CN113512516B - Cooperative swine-origin lactobacillus mucosae and application thereof - Google Patents

Abstract

The invention discloses a cooperative swine lactobacillus mucosae and application thereof. The strain is named as lactobacillus mucosae LM410 with the preservation number of CGMCC No.22828. The strain is separated from the excrement of a healthy cooperative pig, and has good acid production, acid resistance, cholate resistance and pathogenic bacterium antagonism. The lactobacillus mucosae LM410 provided by the invention can inhibit the growth of C-type clostridium perfringens, has an obvious protection effect on the damage of pig intestinal epithelial cells caused by the C-type clostridium perfringens, and has a wide development prospect and an application value in the prevention and treatment of bacterial infectious intestinal diseases of livestock and poultry.

Description

Cooperative swine-origin lactobacillus mucosae and application thereof

Technical Field

The invention relates to a cooperative swine-origin lactobacillus mucosae and application thereof, belonging to the technical field of microorganisms.

Background

The diarrhea of piglets is one of the main diseases which troubles the pig production, and the economic benefit of the pig industry is seriously influenced. The death rate of piglets in China is reported to be 15% -20%, wherein the death rate of piglets caused by diarrhea accounts for about 40% of the total death rate, and the direct economic loss brought to the pig industry every year reaches hundreds of billions of yuan. Clostridium perfringens type C, also known as clostridium welchii type C, is one of the main pathogens causing diarrhea in piglets; the infection mainly occurs in piglets within 2 weeks, and the piglet has the characteristics of acute morbidity and short disease course, the mortality rate of the sick piglets reaches more than 70 percent, and the healthy development of the pig industry is seriously influenced. Clostridium perfringens infectious diarrhea is reported to occur in all swine countries of the world; according to investigation of 33 large-scale pig farms in Guangdong, jiangxi, hubei and Yunnan of China in 2015, the infection rate of clostridium perfringens in the piglet lactation stage is up to 80%.

Although the occurrence of diarrhea diseases is reduced and controlled to a certain extent by measures such as strengthening feeding management, injecting vaccines, using antibiotic medicines for prevention and treatment and the like, the problems of pathogenic bacteria drug resistance enhancement, antibiotic residue and the like are caused by long-term use of antibiotics. At the same time, the efficacy of a toxoid vaccine to prevent infection by the pathogen is susceptible to the antigenic component of the toxoid. These problems make the prevention and treatment of C-clostridium perfringens infectious diarrhea of piglets more and more difficult, and antibiotic-free and green healthy breeding also requires that the use of antibiotics be minimized or even prohibited. Therefore, a strategy to find an alternative antibiotic is an urgent need to prevent C-type clostridium perfringens diarrhea in piglets.

Lactobacillus mucosae: (A)Lactobacillus mucosae) Is one of the probiotics in the lactobacillus in the intestinal tract of the animals. In recent years, researches show that lactobacillus mucosae has important functions in cell adhesion, acid production by carbohydrate metabolism, pathogenic bacterium antagonism and the like, and can influence the integrity and the function of intestinal barrier through dephosphorylation of tight junction protein, actin regulation and the like. The research also finds that the lactobacillus mucosae can adhere to the intestinal epithelial tissue to form a biofilm and simultaneously generate antibiotics, and has the probiotic effects of inhibiting the colonization of pathogenic bacteria and regulating the intestinal immune system. In addition to this, the present invention is,Lactobacillus mucosaeNK41 and Bifidobacterium longum (Bifidobacterium longum) Synergistic effect, by inhibiting Nuclear factor kappa-B (NF-kappa B) protein activationTumor necrosis factor-alpha (TNF-alpha) expression and bacterial lipopolysaccharide production to alleviate colitis symptoms in mice. The development and the utilization of the lactobacillus mucosae as an antibiotic substitution strategy for preventing and treating the bacterial infectious intestinal diseases of the livestock and the poultry are demonstrated, and the method has wide development prospect and application value.

Disclosure of Invention

The first purpose of the invention is to provide a lactobacillus mucosae of cooperative porcine origin, which has good acid-producing, acid-and bile salt-resistant capabilities and an antagonistic capability to the growth of clostridium perfringens type C.

