Strain for preventing diarrhea of weaned piglets and application thereof

Abstract

The invention discloses a strain for preventing diarrhea of weaned piglets and application of the strain, relates to the field of animal husbandry, and aims to solve the problem of frequent weaning diarrhea of piglets in the current pig raising industry. The enterococcus monteilii PL86 disclosed by the invention is currently preserved in the China Center for Type Culture Collection, the preservation address is Luojia Mountain, Luojia Mountain, eight road, Wuchang District, Wuhan City, Hubei Province, the preservation date is July 10, 2023, and the preservation number is CCTCC NO: M 20231246. On the basis of research on the occurrence mechanism of piglet weaning diarrhea, main dominant microorganisms in excrement of weaning piglets are separated and identified, biological characteristic research is carried out on the dominant microorganisms, a scientific theoretical basis is provided for production of probiotic preparations capable of preventing weaning piglet diarrhea, and the beneficial effects of reducing use of drugs in the animal breeding process and improving the production efficiency are achieved. And the production of functional animal products has great significance in protecting human health.

Description

Technical Field

[0001] The invention relates to the field of animal husbandry, and in particular to a strain capable of preventing diarrhea in weaned piglets and an application thereof. Background Art

[0002] Piglet diarrhea is a common disease in large pig farms in my country, with a high incidence and mortality rate, causing serious economic losses to the pig farming industry. Intestinal flora can promote intestinal development, enhance digestion and absorption of nutrients, and regulate the body's immune function. The intestinal microecology and immune system of lactating piglets are not yet perfect, and they are easily affected by pathogens, which can easily cause diarrhea. In 2019, my country issued relevant regulations: All feed production companies are prohibited from producing and selling commercial feed containing growth-promoting additives (except traditional Chinese medicine) from July 1, 2020, which poses a severe challenge to the survival and development of feed production companies. At present, the livestock and poultry farming industry is faced with the search for new antibiotic alternatives to adjust the balance of intestinal flora and inhibit the occurrence of intestinal inflammation, and the development of highly efficient disease-resistant probiotic strains has always been the focus of attention in the breeding industry.

[0003] In practical production, piglet diarrhea is the most common and dangerous disease in my country's pig farming industry. In fact, the causes of piglet diarrhea include feeding system, breeding environment, infectious diseases and eating habits. The occurrence of diarrhea is also related to the time after birth. The incidence of diarrhea in piglets within 0 to 7 days after birth is 0.6%. When it is 8 to 13 days after birth, the incidence of diarrhea suddenly increases to 32%. The incidence rate is as high as 41.4% from 14 to 17 days. On the contrary, the incidence rate drops to 8.4% within 22 to 28 days, and the mortality rate rises sharply to 20 to 30%. The diarrhea of ​​piglets not only inhibits their own growth and development, but also causes the death of piglets when the diarrhea is severe. Previous studies have found that even if piglets rely on their own immune tolerance, there is a great possibility of stunted development and thus become dead pigs. In general, the disease of piglet diarrhea has seriously troubled pig farmers and caused significant economic losses to my country's pig farming industry.

[0004] Lactic acid bacteria is one of the most commonly used probiotics. It can adjust the structure of intestinal flora, reduce the infection of pathogenic bacteria in the intestine, thereby improving immune function. It can also improve the diarrhea of ​​weaned piglets caused by pathogenic bacteria to a certain extent. Excellent lactic acid bacteria strains are also being developed continuously. Lactic acid bacteria can be used alone or with feed to feed animals to prevent and treat diarrhea in weaned piglets. It has a low cost and can be used as a substitute for antibiotics when widely used with feed. It has a great advantage in preventing diarrhea in weaned piglets. Summary of the invention

[0005] The purpose of the present invention is to solve the problem of frequent diarrhea in weaned piglets in the current pig farming industry, and to provide a strain for preventing diarrhea in weaned piglets and its application.

[0006] A strain for preventing diarrhea in weaned piglets of the present invention is Enterococcus mundtii PL86, which is deposited in the China Center for Type Culture Collection, with the deposit address being Luojia Mountain, Bayi Road, Wuchang District, Wu'an City, Hubei Province, the deposit date being July 10, 2023, and the deposit number being CCTCC NO:M 20231246.

[0007] The present application discloses an application of a strain for preventing diarrhea in weaned piglets, which uses Enterococcus mundtii PL86 to prevent the occurrence of diarrhea in weaned piglets.

[0008] Furthermore, the prevention of diarrhea in weaned piglets refers to reducing the incidence of diarrhea under E. coli invasion after feeding the animals.

[0009] The present application discloses a strain for preventing diarrhea in weaned piglets, which is Enterococcus mundtii PL86 and has no toxic effect on animals.

[0010] Furthermore, the "no toxic effect" means that after daily feeding of animals with Enterococcus mundtii PL86, there is no significant change in body weight, and the animals are in good physical and mental condition.

[0011] The invention discloses a strain capable of preventing diarrhea in weaned piglets. The strain is Enterococcus mundtii PL86, which can inhibit the number of diarrhea-causing bacteria in the intestines of animals and increase the content of lactic acid bacteria, thereby preventing diarrhea in piglets.

[0012] Furthermore, the intestinal diarrhea-causing bacteria is pathogenic Escherichia coli ATCC 25922.

[0013] The present invention has the following beneficial effects:

[0014] The Enterococcus mundtii PL86 of the present invention has good biological characteristics in vitro culture, has the strongest ability to produce protease and amylase, can effectively inhibit the growth of pathogenic Escherichia coli, and daily feeding of mice does not affect the weight and food intake of mice. The mice are in good physical and mental conditions and have no obvious toxic effects. The content of lactic acid bacteria in the feces of mice increases during the feeding period, and the growth of pathogenic Escherichia coli can be inhibited after the pathogenic Escherichia coli is attacked, and the content of Escherichia coli in the feces is significantly reduced. No diarrhea occurs during the test period.

