Lactobacillus reuteri SKLAN202402ZF and application thereof

Abstract

The invention relates to microorganisms, in particular to lactobacillus reuteri SKLAN202402ZF and application of the lactobacillus reuteri SKLAN202402ZF. The preservation number of the lactobacillus reuteri SKLAN202402ZF is CGMCC (China General Microbiological Culture Collection Center) No.30023, and the lactobacillus reuteri SKLAN202402ZF can be used for preparing the lactobacillus reuteri. Through screening and identification, the strain has excellent antibacterial and immune regulation functions. By adding the strain into piglet feed, the intestinal health of piglets can be remarkably improved, the diarrhea occurrence rate is reduced, and the feed conversion rate and the growth speed are increased. The application of the strain not only helps to improve pig breeding benefits, but also conforms to the trend of antibiotic-free breeding and green breeding.

Description

Technical Field

[0001] The present invention relates to microorganisms, specifically Lactobacillus reuteri SKLAN202402ZF and applications thereof. Background Art

[0002] Antibiotics are commonly used drugs in production to prevent and treat diarrhea in piglets. However, long-term and widespread use of antibiotics not only leads to increased resistance of pathogens, but also may remain in animal products, posing a potential threat to human health. Antibiotic exposure can also cause intestinal damage and intestinal flora disorders in piglets, forming long-term chronic inflammation, affecting nutrient absorption and piglet growth.

[0003] Lactic acid bacteria, especially Lactobacillus reuteri ( Lactobacillus reuteri ), as a common probiotic, has received extensive attention in animal husbandry in recent years. Lactobacillus reuteri has multiple biological functions, such as inhibiting pathogens, promoting the growth of beneficial bacteria, enhancing immune function and improving intestinal microecological balance. Studies have shown that using specific lactic acid bacteria strains as feed additives can effectively reduce the incidence of diarrhea in piglets and improve the survival rate and growth performance of piglets. Summary of the invention

[0004] The present invention aims to provide a strain of Lactobacillus reuteri ( Lactobacillus reuteri )SKLAN202402ZF.

[0005] Another object of the present invention is to provide the above-mentioned Lactobacillus reuteri ( Lactobacillus reuteri )Application of SKLAN202402ZF.

[0006] Another object of the present invention is to provide a biological bacterial agent.

[0007] Another object of the present invention is to provide a feed additive.

[0008] Lactobacillus reuteri of the present invention ( Lactobacillus reuteri ) SKLAN202402ZF, its deposit number is CGMCC No.30023.

[0009] The present invention provides a method containing Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF. The bacterial agent can be a liquid bacterial agent or a solid bacterial agent, and can be prepared by conventional technical means and adding auxiliary materials allowed in the field of microbial preparations.

[0010] The present invention provides Lactobacillus reuteri ( Lactobacillus reuteri ) Application of SKLAN202402ZF for preparing the following preparations: Preparations that inhibit pathogenic microorganisms; Preparations for promoting animal growth; Preparations to improve intestinal immunity; Preparations to repair intestinal damage; Or preparations that relieve intestinal inflammation.

[0011] Preferably, the pathogen is a bacterium, more preferably a Gram-negative bacterium.

[0012] As an embodiment of the present invention, the pathogenic microorganisms are Escherichia coli and Salmonella.

[0013] According to the animal feed additive of the present invention, it contains the above-mentioned Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF or Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF was prepared.

[0014] The above-mentioned animal feed additive or animal feed can be made from Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF is prepared, specifically, from Lactobacillus reuteri ( Lactobacillus reuteri )SKLAN202402ZF obtained by fermentation.

[0015] The beneficial effects of the present invention are: The invention provides a novel Lactobacillus reuteri SKLAN202402ZF, which has excellent antibacterial and immunomodulatory functions after screening and identification. By adding the strain to piglet feed, the intestinal health of piglets can be significantly improved, the incidence of diarrhea can be reduced, and the feed conversion rate and growth rate can be increased. The application of the strain not only helps to improve the efficiency of pig farming, but also conforms to the trend of antibiotic-free farming and green farming.