The second purpose of the invention is to provide the application of the lactobacillus mucosae in preventing and treating pig intestinal epithelial cell IPEC-J2 injury caused by C-type clostridium perfringens infection, and the lactobacillus mucosae has wide development prospect and application value in the prevention and treatment of bacterial infectious intestinal diseases of livestock and poultry.

In order to solve the problems, the invention firstly provides a lactobacillus mucosae of cooperative porcine origin, which is the lactobacillus mucosae preserved in the China general microbiological culture Collection center (CGMCC) at 7-6.7-6.2021Lactobacillus mucosaeThe preservation number is CGMCC No.22828. The address of the depository: xilu No. 1, beijing, chaoyang, beijing, and institute for microbiology, china academy of sciences.

The lactobacillus mucosaeLactobacillus mucosaeIs obtained by separating and purifying healthy cooperative pig excrement samples.

The lactobacillus mucosaeLactobacillus mucosaeThe 16S rDNA sequence of (1) is GenBank No. MZ047317.

The lactobacillus mucosaeLactobacillus mucosaeHas good acid, acid and bile salt resistance and pathogenic bacteria antagonistic capability. Survival rates of greater than 80.00% in MRS broth at pH 4.0, 3.0 and 2.0; the survival rate of MRS broth with the added amount of the pig bile salt of 0.10%, 0.20% and 0.30% is more than 90.00%; significantly inhibits the growth of clostridium perfringens type C when co-cultured with clostridium perfringens type C.

The second purpose of the invention is to provide the application of the strain in preventing and treating pig IPEC-J2 cell injury caused by C type clostridium perfringens.

The lactobacillus mucosaeLactobacillus mucosaeThe number of viable bacteria is 10 ⁷ CFU/mL and 10 ⁸ The cell morphology and the survival rate of the IPEC-J2 cells are not obviously influenced in CFU/mL, the damage of the IPEC-J2 cell morphology caused by C-type clostridium perfringens infection can be effectively relieved, and the activity of the IPEC-J2 cells after the C-type clostridium perfringens infection is obviously increased.

The lactobacillus mucosaeLactobacillus mucosaeCan obviously inhibit the increase of the expression of inflammatory factors after C-type clostridium perfringens infects IPEC-J2 cells and obviously increase the reduction of the expression of C-type clostridium perfringens infects IPEC-J2 cell tight junction protein.

The invention has the beneficial effects that: the lactobacillus mucosae LM410 separated from the cooperative pig manure for the first time has good in-vitro probiotic potential, can inhibit the growth of C-type clostridium perfringens, has an obvious protection effect on IPEC-J2 cell damage caused by C-type clostridium perfringens, and can be used for preventing and treating pig bacterial infectious diarrhea diseases.

Drawings

FIG. 1 is a colony morphology of the isolated strain in example 1 of the present invention;

FIG. 2 shows the results of the catalase test of the isolated strain in example 1 of the present invention. A is a positive control (clostridium perfringens type C), B is an isolated strain;

FIG. 3 shows the results of the staining microscopy of the isolated strain of example 1 of the present invention (1000X);

FIG. 4 is a diagram showing PCR amplification of the isolated strain in example 1 of the present invention. M is 2000 bp DNA marker;1 is negative control; 2 is an isolated strain;

FIG. 5 shows phylogenetic trees of isolated strains in example 1 of the present invention;

FIG. 6 shows Lactobacillus mucosae strain in example 2 of the present inventionLactobacillus mucosaeGrowth curve and acid production curve of (a);

FIG. 7 shows Lactobacillus mucosae strain in example 2 of the present inventionLactobacillus mucosaeInhibiting the growth pattern of clostridium perfringens type C. LM is Lactobacillus mucosaeLactobacillus mucosaePure culture viable count; LM in co-cThe ulture is Lactobacillus mucosaeLactobacillus mucosaeViable count in co-culture with clostridium perfringens type C; CP is the pure culture viable count of the clostridium perfringens type C; CP in co-culture is C-type clostridium perfringens and lactobacillus mucosaeLactobacillus mucosaeViable count in co-culture;