[0015] The present invention is based on the study of the occurrence mechanism of diarrhea in weaned piglets, separates and identifies the main dominant microorganisms in the feces of weaned piglets, and studies the biological characteristics of the dominant microorganisms, providing a scientific theoretical basis for the production of probiotic preparations that can prevent diarrhea in weaned piglets, and is of great significance for reducing the use of drugs in the animal breeding process, producing functional livestock products, and protecting human health. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the technical roadmap of the present invention;

[0017] Figure 2 This is a partial Gram staining result diagram of the present invention;

[0018] Figure 3 The electrophoresis diagram of 16S rRNA sequence amplification of isolated strains; M: DL 2 000; 1-12 are PCR products of PL2, PL 6, PL 9, PL 12, PL 24, PL 84, PL 86, PL 92, PL 96, PL 104, PL 106, PL 110 strains, respectively; 13: negative control;

[0019] Figure 4 Phylogenetic tree diagram of isolated strains based on 16S rRNA sequences;

[0020] Figure 5-1 The effect of different acid concentrations on lactic acid bacteria strains;

[0021] Figure 5-2 The effect of different acid concentrations on Bacillus strains;

[0022] Figure 6-1 The effect of different bile salt concentrations on lactic acid bacteria strains;

[0023] Figure 6-2 The effect of different bile salt concentrations on Bacillus strains;

[0024] Figure 7 The effect of different temperatures on lactic acid bacteria strains;

[0025] Figure 8-1 is the antibacterial activity of lactic acid bacteria strains;

[0026] Figure 8-2 for the antibacterial activity of Bacillus strains;

[0027] Fig. 9 Schematic diagram for rating loose stools. DETAILED DESCRIPTION

[0028] In order to make the objectives, technical solutions and advantages of the embodiments of the present invention more clearly understood, the spirit of the contents disclosed by the present invention will be described in detail below. After understanding the embodiments of the contents of the present invention, any technician in the relevant technical field can change and modify them according to the techniques taught by the contents of the present invention without departing from the spirit and scope of the contents of the present invention.

[0029] The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention. Figure 1 The experiment was conducted as follows.

[0030] Example 1: Isolation and identification of strains from the dominant flora in the intestines of healthy piglets:

[0031] 1. Materials and methods

[0032] 1.1 Test materials:

[0033] Fecal samples of the tested piglets were collected from large-scale pig farms in the three northeastern provinces by rectal sampling.

[0034] ①On December 9, 2021, Weijia Village, Niuzhuang Town, Haicheng City, Anshan City, Liaoning Province provided fecal samples of 12-day-old Sanyuan piglets, collecting three healthy piglets and three diarrhea piglets;

[0035] ②On December 11, 2021, Lvkang Animal Husbandry in Baicheng City, Jilin Province, feces of 12-day-old Sanyuan piglets, 2 healthy feces and 3 diarrhea feces were collected;

[0036] ③On December 11, 2021, at the Da Bei Nong Qian'an Farm in Songyuan City, Jilin Province, 15-day-old Sanyuan piglets were excreted, and 5 healthy excrements were collected;

[0037] ④On December 13, 2021, at Gan Yiyuan Farm in Fuxin City, Liaoning Province, feces of 10-day-old Sanyuan piglets were collected, and 3 healthy feces were collected;

[0038] ⑤On December 17, 2021, at Qian'an Farm, Zhaozhou County, Daqing City, Heilongjiang Province, feces of 32-day-old Sanyuan piglets were collected, and 5 cases of diarrhea feces were collected;

[0039] ⑥On December 23, 2021, Weijia Village, Niuzhuang Town, Haicheng City, Anshan City, Liaoning Province and Gan Yiyuan Breeding Farm in Fuxin City, Liaoning City provided the second batch of 7-day-old Sanyuan piglet feces, collecting 5 diarrhea feces and 3 healthy feces.

[0040] 1.2. Main instruments and equipment: see Table 1.

[0041] Table 1 Main instruments and equipment

[0042]

[0043] 1.3 Test methods and results:

[0044] 1.3.1, strain isolation and purification:

[0045] Using MRS, NA, and CCA solid culture media, 133 suspected lactic acid bacteria, 97 suspected Bacillus, and 18 suspected Clostridium butyricum were preliminarily isolated from the feces of healthy weaned piglets for binary streak test. Single colonies were preliminarily screened and identified from the three culture media, and the colonies were streaked and purified on the three culture media 2 to 3 times to select single colonies. Subsequently, after considering conditions such as color, size, gloss, and shape, representative strains were selected for binary streak, Gram staining, oil microscopy, and catalase test.

[0046] Binary streak method: This method uses MRS solid culture medium, divides the culture medium into two equal parts with a marker pen, and streaks the selected strains on the culture medium in pairs, and then cultures them anaerobically at 30°C for 48 hours. The strains are observed, and strains with different colony morphology are selected for the next screening test. After preliminary screening by binary streak, 48, 47 and 12 strains of suspected lactic acid bacteria, Bacillus and Clostridium butyricum with different colony morphology were obtained, respectively.

[0047] Catalase test: This method uses an inoculation loop to dip a single strain and apply it to the catalase solution. If bubbles appear, it is positive, otherwise it is negative. After the catalase test, 35 catalase-negative suspected lactic acid bacteria strains and 26 catalase-positive suspected Bacillus strains were screened, of which 9 were suspected Clostridium butyricum strains for Gram staining microscopy test.

[0048] Gram staining: This method is to perform Gram staining on the strains screened by the above method.

