Strain for preventing weaned piglet diarrhea and application thereof

Abstract

This invention relates to a strain for preventing weaned piglet diarrhea and its application, and aims to address the frequent occurrence of weaned diarrhea in piglets in the current pig farming industry. The strain, *Enterococcus mundtii* PL86, is currently deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wu'an City, Hubei Province, on July 10, 2023, with accession number CCTCC NO: M 20231246. Based on research into the pathogenesis of weaned piglet diarrhea, this invention isolates and identifies the main dominant microorganisms in the feces of weaned piglets and studies their biological characteristics. This provides a scientific theoretical basis for the production of probiotic preparations that can prevent weaned piglet diarrhea, and is of great significance for reducing drug use in animal husbandry, producing functional livestock products, and protecting human health.

Description

Technical Field

[0001] This invention relates to the field of animal husbandry, specifically to a strain of bacteria that can prevent diarrhea in weaned piglets and its application. Background Technology

[0002] Piglet diarrhea is a common and prevalent disease in large-scale pig farms in my country, characterized by high morbidity and mortality, causing severe economic losses to the pig farming industry. Gut microbiota promotes intestinal development, enhances the digestion and absorption of nutrients, and regulates the body's immune function. The gut microbiota and immune system of suckling piglets are not yet fully developed, making them susceptible to pathogens and prone to diarrhea. In 2019, my country issued regulations prohibiting feed production enterprises from producing and selling commercial feed containing growth-promoting additives (excluding traditional Chinese medicine) starting July 1, 2020. This poses a serious challenge to the survival and development of feed production enterprises. Currently, the livestock and poultry farming industry faces the challenge of finding new antibiotic alternatives to adjust the balance of gut microbiota and inhibit intestinal inflammation, and the development of highly effective disease-resistant probiotic strains has been a focus of attention in the livestock industry.

[0003] In practice, piglet diarrhea is one of the most common and dangerous diseases in my country's pig industry. The causes of piglet diarrhea include feeding practices, farming environment, infectious diseases, and dietary habits. The occurrence of diarrhea is also related to the time since birth. The incidence of diarrhea in piglets within 0-7 days of birth is 0.6%, but it surges to 32% between 8-13 days, reaching as high as 41.4% between 14-17 days. Conversely, the incidence drops back to 8.4% between 22-28 days, while the mortality rate jumps to 20-30%. Piglet diarrhea not only inhibits their growth and development but can also lead to death in severe cases. Previous studies have found that even if piglets survive due to immune tolerance, there is a high probability of stunted growth, resulting in runts. In summary, piglet diarrhea seriously troubles pig farmers and causes significant economic losses to my country's pig industry.

[0004] Lactic acid bacteria are among the most commonly used probiotics. They can adjust the intestinal flora structure, reduce the infection of pathogenic bacteria in the intestine, thereby enhancing immune function. They can also, to some extent, improve diarrhea in weaned piglets caused by pathogenic bacteria. Superior lactic acid bacteria strains are continuously being developed. Lactic acid bacteria can be used alone or fed with animal feed to prevent and treat diarrhea in weaned piglets. They are relatively inexpensive and, when widely used in feed, can serve as an alternative to antibiotics, offering significant advantages in preventing diarrhea in weaned piglets. Summary of the Invention

[0005] The purpose of this 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] The present invention discloses a strain of Enterococcus mundtii PL86 for preventing diarrhea in weaned piglets. It is deposited at the China Center for Type Culture Collection (CCTCC), located at Luojia Mountain, Bayi Road, Wuchang District, Wu'an City, Hubei Province, on July 10, 2023, with accession number CCTCC NO:M 20231246.

[0007] The application of a strain for preventing diarrhea in weaned piglets in this application involves using Enterococcus mundtii PL86 to prevent the occurrence of weaned diarrhea in piglets.

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

[0009] The application of the strain of Enterococcus mundtii PL86 used in this application to prevent diarrhea in weaned piglets showed no toxicity after being used on animals.

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

[0011] The present invention relates to the application of a strain of Enterococcus mundtii PL86 that can prevent diarrhea in weaned piglets. When used in animals, it can inhibit the number of diarrhea-causing bacteria in the intestine and increase the content of lactic acid bacteria, thereby preventing the occurrence of diarrhea in piglets.

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

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

[0014] The Enterococcus mundtii PL86 strain of this invention exhibits excellent biological characteristics in vitro, demonstrating the strongest ability to produce protease and amylase. It effectively inhibits the growth of pathogenic Escherichia coli. Daily feeding to mice does not affect their weight or food intake, and the mice remain in good physical and mental condition with no significant toxic effects. During the feeding period, the content of lactic acid bacteria in the mice's feces increases. After challenge with pathogenic Escherichia coli, the strain inhibits the growth of Escherichia coli, and the content of Escherichia coli in the feces is significantly reduced. No diarrhea occurred during the experiment.