FIG. 8 shows different concentrations of Lactobacillus mucosae in example 3 of the present inventionLactobacillus mucosaeEffect on IPEC-J2 cell viability;

FIG. 9 shows Lactobacillus mucosae used in example 3 of the present inventionLactobacillus mucosaeEffect on the morphology of clostridium perfringens type C infected IPEC-J2 cells (100 ×); control is blank Control group; LM is a lactobacillus mucosae LM410 culture group; CP is C-type clostridium perfringens culture group; LM + CP is Lactobacillus mucosaeLactobacillus mucosaeA co-cultured group with clostridium perfringens type C; FIG. 10, FIG. 11, FIG. 12 are the same;

FIG. 10 shows Lactobacillus mucosae strain in example 3 of the present inventionLactobacillus mucosaeEffect on the viability of clostridium perfringens type C infected IPEC-J2 cells;

FIG. 11 shows Lactobacillus mucosae strain in example 3 of the present inventionLactobacillus mucosaeEffect on clostridium perfringens type C infection IPEC-J2 cytokine expression;

FIG. 12 shows Lactobacillus mucosae strain in example 3 of the present inventionLactobacillus mucosaeEffect on C-type perfringolysis factor infection of IPEC-J2 cells on claudin expression.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples. These examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention.

Example 1 Lactobacillus mucosaeLactobacillus mucosaeSeparation and identification of

1. Separation and purification of bacterial strains

Taking about 0.5g of healthy cooperative pig manure sample, diluting the healthy cooperative pig manure sample into 10 parts by using 1 XDPBS buffer solution ^-3 、10 ^-4 、10 ^-5 3 gradients. 70.00 mu L of fecal diluent with different concentrations are respectively taken and coated on Ca containing vancomycin (filter sterilization, final concentration of 2.00 mg/mL)CO ₃ MRS plates, anaerobic culture at 37 ℃ 48 h. Selecting single colony with obvious calcium dissolving ring and according with typical colony morphological characteristics of lactobacillus, streaking and culturing on MRS plate, and purifying repeatedly for three times. 70 suspected strains which accord with the colony morphological characteristics of lactobacillus, such as milky white, round, smooth edge, full overall, and the like, are selected (figure 1).

The CaCO3-MRS culture medium used by the invention comprises the following components: peptone 10.0 g/L, beef extract 5.0 g/L, glucose 20.0 g/L, dipotassium hydrogen phosphate 2.0 g/L, triammonium citrate 2.0 g/L, sodium acetate 5.0 g/L, magnesium sulfate 0.2 g/L, manganese sulfate 0.05 g/L, tween-80.0 g/L, agar 15.0 g/L, caCO ₃ 15.0 g/L. Adjust to final pH 6.2. + -. 0.1.

2. Identification of strains

The strain identification comprises hydrogen peroxide catalase test, gram staining microscopy observation of thallus morphology, biochemical identification and 16S rDNA molecular identification.

(1) Catalase test

1 clean glass slide is taken, a single colony is picked by an inoculating loop and coated on the glass slide, a drop of 3% hydrogen peroxide (prepared at present) is dripped, and whether bubbles are produced or not is immediately observed. If there are bubbles, the cells were catalase positive and no bubbles, catalase negative strains 52 were selected using E.coli as a positive control (FIG. 2).

(2) Microscopic examination of gram stain

After staining the catalase negative strains according to the gram staining kit specification, the morphological characteristics of the strains are observed by a microscope, and gram positive strains 33 which accord with the typical bacterial characteristics of the lactobacillus are screened out (figure 3).

(3) Biochemical identification

The selected strain was inoculated into MRS broth and shake-cultured at 37 ℃ for 24 h. HBI lactobacillus biochemical identification strip (GB) is adopted for culture and identification, identification results are compared with biochemical characteristics of lactobacillus mucosae in Vol.3 (2009) of Bergey's Manual of systematic bacteriology, and 7 suspected strains of the lactobacillus mucosae are screened (Table 1). + positive and-negative.

TABLE 1 Biochemical identification of isolated strains

The MRS broth used in the invention comprises the following components: weighing 48.30 g, heating and dissolving in 1L distilled water, autoclaving at 121 deg.C for 15min, and adjusting to final pH of 6.2 + -0.1.