[0049] In MRS medium, strains with positive Gram staining and negative catalase were preliminarily identified as Lactobacillus; in NA medium, strains with positive Gram staining and positive catalase were preliminarily identified as Bacillus; in CCA medium, strains with positive Gram staining and negative catalase were preliminarily identified as Clostridium butyricum. MRS, NA, and CCA liquid culture media without agar were enriched at 30°C for 24 hours, and then an equal amount of 40% glycerol was added. After aliquoting, they were stored in an environment of -80°C. By Gram staining microscopy test, 12 Gram-positive lactic acid bacteria strains, 9 Gram-positive Bacillus strains and 3 Gram-positive Clostridium butyricum strains were screened. The lactic acid bacteria were named PL 2, PL 6, PL 9, PL 12, PL 24, PL 84, PL 86, PL92, PL 96, PL 104, PL 106, PL 110; the Bacillus were named PB 3, PB 11, PB 27, PB 43, PB 58, PB71, PB 76, PB 89, PB 96; the Clostridium butyricum were named PC 5, PC 10, PC 17.

[0050] After observing the morphology of each colony, performing the catalase test and performing Gram staining, the experimental results are shown in Table 2. The results of Gram staining showed that the bacteria of PL 24, PL 84, PL 86, PL 104, PB 96 and PC 10 were spherical, while the remaining strains were rod-shaped. Figure 2 Some of the results of Gram staining are shown in .

[0051] Table 2 Preliminary isolation and screening of strains

[0052]

[0053]

[0054] Note: “-” indicates negative, “+” indicates positive, “L” indicates large, “M” indicates medium, and “S” indicates small.

[0055] 1.3.2 Physiological and biochemical identification of dominant strains:

[0056] The biochemical identification tubes of lactic acid bacteria include arabinose biochemical tube, cellobiose biochemical tube, fructose biochemical tube, glucose biochemical tube, lactose biochemical tube, maltose biochemical tube, mannitol biochemical tube, mannose biochemical tube, melezitose biochemical tube, ribose biochemical tube, salicin biochemical tube, sorbitol biochemical tube, rhamnose biochemical tube, sucrose biochemical tube and xylose biochemical tube.

[0057] The biochemical identification tubes of Bacillus include contact enzyme biochemical tubes, gelatin liquefaction biochemical tubes, VIP test biochemical tubes, nitrate reduction biochemical tubes, glucose biochemical tubes, mannitol biochemical tubes, xylose biochemical tubes, arabinose biochemical tubes and indole test biochemical tubes.

[0058] According to the detection methods of strain physical and chemical characteristics in the Manual of Identification of Common Bacteria Systems and Bergey's Manual of Bacterial Identification, the physiological and biochemical identification results of the 24 strains isolated in the experiment are shown in Tables 3 and 4. Preliminary judgment showed that PL 2, PL 6, PL 9, PL 12, PL 24, PL 84, PL 86, PL 92, PL 96, PL 104, PL 106, and PL 110 were lactic acid bacteria; PB 3, PB 11, PB27, PB 43, PB 58, PB 71, PB 76, PB 89, and PB 96 were Bacillus.

[0059] Table 3 Biochemical characteristics of lactic acid bacteria

[0060]

[0061]

[0062] Note: “+” indicates growth, “-” indicates no growth, the same as in the following table.

[0063] Table 4 Biochemical characteristics of Bacillus

[0064]

[0065] 1.3.3. This application uses a bacterial genome kit to extract the DNA of lactobacillus, and then uses molecular biological techniques such as 16SrRNA gene sequence analysis and DNA hybridization to identify the genus and species of anti-diarrhea probiotics. DNA extraction: First, the strain is subjected to Gram staining microscopy and plate colony morphology observation. After confirming that the strain has been purified, a single colony is selected from it and inoculated into MRS liquid culture medium. It is cultured at a constant temperature of 37°C for 24 hours, and then the DNA of the strain is extracted and stored in a -20°C refrigerator.

[0066] PCR amplification: Using the bacterial genomic DNA isolated from the piglet intestine as a template, universal primers were used to amplify the 16SrDNA gene fragment, with primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3') upstream and 1492R (5'-GGCTACCTTGTTACGACTT-3') downstream. Primer synthesis and PCR product sequencing were completed by Beijing Liuhe Huada Co., Ltd.

[0067] The PCR amplification system was a 25 μL reaction system, including 1 μL of upstream and downstream primers, 2 μL of template DNA, 12.5 μL 2× Taqplus Buffer, 8.5 μL sterile dEPC H2O, and sterile ultrapure water was set as a negative control group. PCR amplification conditions: the sample was placed in a 94°C environment for pre-denaturation for 5 min, denatured in a 94°C environment for 30 s, annealed in a 55°C environment for 30 s, extended in a 72°C environment for 1 min 30 s, and after 30 cycles, finally extended in a 72°C environment for 5 min. After the PCR amplification was completed, 5 μL of the above PCR amplification product was extracted, and the electrophoresis voltage was 110 V for 30 min. After completion, the band size and brightness intensity of all PCR products were measured using the SDS-PAGE UV gel imaging analysis system, and the PCR product stock solution that met the expected results was sent to Beijing Liuhe BGI Gene Technology Co., Ltd. for sequence determination.

[0068] Results of 16S rRNA sequence amplification of each strain: This experiment found that 12 lactic acid bacteria and 9 Bacillus strains showed specificity at 1450bp of the target band of the 16SrRNA gene sequence. All 12 lactic acid bacteria strains and 9 Bacillus strains showed bands consistent with the expected results. Figure 3 .

[0069] 1.3.4 Data Analysis:

[0070] After the sequencing results were spliced, NCBI was used for Blast analysis, and then the sequences were aligned and compared with the relevant sequences retrieved in GenBank using the tool MEGA 11.0, and the phylogenetic tree was constructed using the neighbor-joining method. Finally, the sequence data was subjected to boot strap analysis with 1,000 random repeated samplings to evaluate the topological structure of the tree.

