CN113512516A

CN113512516A - Cooperative swine-origin lactobacillus mucosae and application thereof

Info

Publication number: CN113512516A
Application number: CN202110927936.9A
Authority: CN
Inventors: 杨巧丽; 张生伟; 裴利君; 高小莉; 王鹏飞; 滚双宝; 马艳萍
Original assignee: Gansu Farmer Education And Training Station Gansu Agricultural Radio And Television School; Gansu Agricultural University
Current assignee: Gansu Farmer Education And Training Station Gansu Agricultural Radio And Television School; Gansu Agricultural University
Priority date: 2021-08-13
Filing date: 2021-08-13
Publication date: 2021-10-19
Anticipated expiration: 2041-08-13
Also published as: CN113512516B

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 of age, and has the characteristics of acute morbidity and short course of disease, 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 antibiotics for prevention and treatment and the like, the problems of pathogenic bacteria drug resistance enhancement, antibiotic residue and the like occur when the antibiotics are used for a long time. 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 have made the control 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 plays an important role in cell adhesion, carbohydrate metabolism for acid production, pathogenic bacteria antagonism and the like, and can influence the integrity and the function of intestinal barriers through tight junction protein dephosphorylation, actin regulation and the like. It is also found that lactobacillus mucosae can adhere to intestinal epithelial tissue to form 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 mucosae NK41 and Bifidobacterium longum (B.) (Bifidobacterium longum) Synergistically, by inhibiting Nuclear Factor kappa-B (NF-kappa B) protein activation, Tumor 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 number MZ 047317.

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 clostridium perfringens type C.

The lactobacillus mucosaeLactobacillus mucosaeThe number of viable bacteria is 10⁷CFU/mL and 10⁸The CFU/mL does not have obvious influence on the cell morphology and the survival rate of IPEC-J2, and can effectively relieve IPEC-J2 cell morphology damage caused by C-type clostridium perfringens infection and obviously increase the activity of IPEC-J2 cells after C-type clostridium perfringens infection.

The lactobacillus mucosaeLactobacillus mucosaeCan obviously inhibit the increase of the expression of inflammatory factors after the C-type clostridium perfringens is infected with IPEC-J2 cells and obviously increase the reduction of the expression of the C-type clostridium perfringens infected with IPEC-J2 cell claudin.

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 the C-type clostridium perfringens, and can be used for preventing and treating pig bacterial infectious diarrhea diseases.

Drawings

FIG. l 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-culture is lactobacillus mucosaeLactobacillus mucosaeViable count in coculture 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 cells infected with IPEC-J2 by clostridium perfringens type C;

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 mucosaeFor clostridium perfringens type CEffects of infection of IPEC-J2 cells on expression of claudin.

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^-53 gradients. 70.00 mu L of different concentrations of fecal dilutions were applied to CaCO containing vancomycin (filter sterilized, final concentration 2.00 mg/mL)₃MRS plates, anaerobic culture at 37 ℃ for 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: 10.0 g/L of peptone, 5.0 g/L of beef extract, 20.0 g/L of glucose, 2.0 g/L of dipotassium phosphate, 2.0 g/L of ammonium citrate tribasic, 5.0 g/L of sodium acetate, 0.2 g/L of magnesium sulfate, 0.05 g/L of manganese sulfate, 801.0 g/L of Tween-agar, 15.0 g/L, CaCO of agar₃15.0 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 strains were inoculated into MRS broth and shake-cultured at 37 ℃ for 24 hours. 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 of the product, heating and dissolving in 1L of distilled water, autoclaving at 121 ℃ for 15min, and adjusting to a final pH of 6.2 +/-0.1.

(4) 16S rDNA molecular identification

The selected strains were 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 the fragment size was 1261bp by PCR amplification and detection by 1% agarose gel electrophoresis (FIG. 4). The PCR reaction program is: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30 s; annealing at 58 ℃ for 30 s, extending at 72 ℃ for 2 min, and performing 35 cycles; extension at 72 ℃ for 10 min.

Sequencing the PCR product with Yangtianluoae Oako Biotechnology GmbH, comparing the obtained sequence with the standard strain in NCBI database by BLAST, constructing phylogenetic tree according to the reference sequence, and separating strain 2 from strain 2Lactobacillus mucosae LM011 (NZ _ CP 062966.1) has the closest relationship, sequence coverage of 100%, homology of 99% of (FIG. 5), isolate 2 was determined to be a species or variant of Lactobacillus mucosae, designatedLactobacillus mucosae LM410。

Lactobacillus mucosaeLactobacillus mucosaeThe 16S rDNA gene sequence of (2) was submitted to the NCBI database, GenBank number MZ 047317.

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 1.00% of the inoculum size into MRS broth, culturing at 37 deg.C for 24 hr in a shaker, sampling every 2 hr, and determining the absorbance (OD) of the culture medium₆₀₀) And pH value, and drawing a growth curve and an acid production curve by taking the culture time as an abscissa and the light absorption value and the pH value as an ordinate respectively. The result shows that the growth is relatively slow in 0-8 h, the logarithmic growth phase is 8-20 h, and the stationary phase and the decline phase are started after 20 h; the pH value of the MRS broth is gradually reduced in the culture process, and the pH value of the MRS broth is reduced to be below 4.50 and tends to be stable after the MRS broth is cultured for 22 hours (figure 6), which shows that the MRS broth has good acid production performance.