(4) 16S rDNA molecular identification

The selected strain was inoculated into MRS broth and shake-cultured at 37 ℃ for 24 h. Specific primers (LabM-F: 5'-TGAGTAACACGTAGGTAACCTG-3'; labM-R: 5'-ATGCTGATCCGCGATTACT-3') were designed based on the Lactobacillus mucosae 16S rRNA gene, and genomic DNA of each strain was extracted according to the procedures described in the protocol of the plasmid miniprep kit, and subjected to PCR amplification and 1% agarose gel electrophoresis detection to amplify a fragment having a size of 1261bp (FIG. 4). The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ 30 s; annealing at 58 ℃ for 30 s, extending at 72 ℃ for 2 min, and performing 35 cycles; extension for 10min at 72 ℃.

The PCR product is sent to Yang Lingtian Olympic Biotechnology GmbH for sequencing, the obtained sequence is compared with the standard strain in NCBI database by BLAST, phylogenetic tree is constructed according to the reference sequence, and the strain 2 and the NCBI database are separatedLactobacillus mucosaeLM011 (NZ _ CP 062966.1) has the closest relationship, sequence coverage of 100% and homology of 99% (FIG. 5), isolate 2 was identified as a species or variant of Lactobacillus mucosae, designated as Lactobacillus mucosaeLactobacillus mucosaeLM410。

Lactobacillus mucosaeLactobacillus mucosaeThe 16S rDNA gene sequence of (A) was submitted to the NCBI database, genBank No. MZ047317.

Lactobacillus mucosaeLactobacillus mucosaeAdding sterilized glycerol with final concentration of 30%, and storing at-80 deg.C.

Example 2 Lactobacillus mucosaeLactobacillus mucosaeAnalysis of biological Properties

1. Growth curve and acid production curve

Activated lactobacillus mucosaeLactobacillus mucosaeInoculating MRS broth with 1.00% of inoculum size,culturing 24 h in a shaker at 37 deg.C, sampling every 2h, and determining absorbance (OD) of the culture medium ₆₀₀ ) And pH value, taking the culture time as an abscissa, and respectively taking the light absorption value and the pH value as an ordinate to draw a growth curve and an acid production curve. The result shows that the growth of 0-8 h is relatively slow, 8-20 h is in logarithmic growth phase, and 20 h enters a stabilization phase and a decay phase later; the pH value of the MRS broth is gradually reduced to be below 4.50 and tends to be stable after the 22 h is cultured (figure 6), which shows that the MRS broth has good acid production performance.

2. Acid resistance measurement

Using 6.00 mol.L ^-1 Hydrochloric acid adjusted the MRS broth pH to 4.00, 3.00 and 2.00, respectively. Activated lactobacillus mucosaeLactobacillus mucosaeInoculating to MRS liquid culture medium with pH of 4.00, 3.00 and 2.00 according to 1.00%, anaerobically culturing at 37 deg.C for 24 h, using uninoculated MRS broth with corresponding pH as blank control, and counting viable bacteria of bacteria liquid by plate coating method to calculate survival rate. The results showed that Lactobacillus mucosaeLactobacillus mucosaeThe survival rates of MRS broth at pH 4.00, 3.00 and 2.00 were all above 80.00% (table 2), indicating that it has strong acid tolerance.

TABLE 2 Lactobacillus mucosaeLactobacillus mucosaeAcid resistance

pH value	Viable count (lgCFU/mL)	Survival rate (%)
			6.20	9.32	100.00
4.00	8.26	88.63
			3.00	7.88	84.55
2.00	7.78	83.48

3. Determination of the bile salt resistance

Activated lactobacillus mucosaeLactobacillus mucosaeInoculating to MRS broth containing 0.10%, 0.20% and 0.30% of pig bile salt according to the inoculation amount of 1.00%, culturing at 37 deg.C under anaerobic condition for 24 h, taking MRS broth without pig bile salt as blank control, performing viable bacteria counting on the bacterial liquid by using plate coating method, and calculating the survival rate. The results showed that Lactobacillus mucosaeLactobacillus mucosaeThe survival rate of MRS broth with the added amount of pig bile salt of 0.10%, 0.20% and 0.30% is more than 90.00% (Table 3), which shows that the meat has stronger bile salt resistance.