[0071] 16S rRNA sequence analysis of each strain: BLAST comparison was performed in Genbank to draw a genetic purification tree. The results are as follows Figure 4 As shown, PL 2, PL 12, and PL 96 are in the same evolutionary branch with Lactobacillus plantarum, and the evolutionary affinity is 89%; PL 9 is in the same evolutionary branch with Lactobacillus plantarum, and the affinity is 84%; PL 6, PL 92, PL 106, and PL 110 are in the same evolutionary branch with Lactobacillus plantarum, and the affinity is 63%; PL 24 is in the same evolutionary branch with Enterococcus lactis, and the affinity is 100%; PL 86 is in the same evolutionary branch with Enterococcus monnieri, and the evolutionary affinity is 100%; PL 84 and PL 104 are in the same evolutionary branch with Enterococcus faecium, and the evolutionary affinity is 99% respectively;

[0072] BLAST comparison was performed in Genbank and a genetic purification tree was drawn. PB 3 and PB 27 were located in the same evolutionary branch with Bacillus subtilis, with evolutionary affinities of 92%, PB 43 and PB 96 were located in the same evolutionary branch with Bacillus subtilis, with evolutionary affinities of 93%, PB 11, PB 58 and PB 76 were located in the same evolutionary branch with Bacillus subtilis, with evolutionary affinities of 92%, PB89 was located in the same evolutionary branch with Bacillus subtilis, with a affinity of 90%, and PB 71 was related to Bacillus halodurans with a affinity of 97%.

[0073] 2. Conclusion

[0074] A total of 21 dominant bacteria were isolated and identified from the intestines of healthy piglets. The results showed that PL 2, PL 6, PL 9, PL 12, PL 92, PL 96, PL 106 and PL 110 were Lactobacillus plantarum, PL 24 was Enterococcus lactis, PL 86 was Enterococcus monnieri, PL84 and PL 104 were Enterococcus faecium; PB 3 and PB 27 belonged to Bacillus subtilis, PB 11, PB 43, PB 58 and PB 76, PB89 and PB 96 were Bacillus subtilis, and PB 71 was a halotolerant Bacillus.

[0075] Example 2: Biological characteristics of strains in the dominant flora of the intestines of healthy piglets:

[0076] 1. Materials and methods

[0077] 1.1 Test materials:

[0078] Lactobacillus plantarum PL 2, PL 6, PL 9, PL 12, PL 92, PL 96, PL106 and PL 110 isolated from the intestines of healthy piglets, Enterococcus lactis PL 24, Enterococcus monnieri PL 86, Enterococcus faecium PL 84 and PL 104, Bacillus subtilis PB 3, PB 27, Bacillus subtilis PB 11, PB 43, PB 58 and PB 76, PB 89 and PB 96, Bacillus halobacterium PB 71.

[0079] Different pH broths: Weigh MRS liquid culture medium and NA broth respectively, use double distilled water to make the two types of broths to 1000mL, and divide them into 5 conical flasks, adjust the broth with 2mol hydrochloric acid, and adjust the pH value to pH3.0, pH3.5, pH4.0, pH4.5 and pH5.0 respectively, then sterilize the conical flasks with high pressure steam (115℃, 30min), put them into the clean bench, and let them cool for use;

[0080] Broths with different bile salt concentrations: Weigh MRS liquid culture medium and NA broth respectively, make up to 200 mL with double distilled water in conical flasks, add 0, 0.10, 0.20, and 0.30 g of ox bile salts to the subpackaged culture medium, respectively, so that the bile salt solubility in MRS and NA liquid culture media is 0%, 0.2%, 0.4%, and 0.6%. Then, sterilize the conical flasks with high-pressure steam (115°C, 20 min), put them in a clean bench, and let them cool for later use.

[0081] 1.2. Main instruments and equipment: Same as Table 1.

[0082] 1.3. Data processing:

[0083] In this experiment, the software Excel 2021 was used to first calculate and organize the experimental data, and then the statistical software SPSS23.0 was used to perform variance analysis on the experiment, and the Tukey method was used to carry out multiple comparisons. The significance level of the difference was P<0.05. Finally, Graphpad prism 8 was used to plot the experimental results.

[0084] 1.4. Test methods and results:

[0085] 1.4.1. Test on the effect of different acid concentrations on strain growth:

[0086] In this study, hydrochloric acid was used to adjust the pH of the liquid culture medium to five levels: pH 3.0, pH 3.5, pH 4.0, pH 4.5, and pH 5. The prepared culture medium was then divided into 5 mL portions per test tube and sterilized at 121°C under high pressure for 15 min. The culture medium of the strain to be tested was added to the sterilized culture medium at the same dose of 0.1 mL. The medium was shaken at 37°C at 160 rpm for 24 h. The acid and salt resistance of the test strain was observed. The OD value of each group of bacterial liquid was measured, with 3 replicates per group, and the average value was taken.

[0087] Effect of different acid concentrations on strain growth: The OD values ​​of 12 lactobacilli cultured in pH 5.8 MRS liquid for 24 hours and inoculated into pH 3-pH 5 MRS liquid medium for 24 hours were measured and analyzed. The results are as follows: Figure 5-1 As shown in the figure, a total of 3 strains of lactic acid bacteria were screened that could tolerate pH 3 and pH 3.5 in vitro. Their survival in MRS liquid culture medium was as follows: Figure 5-1. In MRS liquid medium at pH=3, the survival rate of PL 96 was significantly higher than that of other strains (P<0.05), and there was no significant difference with PL86. At pH 3.5, the survival rate of PL 86 was significantly higher than that of other strains (P<0.05), and there was no significant difference with PL 96. At pH 4, the survival rates of PL 86, PL 92, PL 96 and PL 104 were significantly higher than those of other strains (P<0.05), indicating that they have better tolerance to acid.