[0016] Compared with the existing Lactobacillus reuteri, the strain of the present application has the following beneficial properties: 1. Specificity of the inhibitory effect on pathogenic bacteria: Although there are reports in the prior art that Lactobacillus reuteri inhibits pathogenic bacteria and regulates immune function, the SKLAN202402ZF strain of the present application shows a significant inhibitory effect on Salmonella and Escherichia coli, especially in the application in animals, and can effectively prevent the growth and colonization of these two pathogens in the body; 2. Relief of intestinal inflammation after antibiotic exposure: The test results show that oral administration of Lactobacillus reuteri SKLAN202402ZF can significantly reduce the susceptibility of mice after antibiotic exposure. Therefore, the SKLAN202402ZF strain of the present application has a significant effect in alleviating intestinal inflammation caused by antibiotic exposure, which makes this strain have a unique advantage in the application scenario of antibiotic replacement and can effectively reduce the side effects caused by long-term use of antibiotics. 3. Repair and enhancement of intestinal barrier function: After oral administration of SKLAN202402ZF, the mRNA expression levels of ZO-1 and Occludin in the mouse colon were significantly higher than those in the LC group ( p < 0.05), indicating that oral administration of Lactobacillus reuteri SKLAN202402ZF can significantly improve the intestinal barrier damage caused by antibiotic exposure, help restore the integrity of the intestinal mucosal barrier, thereby protecting animals from pathogens and promoting intestinal health; 4. Tolerance and colonization ability: SKLAN202402ZF strain has strong tolerance to artificial bile salts, artificial gastric juice, and artificial intestinal juice, and can survive in complex digestive environments and colonize in animals. This feature significantly enhances the application potential of the strain, especially in the actual feeding process, the strain can play a role for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Lactobacillus reuteri of the present invention ( Lactobacillus reuteri ) Growth curve of SKLAN202402ZF; Figure 2A Lactobacillus reuteri of the present invention ( Lactobacillus reuteri ) Oil immersion microscopy image of SKLAN202402ZF after malachite green staining; Figure 2B Lactobacillus reuteri of the present invention ( Lactobacillus reuteri ) Colony morphology of SKLAN202402ZF after culture on MRS agar medium; Figure 3 This is a graph showing the weight changes of mice in the experiment in Example 3 of the present invention; Figure 4 The paraffin sections of the colon of mice in Example 3 of the present invention, wherein A: HE staining image and B: mouse colon image; Figure 5 The colon changes of mice in Example 3 of the present invention, wherein A: colon length, B: colon paraffin section inflammation score, C: colon paraffin section intestinal epithelial injury score; Figure 6 The relative mRNA expression levels of the intestinal barrier genes ZO-1 and Occludin and the inflammatory factors in mice in Example 3 of the present invention are TNF-α , IL-1β , IL-18 , IL17 and IL-23 Relative mRNA expression level; Figure 7 This is a graph showing the weight change of piglets in Example 4 of the present invention; Figure 8 It is the diarrhea rate of the piglet test in Example 4 of the present invention.

[0018] Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF, deposited on March 13, 2024 in the General Microbiology Center of China Microorganism Culture Collection Administration (referred to as CGMCC, address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postal Code 100101), and classified as Lactobacillus reuteri Lactobacillus reuteri , the deposit number is CGMCC No.30023. DETAILED DESCRIPTION

[0019] The technical scheme of the present invention is described clearly and completely below in conjunction with specific embodiments. Lactobacillus reuteri ) strain SKLAN202402ZF, all reagents and raw materials used were commercially available. Example 1 Isolation and identification of Lactobacillus reuteri SKLAN202402ZF strain

[0020] Lactobacillus reuteri ( Lactobacillus reuteri ) Isolation and identification of SKLAN202402ZF: Isolated from the colonic digesta of healthy Duroc×Chang×Day piglets at 28 days old.

[0021] (1) Colonic chyme collection: Ten healthy and strong 28-day-old piglets of Duroc×Chang×Da were selected and their colons were isolated. The intestinal segments were opened and 1 g of colonic chyme was collected from each piglet. The chyme was added to 20 mL of sterile saline containing 20% ​​glycerol and vortexed to mix. The chyme was then dispensed into sterile cryovials, 1.5 mL per tube. The chyme was quickly frozen in liquid nitrogen and stored in a -80°C refrigerator.

[0022] (2) Strain isolation: Take 1 mL of the piglet colon chyme preserved in step (1), dilute it 10-fold with sterile saline, and evenly spread 0.1 mL of the dilution on MRS agar medium. Incubate at 37°C for 48 hours. Inoculate the grown colonies onto new MRS agar medium by streaking and purify them for 48 hours.

[0023] (3) Strain enrichment: The purified strain was inoculated into sterile MRS broth medium using a sterile inoculation loop and placed in a shaker. After culturing at 37°C for 48 hours, 16S rDNA identification was performed and the remaining portion was diluted with physiological saline containing 20% ​​glycerol and stored in a -80°C refrigerator.