TABLE 3 Lactobacillus mucosaeLactobacillus mucosaeCapacity of resisting bile salt

Amount of added pig bile salt (%)	Viable count (lgCFU/mL)	Survival rate (%)
			0.00	9.32	100.00
0.10	8.92	95.70
			0.20	8.84	94.85
0.30	8.73	93.67

4. Bacteriostatic ability

Activated lactobacillus mucosaeLactobacillus mucosaeAnd C type Clostridium perfringens, centrifuging at 5000 × g at 4 deg.C for 10min, washing with PBS, resuspending the cell particles in MRS broth, adjusting to final concentration of 2 × 10 ⁷ CFU/mL. The two strain suspensions were mixed in equal volumes and incubated at 37 ℃ for 8h. And respectively coating the co-culture samples on LB agar and MRS agar for viable bacteria counting, and calculating the survival rate. The results show that Lactobacillus mucosaeLactobacillus mucosaeCan obviously inhibit the growth of clostridium perfringens type C bacteria (figure 7).

The above results all indicate that Lactobacillus mucosaeLactobacillus mucosaeHas good in vitro probiotic function.

Example 3 Lactobacillus mucosaeLactobacillus mucosaeApplication of clostridium perfringens C in preventing and treating pig IPEC-J2 cell damage

1. Different concentrations of Lactobacillus mucosaeLactobacillus mucosaeEffect on IPEC-J2 cell viability

The IPEC-J2 monolayer cells were rinsed 2 times with sterile PBS and then treated as follows: (1) control group: 2mL of DMEM/F12 culture solution is used for culturing IPEC-J2 cells; (2) treatment group: 10 ⁷ 、10 ⁸ 、10 ⁹ CFU/mL lactobacillus mucosaeLactobacillus mucosaeSeparately co-cultured with IPEC-J2. After treating 2h, 4h and 6h, respectively, the supernatants were aspirated into 10mL sterile centrifuge tubes and the cells were washed 3-5 times with sterile PBS followed by digestion with 300 μ L of 0.05% pancreatin-EDTA. After termination of digestion, the cells were resuspended in 500. Mu.L DMEM/F12 basal medium after centrifugation at 309 Xg for 5min, the supernatant discarded. IPEC-J2 cell viability was measured by the CCK-8 method, and the results are shown in FIG. 8. 10 ⁷ CFU/mL and 10 ⁸ Lactobacillus mucosae at CFU/ mL concentrationLactobacillus mucosaeAfter 2h, 4h and 6h of IPEC-J2 cell treatment, the cell viability is not obviously different from that of a control (1)P>0.05）。10 ⁹ Lactobacillus mucosae at CFU/mL concentrationLactobacillus mucosaeAfter 6h of treatment of IPEC-J2 cells, the cell viability is significantly reduced (P<0.01). Demonstration of Lactobacillus MucosaLactobacillus mucosaeA suitable concentration to promote IPEC-J2 cell activity is 10 ⁸ CFU/mL。

2. Lactobacillus mucosaeLactobacillus mucosaeEffect on the morphology of C-type Clostridium perfringens infected IPEC-J2 cells

Rinsing IPEC-J2 monolayer cells with sterile PBS for 2 times, and washing 10 ⁸ CFU/mLL. mucosaeCo-culturing with IPEC-J2 cells 2h, adding 10 ⁸ C.perfringens type CFU/mLC processed cells 4 h. The treatment comprises the following steps: (1) Control: DMEM/F-12 cell culture fluid; (2) LM:10 ⁸ CFU/mLL. mucosae；（3）CP：10 ⁸ CFU/mLCpC；（4）LM+CP：10 ⁸ CFU/mLL. mucosae+10 ⁸ CFU/mLCpC。

After 1h, 2h and 3h of treatment, the cells were washed 3 times with PBS to completely wash the non-adhered bacteria. After 15min fixation with methanol, dyeing is carried out for 30min with Giemsa dye liquor. The cells were washed 2 times with distilled water and observed for cell morphology using an inverted microscope. Each experiment was repeated 3 times. The results are shown in FIG. 9. 10 comparison with the normal cell morphology ⁸ Lactobacillus mucosae at CFU/mL concentrationLactobacillus mucosaeNo significant change occurred in the IPEC-J2 cells treated; 10 ⁸ Significant apoptosis and damage occurred to IPEC-J2 cells treated with C-type Clostridium perfringens at CFU/mL concentration; in LM + CP group, lactobacillus mucosaeLactobacillus mucosaeCan effectively relieve IPEC-J2 cell morphology damage caused by C type clostridium perfringens.