[0088] Depend on Figure 5-2 It can be seen that the 9 strains of Bacillus had poor tolerance to acidity at pH 3-pH 4.5, among which PB 43 had a higher survival rate at pH 4.5, significantly higher than other strains (P<0.05). At pH 5, PB 43 and PB 89 had higher survival rates, and their tolerance to acid was significantly higher than other strains (P<0.05).

[0089] 1.4.2 Effects of different bile salt test concentrations on strain growth:

[0090] The salt concentration (w / v%) of the culture medium was adjusted to three levels of 0.2, 0.4, and 0.6 (w / v%) with analytically pure ox bile salt, and then the prepared culture medium was packaged in an amount of 5 mL per test tube. After sterilization at 121°C under high pressure conditions for 15 minutes, the culture medium of the test strain was added to the sterilized culture medium at the same dose of 0.1 mL. After shaking at 37°C and 160 rpm for 24 hours, the acid and salt resistance of the test strain was observed, and the OD value of each group of bacterial solution was measured respectively, with 3 replicates per group, and the average value was finally taken.

[0091] Effects of different bile salt concentrations on strain growth: Lactic acid bacteria: The experimental results are Figure 6-1 As shown in the figure, the growth of Lactobacillus was inhibited to a certain extent under three different bile salt levels, but the survival rate of strain PL 24 was significantly higher than that of other strains (P<0.05) when the bile salt concentration in the culture medium was 0.2%, and there was no significant difference with PL 12 and PL 96. The survival rate of PL 24 and PL 84 was significantly higher than that of other strains when the bile salt concentration was 0.4% and 0.6% (P<0.05). PL 12, PL 24 and PL 84 strains can grow and reproduce normally, indicating that these strains have relatively good bile salt tolerance.

[0092] Bacillus: Experimental results by Figure 6-2As shown in the figure, at three different bile salt levels, the growth of Bacillus was inhibited to a certain extent, and the activity of Bacillus decreased with the increase of bile salt concentration in the culture medium. When the bile salt concentration was 0.2%, the survival rate of strain PB96 was significantly higher than that of other strains (P<0.05), and there was no significant difference with PB 58. When the bile salt concentration was 0.4%, PB 76 had a higher tolerance to bile salt concentration, which was significantly higher than that of other strains (P<0.05). When the concentration was 0.6%, PB 58 had a significantly higher tolerance to bile salt concentration than PB 27 (P<0.05).

[0093] 1.4.3. Test on sensitivity of strains to different temperatures:

[0094] The temperature was adjusted to 45°C, 50°C, 55°C, and 60°C, and the temperature sensitivity of the test strain was observed after 30 minutes of treatment. The OD value of each group of bacterial liquid was measured at 37°C with a shaker at 160 rpm for 12 hours (the temperature group was set to stand for 6 hours), and 3 replicates were performed for each group, and the average value was finally taken.

[0095] Effects of different temperature levels on strain growth: Lactic acid bacteria: There was no significant difference in the growth and reproduction of strains at different temperatures; as the temperature increased in the range of 45-50°C, the strain showed a relatively high sensitivity, and its growth and reproduction ability would gradually decrease, but it could still grow. At 45°C, the survival rate of PL 12 was significantly higher than that of PL 24 and PL 6 (P<0.05). At 50°C, the growth and reproduction rates of PL 2, PL 9 and PL 86 were significantly higher than those of other strains (P<0.05). At 55-60°C, its reproduction ability decreased and was not suitable for the growth and reproduction of lactic acid bacteria. Strain PL 24 was highly sensitive to temperature (P<0.05). After treatment, its growth was not obvious, and other temperatures had no effect on the growth and reproduction of the strain. For specific data, see Figure 7 .

[0096] Bacillus: When screening strains, they were placed in a water bath at 75°C for 15 minutes. The strains were not sensitive to temperature and there was no obvious change in their growth.

[0097] 1.4.4. Determination of the probiotic ability of dominant strains:

[0098] After centrifugation at 6 000×g for 5 min for the activated strain, the supernatant was filtered with a 0.22 μm sterile filter membrane to remove bacteria. The bacterial solution was taken with an Oxford cup, and the ability of bacteria to produce protease was determined by the casein hydrolysis test, and the ability of bacteria to produce amylase was detected by the starch hydrolysis test; then 4 holes (7 mm in diameter) were evenly punched in the center of the culture plate; 150 μL of cell-free supernatant was added to the wells, and uninoculated culture medium was added as a control. The diameter of the hydrolysis zone was measured after constant temperature culture at 37°C for 12 h.

[0099] The results of protein and starch hydrolysis by the 21 dominant strains are shown in Table 5. The ability of PL 86 to produce protease and amylase was significantly higher than that of other strains (P<0.05), with hydrolysis diameters of 37 mm and 22 mm, respectively. Among them, PL 6, PL 12, PL 24, PL 86, PL 92, PL 96 and PL 106 could produce protease and amylase.

[0100] Table 5 Casein hydrolysis and starch hydrolysis tests of various strains

[0101]

[0102] Note: “-” indicates no hydrolysis reaction occurred.

[0103] 1.4.5. Antibacterial activity test of the strain against pathogenic Escherichia coli ATCC 25922:

[0104] The strain was cultured for 48 hours, during which 5 mL of samples were taken every 4 hours, and the supernatant was centrifuged to detect its pH, and 200 μL was taken for antibacterial test. The 100-fold diluted Escherichia coli liquid was evenly spread on the LB plate, and two Oxford cups with an inner diameter of 6 mm were placed at equal distances in each plate. 200 μL of anti-diarrhea probiotic supernatant was added to the cups, and the plates were placed in a 37°C constant temperature incubator for 24 hours. The diameter of the antibacterial zone of the fermentation supernatant at each time point was measured with a vernier caliper, and 3 parallels were used for each sample.