[0024] (4) 16S rDNA identification: The enriched bacterial solution was subjected to colony PCR amplification using bacterial universal primers, and then 16S rDNA sequencing was performed for identification. The 16S rDNA sequence of each strain was compared with the 16S rDNA sequences of all the determined bacteria in the database on NCBI. Based on the sequencing results, microbiological characteristics, and physiological and biochemical properties, 8 strains were identified as Lactobacillus reuteri. The bacteria selected in the present invention had a similarity of 99.93% with Lactobacillus reuteri and were identified as Lactobacillus reuteri. Example 2 Detection of characteristics of Lactobacillus reuteri SKLAN202402ZF 1. Growth Curve Determination

[0025] Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF was cultured until the concentration reached 4.1×10 8 CFU / mL aspirate the bacterial solution and inoculate it into MRS broth medium at 1% of the medium, and place it in a shaker at 37°C. Use a microplate reader to measure the OD value of the medium every hour. 600 The value of Figure 1 As shown, the results showed that Lactobacillus reuteri ( Lactobacillus reuteri ) The SKLAN202402ZF strain reached the logarithmic growth phase after 9 hours and entered the plateau phase after 15 hours, with the maximum number of viable cells reaching 4.1×10 8 CFU / mL. 2. Morphology and Colony Observation

[0026] Smear the bacteria on a glass slide and dry it, then stain it with 1% malachite green for 3 minutes. After staining, slowly rinse it with deionized water to remove excess staining solution, dry the slide, and observe and take pictures under an oil lens. Lactobacillus reuteri ) SKLAN202402ZF shape Figure 2A As shown, the colony morphology after culture on MRS agar medium is as follows Figure 2B shown.

[0027] The microbiological characteristics of Lactobacillus reuteri SKLAN202402ZF are as follows: Colony morphology: The surface of the colony is smooth, shiny and moist. The colony is round with smooth and neat edges, milky white and slightly sour.

[0028] After staining, Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF bacteria are rod-shaped, non-motile, and have no flagella.

[0029] Growth characteristics: In MRS broth medium, placed in a 37°C shaker culture, it enters the logarithmic growth phase after 9 hours and enters the plateau phase after 12 hours. The maximum number of viable bacteria is 4.1×10 8 CFU / mL. 3. Antibacterial ability test

[0030] Two common pathogenic bacteria were selected as pathogen indicator bacteria: Escherichia coli ( Escherichia coli ) BW25113 and Salmonella typhimurium ( Salmonella typhimurium )ATCC14028.

[0031] The antibacterial activity of the strains was determined by the Oxford cup method. Escherichia coli ) BW25113 and Salmonella typhimurium ( Salmonella typhimurium )ATCC14028 were inoculated into LB broth medium and cultured in a 37°C incubator for 12-18 hours. Escherichia coli ) BW25113 and Salmonella typhimurium ( Salmonella typhimurium )ATCC14028 strain concentration was adjusted to 1×10 8 CFU / mL, take 200μl of each, evenly spread on LB agar medium, put the sterilized Oxford cup evenly on the LB medium, add 100μl of activated bacterial solution with a concentration of 4.1×10 8 CFU / mL of bacterial solution with MRS broth. Place in a 37°C incubator for 12 hours. Use a vernier caliper to measure the size of the inhibition zone and evaluate the antibacterial effect. The results show (Table 1) that compared with the other 7 lactobacilli, Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF can better inhibit the growth of Salmonella and Escherichia coli.

[0032] Table 1 The inhibitory ability of Lactobacillus reuteri against pathogens Strain number salmonella Escherichia coli SKLAN202409ZF 0.19 0.22 SKLAN202412ZF 0.1 0.31 SKLAN202413ZF 0.08 0.25 SKLAN202414ZF 0.12 0.19 SKLAN202402ZF 0.20 0.30 SKLAN202429ZF 0.18 0.24 SKLAN202432ZF 0.03 0.13 SKLAN202420ZF 0.17 0.20 Note: The above values ​​are the diameters of the inhibition zones, in cm. 4. Bile salt tolerance test

[0033] Take 100 uL of enriched culture of Lactobacillus reuteri ( Lactobacillus reuteri) SKLAN202402ZF bacterial solution (concentration of 4.1×10 8 CFU / mL) was added to 900 uL of 0.3% bile salt solution, incubated in a 37°C incubator for 1 hour, and 100 uL of bacterial solution was spread on a plate and counted. The results are shown in Table 2. Compared with other Lactobacillus reuteri, Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF has stronger bile salt tolerance, with a survival rate of 13.0% in 0.3% bile salt.