3. Lactobacillus mucosaeLactobacillus mucosaeEffect on the viability of C-type Clostridium perfringens infected IPEC-J2 cells

Test treatment As in example three 2, cells were collected and Lactobacillus mucosae was detected by CCK-8 methodLactobacillus mucosaeEffect on the viability of cells infected with IPEC-J2 by clostridium perfringens type C. The results are shown in FIG. 10. The results show that Lactobacillus mucosae strains compared to the Clostridium perfringens type C treated group aloneLactobacillus mucosae(ii) cell viability is significantly increased by co-processing cells with Clostridium perfringens type CP<0.01 Explain the Lactobacillus mucosaeLactobacillus mucosaeCan increase the cell activity of the C-type clostridium perfringens infected IPEC-J2 cells.

4. Lactobacillus mucosaeLactobacillus mucosaeEffect on C-type Clostridium perfringens infection IPEC-J2 cytokine expression

The experimental treatment is the same as example three 2, the cells are collected after the treatment is finished, the cells are washed for 3 times by sterile PBS, 1ml of LTrizol reagent is added into each hole of a 6-hole plate, the cells are cracked for 5min at normal temperature, the cells are completely transferred into a 1.5 mL RNase-free centrifugal tube by blowing, the cells are stored at the temperature of minus 80 ℃, the subsequent RNA extraction is carried out, and the cell factors are detectedIL-8、TNF-αAndIL-1βexpression of mRNA. Each experiment was repeated 3 times. The results are shown in FIG. 11, from which it can be seen that IPEC-J2 cytokine inflammatory factor is very significantly increased after 4h treatment of C-type C Clostridium perfringens cells compared to the control groupIL-8、TNF-αAndIL-1βexpression of (A), (B)P<0.01 ); lactobacillus mucosaeLactobacillus mucosaeThe cell is co-processed with the C type clostridium perfringens for 4h, so that inflammatory factors after the C type clostridium perfringens infects IPEC-J2 cells can be remarkably inhibitedIL-8AndIL-1βexpression of (A), (B)P<0.01）。

5. Lactobacillus mucosaeLactobacillus mucosaeEffect on C-type Clostridium perfringens infection of IPEC-J2 cell Tight connexin expression

Experimental treatment As in example III 2, after the treatment, the cells were collected and washed 3 times with sterile PBS, and 6-well plates were used for each wellAdding 1ml of Trizol reagent, cracking at normal temperature for 5min, blowing and completely transferring into a 1.5 mL non-RNA enzyme centrifugal tube, preserving at-80 ℃, performing subsequent RNA extraction, and detecting cell tight junction proteinOCLNAndCLDN1expression of mRNA. The test was repeated 3 times. The results are shown in FIG. 12, from which it can be seen that IPEC-J2 cells are significantly reduced after 4h treatment of C-type C Clostridium perfringens cells compared to the control groupOCLNAndCLDN1expression of (A), (B)P<0.05 ); lactobacillus mucosaeLactobacillus mucosaeThe cells are co-processed with the clostridium perfringens type C for 4h, so that the IPEC-J2 cells infected by the clostridium perfringens type C can be remarkably increasedOCLNAndCLDN1expression of (A), (B)P<0.05）。

Claims (2)

1. The lactobacillus mucosae strain is a lactobacillus mucosae strain preserved in China general microbiological culture Collection center at 7/6/2021Lactobacillus mucosae The preservation number is CGMCC No.22828.

2. Use of lactobacillus mucosae according to claim 1 for the preparation of a medicament for the control of clostridium perfringens type C infectious intestinal disease in pigs.

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