[0105] Results of the antibacterial activity test of the strains against pathogenic Escherichia coli ATCC 25922: 21 dominant strains were cultured for 48 hours, and the changes in the antibacterial diameter of the supernatant of the fermented bacterial solution against pathogenic Escherichia coli ATCC 25922 were recorded. The experimental results are as follows: Figure 8-1 and Figure 8-2As shown in the figure, after 48 hours of culture, the antibacterial diameter of PL 9 against pathogenic Escherichia coli ATCC25922 was significantly larger than that of other lactic acid bacteria strains (P<0.05), which was 26 mm, and there was no significant difference with PL 96 (P>0.05), but there was no significant difference in pH between PL 6 and PL 86 after 48 hours of culture (P>0.05). The antibacterial diameter of PB 96 in the Bacillus group was significantly larger than that of other Bacillus strains (P<0.05), which was 11 mm, and there was a significant difference with PB 89 (P<0.05).

[0106] 1.4.6. In vitro co-culture test of the strain and pathogenic Escherichia coli ATCC 25922:

[0107] The dominant strain and E. coli culture were diluted with sterile water to about 1.0×10 -8 CFU / ml, take 2% volume of each and inoculate in MRS liquid culture medium, 37℃, 150rpm shaking culture. Take 5mL of bacterial solution at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, test pH, then dilute stepwise and take 200μL of dilution evenly spread on EMB plate, incubate at 37℃ for 24h, count the number of residual viable E. coli, and calculate the survival rate at each treatment time point.

[0108] Results of in vitro co-culture test of strains and pathogenic Escherichia coli ATCC 25922: The pH results of the co-culture of 21 strains with pathogenic Escherichia coli ATCC 25922C are shown in Table 6. After co-culture, the pH value first decreased with the increase of culture time at 0-24h, increased at 48h, and decreased at 72h. The pH of the co-cultured culture solution of the Bacillus group decreased slowly with the culture time, and then reached equilibrium. At 8h, PB 3 and PB 9 were significantly lower than those of other strains (P<0.05). At 12h, PB 3, PB 11, and PB27 were significantly lower than those of other strains (P<0.05). At 24-72h, PB 3 mixed culture was significantly lower than those of other strains (P<0.05). The pH of the co-cultured culture solution of the lactic acid bacteria group gradually decreased with the culture time, and then stabilized. At 12h, PL 86 and PL The acid production capacity of PL 92 was significantly lower than that of other strains (P<0.05). At 24-48h, the acid production capacity of PL 86 mixed culture was significantly higher than that of other strains (P<0.05). At 72h, the acid production capacity of PL9 mixed culture was significantly higher than that of other strains (P<0.05).

[0109] Table 6 pH value of mixed culture

[0110]

[0111]

[0112] The results of the residual colony counts of 21 Bacillus strains mixed with pathogenic Escherichia coli ATCC 25922C culture are shown in Table 7. The log value of the number of residual live E. coli first increased with the extension of the culture time, reached an equilibrium when the culture time reached 4-8h, and then decreased sharply. After the culture time reached 24h, there was no surviving E. coli in the EMB culture medium; in the Bacillus group, the strains PB 3 and PB 11 were significantly lower than other strains at 8h of culture time (P<0.05). The pH values ​​of strains PL 12, PL 24, PL 84, PL 86, and PL 104 in the lactic acid bacteria group were similar to the pH values ​​of the strains cultured alone, which means that the acid production ability of these dominant strains is relatively strong overall. Although the strains produce acid very slowly, they are basically not affected by the E. coli co-cultured in the culture medium.

[0113] Table 7 Residual colony counts of Escherichia coli in mixed culture

[0114]

[0115] 2. Conclusion

[0116] In summary, 12 strains of lactic acid bacteria and 9 strains of Bacillus were isolated from the dominant flora in the feces of healthy piglets, among which Enterococcus mundtii PL 86 had good biological characteristics, the strongest ability to produce protease and amylase, and could effectively inhibit the growth of pathogenic Escherichia coli.

[0117] Example 3: Effects of Enterococcus monteri PL 86 from pigs on growth performance and diarrhea in mice:

[0118] 1. Materials and methods

[0119] 1.1 Test materials:

[0120] A total of 24 8-week-old SPF male Bal b / c mice were purchased from Changsheng Biotechnology Co., Ltd., weighing 20-22 g, with 12 h of light per day, temperature of 21±2°C, and relative humidity of 45%±10%. They were adaptively raised in an isolated environment for 7 days, and had free access to food and water during the adaptive feeding period. Enterococcus monteri PL 86 and pathogenic Escherichia coli ATCC 25922 were stored in the Key Laboratory of Efficient Utilization and Nutritional Regulation of Cold Region Feed Resources, Heilongjiang Bayi Agricultural University.

[0121] 1.2. Main instruments and equipment: see Table 8.

[0122] Table 8 Main instruments and equipment

[0123]

[0124] 1.3 Test methods and results

[0125] 1.3.1 Effects of Enterococcus monnieri PL 86 on body weight and food intake of mice:

[0126] 24 mice were divided into four groups: control group (CON), diarrhea group (EcN), probiotic group (PL 86), and probiotic prevention group (PL 86+EcN). The experiment was divided into two stages. The first stage was the construction of the probiotic prevention barrier, and the second stage was the challenge of pathogenic Escherichia coli ATCC 25922.

[0127] The first stage lasted for 21 days. During this period, the PL 86 group and the PL 86+EcN group were gavaged daily with 200 μL of 1.0×10 8 CFU / mL of Enterococcus monteri PL 86, the CON group and the EcN group were gavaged with the same dose of MRS liquid culture medium every day.