2. Acid resistance measurement

Using 6.00 mol.L^-1Hydrochloric 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, performing viable bacteria count on the bacteria liquid by plate coating method, and calculating 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 acid resistance of Lactobacillus mucosae

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, counting viable bacteria of the bacteria liquid by plate coating method with MRS broth without pig bile salt as blank control, and calculating survival rate. The results showed that Lactobacillus mucosaeLactobacillus mucosaeThe 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% (table 3), which indicates that the pig bile salt has stronger bile salt resistance.

TABLE 3 Lactobacillus mucosae bile salt resistance

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 × 10⁷CFU/mL. The two strain suspensions were mixed in equal volumes and incubated at 37 ℃ for 8 h. And respectively coating the co-culture samples on LB agar and MRS agar for viable bacteria counting, and calculating the survival rate. The results showed 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 preventing and treating pig IPEC-J2 cell injury caused by C-type clostridium perfringens

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

After rinsing 2 times the IPEC-J2 monolayer cells with sterile PBS, the following treatments were performed: (1) control group: 2mL of DMEM/F12 culture solution is used for culturing IPEC-J2 cells; (2) treatment group: 10⁷、10⁸、10⁹CFU/mL lactobacillus mucosaeLactobacillus mucosaeSeparately co-cultured with IPEC-J2. After 2h, 4h and 6h of treatment, 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. The CCK-8 method is adopted to detect the activity of IPEC-J2 cells, and the result is shown in figure 8. 10⁷CFU/mL and 10⁸Lactobacillus mucosae at CFU/

mL concentrationLactobacillus mucosaeAfter

2h, 4h and 6h of IPEC-J2 cell treatment, the cell viability is not significantly different from that of the control (the cell viability is not different from that of the control: (P>0.05）。10⁹CFU/mLConcentration of Lactobacillus mucosaeLactobacillus mucosaeAfter 6h of treatment of IPEC-J2 cells, cell viability was significantly reduced (P<0.01). Demonstration of Lactobacillus MucosaLactobacillus mucosaeA suitable concentration for promoting IPEC-J2 cell activity is 10⁸CFU/mL。

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

After rinsing IPEC-J2 monolayer cells with sterile PBS 2 times, 10 cells were washed⁸CFU/mLL. mucosaeCo-culturing with IPEC-J2 cells for 2h, adding 10⁸The cells were processed for 4h with C.perfringens type CFU/mLC. The treatment comprises the following steps: (1) control: DMEM/F-12 cell culture fluid; (2) LM: 10⁸ CFU/mL L. mucosae；（3）CP：10⁸ CFU/mL CpC；（4）LM+CP：10⁸CFU/mL L. mucosae+10⁸ CFU/mL CpC。

After treatment for 1h, 2h and 3h, the cells were washed 3 times with PBS, and the non-adhered cells were washed off completely. 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 then 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 morphology⁸Lactobacillus mucosae at CFU/mL concentrationLactobacillus mucosaeNo significant change occurred in the IPEC-J2 cells treated; 10⁸Significant 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 mucosaeWith Clostridium perfringens type C bacteriaThe cell activity can be obviously increased by treating the cells together (P<0.01), indicating Lactobacillus mucosaeLactobacillus mucosaeCan increase the cell activity of the clostridium perfringens type C after infecting IPEC-J2 cells.

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

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 at normal temperature for 5min, the cells are completely transferred into a 1.5 mL RNA-free enzyme 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 4h after C-type C Clostridium perfringens treatment of the cells, IPEC-J2 cytokine is very significantly increased compared to the control groupIL-8、TNF-αAndIL-1βexpression of (A), (B)P<0.01); lactobacillus mucosaeLactobacillus mucosaeThe cell is co-processed with the clostridium perfringens type C for 4h, so that inflammatory factors after the clostridium perfringens type C infects IPEC-J2 cells can be obviously inhibitedIL-8AndIL-1βexpression of (A), (B)P<0.01）。

5. Lactobacillus mucosaeLactobacillus mucosaeEffect on the expression of the claudin by C-type Clostridium perfringens infected IPEC-J2 cells

Test treatment the same as example three 2, collecting cells after treatment, washing 3 times with sterile PBS, adding 1mL of Trizol reagent into each hole of 6-hole plate, splitting at normal temperature for 5min, blowing and transferring completely into 1.5 mL of RNA-free enzyme centrifugal tube, preserving at-80 ℃, performing subsequent RNA extraction, detecting cell tight junction proteinOCLNAndCLDN1expression of mRNA. The experiment was repeated 3 times. The results are shown in FIG. 12, from which it can be seen that IPEC-J2 cells were significantly reduced 4h after C-type C Clostridium perfringens treatment 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

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 preventing and treating pig intestinal epithelial cell IPEC-J2 injury caused by clostridium perfringens type C infection.