[0034] Table 2 Bile salt tolerance of Lactobacillus reuteri Strain number 0.3% bile salt survival rate SKLAN202409ZF 2.39 SKLAN202412ZF 6.12 SKLAN202413ZF 3.01 SKLAN202414ZF 1.63 SKLAN202402ZF 13.0 SKLAN202429ZF 2.33 SKLAN202432ZF 0.48 SKLAN202420ZF 3.77 . 5. Artificial gastric juice resistance test

[0035] Take 10uL of enriched culture of Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF bacterial solution (concentration of 4.1×10 8 CFU / mL) was added to 990uL of artificial gastric juice with a pH value of 1.2, and cultured in a 37°C incubator for 2.5 hours. 100uL of the bacterial solution was spread on a plate and counted. The results are shown in Table 3. In artificial gastric juice with a pH value of 1.2, Lactobacillus reuteri ( Lactobacillus reuteri ) The survival rates of SKLAN202402ZF, SKLAN202412 and SKLAN202413 were higher than those of the other five lactobacilli strains.

[0036] Table 3 Resistance of Lactobacillus reuteri to artificial gastric juice Strain number Survival rate in artificial gastric juice SKLAN202409ZF 9.65 SKLAN202412ZF 75.34 SKLAN202413ZF 50.12 SKLAN202414ZF 9.93 SKLAN202402ZF 31.47 SKLAN202429ZF 4.04 SKLAN202432ZF 15.6 SKLAN202420ZF 10.81 . 6. Artificial intestinal fluid resistance test

[0037] Take 10uL of enriched culture of Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF bacterial solution (concentration of 4.1×10 8 CFU / mL) were added to 990uL of artificial intestinal fluid with a pH value of 4, and cultured in a 37°C incubator for 4 hours. 100uL of the bacterial solution was spread on a plate and counted. The results are shown in Table 4. After 4 hours in artificial intestinal fluid, Lactobacillus reuteri ( Lactobacillus reuteri ) The survival rate of SKLAN202402ZF was higher than that of the other 7 strains, reaching 79.7%.

[0038] Table 4 Resistance of Lactobacillus reuteri to artificial intestinal fluid Strain number Survival rate in artificial intestinal fluid SKLAN202409ZF 71.83 SKLAN202412ZF 16.38 SKLAN202413ZF 73.52 SKLAN202414ZF 31.96 SKLAN202402ZF 79.7 SKLAN202429ZF 68.81 SKLAN202432ZF 14.68 SKLAN202420ZF 73.95 . Example 3 Effects of Lactobacillus reuteri SKLAN202402ZF on intestinal damage and inflammation in antibiotic-exposed mice

[0039] In this study, Lactobacillus reuteri ( Lactobacillus reuteri ) The concentration of SKLAN202402ZF bacterial solution is 4.1×10 8 CFU / mL. 1. Test methods

[0040] Forty-eight 21-day-old female ICR mice were randomly divided into four groups: control group (CON), intestinal inflammation group (LPS), antibiotic group (LC), antibiotic plus Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF group (LC+ L.r LC and LC+ L.r The group was fed with water containing 1 g / L lincomycin every day, LC+ L.r The mice in the control group were gavaged with 0.2 mL of Lactobacillus reuteri solution every day, and the mice in the other groups were gavaged with an equal amount of saline. L.r The mice in the control group were injected with LPS, and the injection amount was calculated based on 10 mg / kg of the mouse body weight. The control group was injected with an equal amount of normal saline. Samples were collected 24 hours after LPS injection. The mice ate and drank water normally during the experiment. The mice were observed for one week, and the diarrhea of ​​the mice was recorded every day. The weight of the mice was recorded at the beginning and end of the experiment. 2. Test results (1) Changes in mouse weight

[0041] The weight changes of mice during the whole experimental period were as follows Figure 3 As shown in Figure 2, on day 21 of the experiment, the body weight of mice gavaged with Lactobacillus reuteri SKLAN202402ZF was higher than that of the antibiotic exposure group, but it did not reach a significant level. 24 hours after LPS injection, on day 22 of the experiment, LC+ L.r The body weight of mice in the LPS and LC groups was significantly higher than that in the CON group ( p <0.05). This indicates that oral administration of Lactobacillus reuteri SKLAN202402ZF can significantly reduce the susceptibility of mice after antibiotic exposure. (2) Colon length