[0128] The second stage started from the 22nd day of the experiment and lasted until all the experimental mice in the EcN group developed diarrhea. During this period, the PL 86+EcN group and the EcN group were intragastrically administered with 200 μL of 1.0×10 10 CFU / mL of pathogenic Escherichia coli ATCC 25922, the CON group and PL86 group were gavaged with the same dose of LB liquid culture medium every day.

[0129] In the first stage, the body weight and food intake of mice were measured every 3 days, and in the second stage, the body weight and food intake of mice were measured every day.

[0130] Effects of Enterococcus monteri PL 86 on body weight and food intake of mice:

[0131] As shown in Table 9, the weight of mice in each group showed an upward trend from 0 to 21 days, and the weight of mice gavaged with PL 86 did not decrease. The mental state of mice in each group was good. From 21 to 24 days, the weight of mice in the EcN group showed a downward trend, and the weight of the PL 86+EcN group did not change significantly. At 24 days, the weight of the PL 86+EcN group was significantly higher than that of the EcN group (P<0.05). As shown in Table 10, except for the EcN group, the feed intake of the other groups showed a downward trend from 0 to 6 days. After the challenge of the virus from 21 to 24 days, the feed intake of the EcN group decreased significantly, and the feed intake of mice in the other groups did not change.

[0132] Table 9 Changes in mouse body weight

[0133]

[0134] Note: Superscript letters represent significant differences between groups.

[0135] As shown in Table 10, except for the EcN group, the food intake of the other groups showed a downward trend from 0 to 6 days. The food intake of the EcN group decreased significantly from 21 to 24 days after the virus attack. There was a significant difference in the food intake of the EcN group and the other groups on days 21 and 22 (P<0.05), and the food intake of the other groups did not change.

[0136] Table 10 Changes in food intake of mice

[0137]

[0138] 1.3.2 Effect of Enterococcus monteri PL 86 on the number of lactic acid bacteria and Escherichia coli in the intestines of mice:

[0139] In the first stage, 1 g of feces was collected from the tray 5 hours after oral gavage of mice every 3 days and placed in MRS liquid culture medium. The MRS liquid culture medium was then placed on a shaker at 160 rpm and cultured at 35°C for 24 hours. After the culture was completed, gradient dilutions were performed and plates were spread. The number of lactic acid bacteria was calculated by anaerobically cultured in the MRS culture dish for 24 hours, with 3 replicates per group.

[0140] In the second stage, the number of lactic acid bacteria was measured daily. At the same time, 1 g of feces in the tray was collected 5 hours after the mice were gavaged and placed in LB liquid culture medium. The LB liquid culture medium was then placed in a shaker at 160 rpm and cultured at 35°C for 24 hours. After the culture was completed, gradient dilutions were performed and plates were spread. The number of Escherichia coli was calculated after anaerobically cultured in the LB culture dish for 24 hours. Each group had 3 replicates.

[0141] Results of the effect of Enterococcus monteri PL 86 on the number of lactic acid bacteria and Escherichia coli in the intestines of mice:

[0142] As shown in Table 11, the lactic acid bacteria content in the feces of mice in the PL 86 and PL 86+EcN groups showed an upward trend from 0 to 3 days. During the entire experiment, the average lactic acid bacteria content in the feces of mice in the PL 86 and PL 86+EcN groups was significantly higher than that in the CON and EcN groups (P<0.05). After the challenge, the lactic acid bacteria content in the feces of mice in the PL 86+EcN group decreased significantly at 23 days, but rebounded at 24 days.

[0143] Table 11 Lactobacillus content in mouse feces

[0144]

[0145] Note: Superscript letters represent significant differences among groups.

[0146] The content of E. coli in the feces of mice after infection is shown in Table 12. The content of E. coli in the feces of the EcN group continued to increase, and the average E. coli content on days 21-24 was significantly higher than that of the PL 86+EcN group (P<0.05). The content of E. coli in the feces of the PL 86+EcN group decreased on the 23rd day, and the fecal E. coli at this time was significantly lower than that of all groups (P<0.05). It rebounded on the 24th day but was still significantly different from the EcN group (P<0.05).

[0147] Table 12 Escherichia coli content in mouse feces

[0148]

[0149] 1.3.3 Effects of Enterococcus monteri PL 86 on organ index and diarrhea index of mice:

[0150] In the second phase of the experiment, the amount of feces and loose stools of mice were recorded daily, and the loose stools were graded. Fig. 9 The diarrhea index was calculated as shown. After all mice in the EcN group had diarrhea during the experimental period, all mice were killed on the same day, the colon length was measured, the liver, kidney, and spleen of the mice were weighed, and the index of each organ was calculated. The specific calculation formula is as follows:

[0151] Diarrhea index = loose stool rate × average loose stool grade

[0152] Loose stool rate = number of loose stools per animal ÷ total number of stools for the animal

[0153] The stool looseness grade indicates the degree of loose stool of each animal, which is graded according to the diameter of the stool and is divided into 4 grades: <1 cm as grade 1, 1-1.9 cm as grade 2, 2-3 cm as grade 3, and >3 cm as grade 4.

[0154] Average loose stool grade = total loose stool grades / number of loose stools

[0155] Diarrhea rate (%) = number of animals with diarrhea / total number of animals in the group × 100%

[0156] Organ index = organ weight / mouse body weight × 100%.

[0157] Results of the effects of Enterococcus monteri PL 86 on organ index and diarrhea index of mice:

[0158] All mice in the EcN group had diarrhea on the 24th day of the experiment, while the mice in the other groups did not have diarrhea during the whole experiment and were in good spirits. The organ and diarrhea indexes are shown in Table 13. The diarrhea index of mice in the EcN group was significantly higher than that in other groups, and the difference was extremely significant (P<0.001). After the challenge, the colon length of the EcN group was significantly shortened and significantly different from the PL 86 and PL 86+EcN groups (P<0.05). After feeding PL 86, the liver index of mice increased significantly, and was higher than that of the EcN group, with significant differences between the groups (P<0.05). There was no significant change in the kidney index, and the spleen index of the EcN group increased significantly, which was different from that of the other groups (P<0.05).

