary culture and the tertiary culture are 37 ℃ culture for 17 hours;

preferably, the tertiary culture comprises a culture broth, a culture broth extract, whole bacteria, a whole bacteria extract, a fermentation broth extract, and/or a bacterial powder.

7. The use according to claim 6, wherein the basal medium comprises a carbon source, a nitrogen source, a phosphorus source, trace elements, citric acid monohydrate, sodium acetate and L-malic acid;

preferably, the carbon source is anhydrous glucose; and/or

The nitrogen source is yeast peptone and yeast extract; and/or

The phosphorus source is potassium dihydrogen phosphate; and/or

The microelements are magnesium sulfate and manganese sulfate.

8. The use according to claim 7, wherein the mass to volume ratio of the carbon source is 15-25g/L; the mass volume ratio of the nitrogen source is 10-22g/L; the mass volume ratio of the phosphorus source is 1-5g/L; the mass volume ratio of the trace elements is 0.11-1.05g/L; the mass volume ratio of the citric acid monohydrate is 1-5g/L; the mass volume ratio of the sodium acetate is 2-8g/L; the mass volume ratio of the L-malic acid is 1-5g/L;

preferably, the mass-to-volume ratio of the carbon source is 20g/L; the mass volume ratio of the nitrogen source is 15g/L; the mass volume ratio of the phosphorus source is 2g/L; the mass volume ratio of the trace elements is 0.51g/L; the mass volume ratio of the citric acid monohydrate is 2g/L; the mass volume ratio of the sodium acetate is 5g/L, and the mass volume ratio of the L-malic acid is 3g/L;

preferably, the mass-volume ratio of the yeast peptone is 8-14g/L; the mass-volume ratio of the yeast extract is 2-8g/L; the mass volume ratio of the magnesium sulfate is 0.1-1.0g/L; the mass volume ratio of the manganese sulfate is 0.01-0.05g/L;

more preferably, the mass-to-volume ratio of the yeast peptone is 10g/L; the mass-volume ratio of the yeast extract is 5g/L; the mass volume ratio of the magnesium sulfate is 0.5g/L; the mass volume ratio of the manganese sulfate is 0.01g/L.

9. The use according to claim 6, wherein the optimized medium comprises carbon source, nitrogen source, phosphorus source, trace elements, citric acid monohydrate, sodium acetate, L-malic acid, tween 80 and calcium chloride;

preferably, the carbon source is anhydrous glucose; and/or

The nitrogen source is yeast peptone and yeast extract; and/or

The phosphorus source is potassium dihydrogen phosphate; and/or

The microelements are magnesium sulfate and manganese sulfate.

10. The use according to claim 9, wherein the carbon source has a mass to volume ratio of 20-40g/L; the mass volume ratio of the nitrogen source is 28-54g/L; the mass volume ratio of the phosphorus source is 1-5g/L; the mass volume ratio of the trace elements is 0.31-1.05g/L; the mass volume ratio of the citric acid monohydrate is 1-5g/L; the mass volume ratio of the sodium acetate is 2-8g/L; the mass volume ratio of the L-malic acid is 1-5g/L; the mass volume ratio of the Tween 80 is 0.05-0.3g/L; the mass volume ratio of the calcium chloride is 0.1-1.0g/L;

preferably, the mass-to-volume ratio of the carbon source is 30g/L; the mass volume ratio of the nitrogen source is 40g/L; the mass volume ratio of the phosphorus source is 2g/L; the mass volume ratio of the trace elements is 0.51g/L; the mass volume ratio of the citric acid monohydrate is 2g/L; the mass volume ratio of the sodium acetate is 5g/L; the mass volume ratio of the L-malic acid is 3g/L; the mass volume ratio of the Tween 80 is 0.1g/L; the mass volume ratio of the calcium chloride is 0.5g/L;

preferably, the mass-volume ratio of the yeast peptone is 8-14g/L; the mass-volume ratio of the yeast extract is 20-40g/L; the mass volume ratio of the magnesium sulfate is 0.3-1.0g/L; the mass volume ratio of the manganese sulfate is 0.01-0.05g/L;

more preferably, the mass-to-volume ratio of the yeast peptone is 10g/L; the mass-volume ratio of the yeast extract is 30g/L; the mass volume ratio of the magnesium sulfate is 0.5g/L; the mass volume ratio of the manganese sulfate is 0.01g/L.