[0042] like Figure 4 Figure B and Figure 5 As shown in Figure A, LC + L.r The colon length of mice in the CON group was significantly higher than that in the LC group ( p<0.05). This indicates that oral administration of Lactobacillus reuteri SKLAN202402ZF can improve the shortening of colon length in mice caused by antibiotic exposure. (3) Colon morphology and case scoring

[0043] like Figure 4 As shown in Figure A, the colon of mice in the LC group showed severe inflammatory cell infiltration and epithelial damage. + L.r The colonic inflammation scores and epithelial injury scores of mice in the LPS and LPS groups were significantly lower than those in the LC group, but higher than the CON group ( p <0.05, Figure 5 Figure B and Figure 5 Figure C in Figure 3). This indicates that oral administration of Lactobacillus reuteri SKLAN202402ZF can significantly reduce intestinal inflammation and intestinal damage in antibiotic-exposed mice. (4) Intestinal barrier and inflammatory factors

[0044] like Figure 6 As shown, LC + L.r The mRNA expression levels of ZO-1 and Occludin in the colon of mice in group A were significantly higher than those in group B ( p <0.05), indicating that oral administration of Lactobacillus reuteri SKLAN202402ZF can significantly improve the intestinal barrier damage caused by antibiotic exposure. + L.r Colonic inflammatory factors TNF-α , IL-1β and IL-18 The mRNA expression level of p <0.05), indicating that oral administration of Lactobacillus reuteri SKLAN202402ZF can significantly improve intestinal inflammation caused by antibiotic exposure. Example 4 Effects of Lactobacillus reuteri SKLAN202402ZF on Growth Performance and Diarrhea Rate in Piglets 1. Test methods

[0045] Sixteen 21-day-old weaned piglets were randomly divided into two groups: control group (CON) and Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF group ( L.r ). L.r Piglets in group A were fed with 4.1×10 8CFU / kg of feed was fed for 17 consecutive days, and the control group was fed with basic feed. During the experimental period, the piglets were fed according to the routine feeding and management procedures of the pig farm, and they were free to eat and drink water, and were immunized according to the immunization procedures of the pig farm. The weight of the piglets was recorded at the beginning and end of the experiment. 2. Test results (1) Piglet weight changes

[0046] like Figure 7 As shown in the figure, 7 and 14 days after feeding Lactobacillus reuteri SKLAN202402ZF, the body weight of piglets was higher than that of the control group, but it did not reach a significant level. L.r The weight of piglets in group A was significantly higher than that in group B, indicating that Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF can promote the growth of piglets ( p <0.05). (2) Diarrhea rate

[0047] like Figure 8 As shown, during the entire test period L.r The diarrhea rate of piglets in the group was significantly lower than that in the control group. Lactobacillus reuteri ) SKLAN202402ZF can prevent diarrhea in piglets caused by weaning stress ( p <0.05).

[0048] The above descriptions are only some specific implementation methods of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by any person familiar with the art within the technical scope disclosed in the present invention should be covered within the protection scope of the present invention.

Claims

1. Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF, characterized in that: The Lactobacillus reuteri ( Lactobacillus reuteri ) The deposit number of SKLAN202402ZF is CGMCC No.30023.

2. A method comprising the Lactobacillus reuteri of claim 1 ( Lactobacillus reuteri ) Microbial agent of SKLAN202402ZF.

3. The microbial agent according to claim 2, characterized in that: The microbial agent is a liquid microbial agent or a solid microbial agent.

4. The Lactobacillus reuteri of claim 1 ( Lactobacillus reuteri ) Application of SKLAN202402ZF for preparing the following preparations: Preparations that inhibit pathogenic microorganisms; Preparations for promoting animal growth Preparations for treating inflammation of the colon; Preparations that reduce the rate of diarrhea; or Preparations that improve intestinal immunity.

5. The use according to claim 4, characterized in that: The pathogenic microorganisms are bacteria.

6. The use according to claim 5, characterized in that: The pathogenic microorganism is a Gram-negative bacterium.

7. The use according to claim 6, characterized in that: The pathogenic microorganism is Escherichia coli or Salmonella.

8. An animal feed additive, characterized in that: Containing the Lactobacillus reuteri ( Lactobacillus reuteri ) SKLAN202402ZF, or the Lactobacillus reuteri of claim 1 ( Lactobacillus reuteri ) SKLAN202402ZF is prepared by fermentation.

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