[0159] Table 13 Mouse organ index and diarrhea index

[0160]

[0161] 1.4 Data Processing

[0162] In this experiment, the software Excel 2021 was used to first calculate and organize the experimental data, and then the statistical software SPSS23.0 was used to perform variance analysis on the experiment, and the Tukey method was used to carry out multiple comparisons. The significance level of the difference was P<0.05. Finally, Graphpad prism 8 was used to plot the experimental results.

[0163] 2. Conclusion: Feeding mice with Enterococcus mundtii PL 86 did not cause mental depression or weight loss in mice, but could increase the number of lactic acid bacteria in the intestine to a certain extent, hinder the invasion of bacterial pathogens to the intestine of animals, and effectively prevent the occurrence of diarrhea in animals.

[0164] Example 4: Effect of Enterococcus monteri PL 86 from pigs on diarrhea in piglets:

[0165] 1. Materials and methods:

[0166] 1.1 Test materials:

[0167] Enterococcus monteri PL 86 was deposited in the Key Laboratory of Efficient Utilization and Nutritional Regulation of Cold Region Feed Resources, Heilongjiang Bayi Agricultural University.

[0168] 1.2. Main instruments and equipment: same as Table 8

[0169] 1.3 Test methods:

[0170] 1.3.1. Test time and location:

[0171] On November 13, 2023, feeding experiments were conducted at the following three pig farms: Lvkang Animal Husbandry in Baicheng City, Jilin Province, Da Beinong Qian'an Farm in Songyuan City, Jilin Province, and Qian'an Farm in Zhaozhou County, Daqing City, Heilongjiang Province.

[0172] 1.3.2. Experimental Animal Grouping

[0173] Each of the above pig farms selected male and female piglets of similar age and weight, including 12 healthy piglets (6 male and 6 female piglets) and 36 diarrhea piglets (18 male and 18 female piglets) in each pig farm. The experimental treatments were divided into 4 groups, each with 12 piglets, 6 male and 6 female piglets, including the control group (6 healthy male and female piglets), diarrhea group (6 male and female piglets with diarrhea, no probiotics or drugs), probiotic group (6 male and female piglets with diarrhea, fed with Enterococcus monterelli PL86), and antibiotic group (6 male and female piglets with diarrhea, fed with enrofloxacin, a diarrhea treatment drug). The concentration of Enterococcus monterelli PL 86 was cultured at 1.0×1010 CFU / mL, and 0.2% Enterococcus monterelli PL86 was added to the feed; enrofloxacin was used according to the dosage in the instructions, and each group had free access to food and water, and was managed according to the conventional feeding methods of the pig farm for 7 days.

[0174] 1.3.3 Effect of Enterococcus monteri PL 86 on diarrhea index of piglets:

[0175] Diarrhea index = loose stool rate × average loose stool grade.

[0176] Loose stool rate = number of loose stools excreted by each piglet ÷ total number of stools of the piglet.

[0177] The loose stool grade indicates the degree of loose stool of each piglet. The appearance of stool is used as the scoring standard and is divided into 4 grades: strip or granular, relatively hard is grade 1, normal shape, soft and hard is grade 2, thick, partially shaped, no water separation phenomenon is grade 3, no shape, liquid, feces and water separation is grade 4.

[0178] Average loose stool grade = total loose stool grades ÷ number of loose stools.

[0179] Diarrhea rate (%) = number of piglets with diarrhea / total number of piglets in the group × 100%.

[0180] 1.4 Data Processing

[0181] This study used SPSS23.0 statistical software to perform variance analysis on the experiment, and Tukey's method was used for multiple comparisons. The significance level of the difference was P<0.05.

[0182] 2. Results and Analysis

[0183] After 7 days of the experiment, the diarrhea index was as shown in Table 14. The diarrhea index of the probiotic group, antibiotic group and healthy group piglets in the three pig farms were significantly lower than that of the diarrhea group piglets (P<0.05). The diarrhea of ​​the original diarrhea piglets in each pig farm had been suppressed after feeding Enterococcus monteri PL 86, and their diarrhea index was not significantly different from that of the antibiotic group.

[0184] Table 14 Diarrhea index of piglets

[0185]

[0186] 3. Conclusion: Feeding Enterococcus mundtii PL 86 to piglets with diarrhea can effectively inhibit the occurrence of diarrhea in piglets, and its therapeutic and control effect on diarrhea in piglets is equivalent to that of enrofloxacin.

Claims

1. A strain for preventing diarrhea in weaned piglets, characterized in that: It is Enterococcus mundtii PL86, deposited in the China Center for Type Culture Collection, the preservation address is Luojia Mountain, Bayi Road, Wuchang District, Wu'an City, Hubei Province, the preservation date is July 17, 2023, and the preservation number is CCTCC NO: M 20231246.

2. The use of a strain for preventing diarrhea in weaned piglets according to claim 1, characterized in that Enterococcus mundtii PL86 can prevent the incidence of diarrhea in weaned piglets.

3. The use of a strain for preventing diarrhea in weaned piglets according to claim 2, characterized in that It can inhibit the number of diarrhea-causing bacteria in the intestines and prevent the occurrence of diarrhea in piglets.

4. The use of a strain for preventing diarrhea in weaned piglets according to claim 1, characterized in that Enterococcus mundtii PL86 is not toxic to animals.

5. The use of a strain for preventing diarrhea in weaned piglets according to claim 4, characterized in that The method for preventing diarrhea in weaned piglets refers to reducing the content of Escherichia coli in the intestine, increasing the content of lactic acid bacteria, and preventing the occurrence of diarrhea by feeding Enterococcus mundtii PL86.

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