[0015] This invention is based on research into the pathogenesis of diarrhea in weaned piglets. It isolates and identifies the main dominant microorganisms in the feces of weaned piglets and studies their biological characteristics. This provides a scientific theoretical basis for the production of probiotic preparations that can prevent diarrhea in weaned piglets. It is of great significance for reducing the use of drugs in animal husbandry, producing functional livestock products, and protecting human health. Attached Figure Description

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

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

[0018] Figure 3 Electrophoresis diagram of 16S rRNA sequence amplification of isolated strains; where M: DL 2000; 1-12 are PCR products of strains PL2, PL6, PL9, PL12, PL24, PL84, PL86, PL92, PL96, PL104, PL106, and PL110, respectively; 13: negative control;

[0019] Figure 4 A phylogenetic tree diagram based on the 16S rRNA sequence of the isolated strains;

[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 The antibacterial activity of lactic acid bacteria strains;

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

[0027] Figure 9 This is a diagram illustrating the scarcity rating system. Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the spirit of the contents disclosed in the present invention will be described in detail below. After understanding the embodiments of the present invention, any person skilled in the art can make changes and modifications based on the technology taught in the present invention without departing from the spirit and scope of the present invention.

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

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

[0031] 1. Materials and Methods:

[0032] 1.1 Test Materials:

[0033] Fecal samples from the tested piglets were collected from large-scale pig farms in the three northeastern provinces, and the collection method was rectal sampling.

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

[0035] ②On December 11, 2021, at Lvkang Livestock Farm in Baicheng City, Jilin Province, feces from 12-day-old three-way crossbred piglets were collected, including 2 healthy feces and 3 feces with diarrhea.

[0036] ③ On December 11, 2021, five healthy feces were collected from 15-day-old three-way crossbred piglets at Dabeinong Qian'an Farm in Songyuan City, Jilin Province.

[0037] ④ On December 13, 2021, at Ganyiyuan Livestock Farm in Fuxin City, Liaoning Province, three healthy feces were collected from 10-day-old three-way crossbred piglets.

[0038] ⑤ On December 17, 2021, feces from 5 cases of diarrhea were collected from 32-day-old three-way crossbred piglets at Qian'an Farm, Zhaozhou County, Daqing City, Heilongjiang Province.

[0039] ⑥ On December 23, 2021, Weijia Village, Niuzhuang Town, Haicheng City, Anshan City, Liaoning Province, and Ganyiyuan Livestock Farm, Fuxin City, Liaoning Province, provided the second batch of feces from 7-day-old three-way crossbred piglets. Five cases of diarrheal feces and three cases of healthy feces were collected.

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

[0041] Table 1 Main Instruments and Equipment

[0042]

[0043] 1.3. Experimental methods and results:

[0044] 1.3.1. Strains Isolation and Purification:

[0045] Using MRS, NA, and CCA solid media, 133 suspected lactic acid bacteria, 97 suspected Bacillus, and 18 suspected Clostridium butyricum were initially isolated from the feces of healthy weaned piglets. The streak plate test was then performed. Single colonies were preliminarily screened and identified from the three media. The colonies were streaked and purified 2-3 times on each of the three media. Individual colonies were then selected, and after considering factors such as color, size, luster, and shape, representative strains were selected for streak plate testing, Gram staining, oil immersion microscopy, and catalase test.

[0046] The streak-and-divide method: This method uses MRS solid medium. The medium is divided into two equal parts using a labeling pen. Different selected bacterial strains are streaked in pairs on the two halves of the medium. The culture is then anaerobic at 30°C for 48 hours. The cultures are observed, and strains with different colony morphologies are selected for further screening. Initial screening using the streak-and-divide method yielded 48, 47, and 12 suspected lactic acid bacteria, Bacillus, and Clostridium butyricum strains with different colony morphologies, respectively.

[0047] Catalase test: This method uses an inoculation loop to pick up a single strain and smear it in catalase solution. The presence of bubbles indicates a positive result, otherwise a negative result. After screening using the catalase test, 35 catalase-negative suspected lactic acid bacteria strains and 26 catalase-positive suspected Bacillus strains were identified. Nine of these were suspected Clostridium butyricum strains, which underwent Gram staining and microscopic examination.

[0048] Gram staining: This method involves Gram staining of strains selected through the above methods.

[0049] In MRS medium, strains showing Gram-positive staining and catalase-negative results were preliminarily identified as *Lactobacillus*; in NA medium, strains showing both Gram-positive staining and catalase-positive results were preliminarily identified as *Bacillus*; and in CCA medium, strains showing both Gram-positive staining and catalase-negative results were preliminarily identified as *Clostridium butyricum*. MRS, NA, and CCA liquid media without agar were enriched at 30°C for 24 hours, then an equal volume of 40% glycerol was added, and after aliquoting, the media were stored at -80°C. Twelve Gram-positive lactic acid bacteria strains, nine Gram-positive Bacillus strains, and three Gram-positive Clostridium butyricum strains were screened by Gram staining microscopy. The lactic acid bacteria strains were named 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, respectively; the Bacillus strains were named PB 3, PB 11, PB 27, PB 43, PB 58, PB 71, PB 76, PB 89, and PB 96; and the Clostridium butyricum strains were named PC 5, PC 10, and PC 17.

[0050] The experimental results obtained after observing the morphology of each colony, performing the catalase experiment, and conducting Gram staining microscopy are shown in Table 2. The Gram staining microscopy results indicate that PL 24, PL 84, PL 86, PL 104, PB 96, and PC 10 cells are spherical, while the remaining strains are rod-shaped. Figure 2 The results of Gram staining are shown in the image.

[0051] Table 2 Preliminary isolation and screening of each strain

[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] Biochemical identification tubes for lactic acid bacteria include arabinose, cellobiose, fructose, glucose, lactose, maltose, mannitol, mannose, melitriose, ribose, salicin, sorbitol, rhamnose, sucrose, and xylose.

[0057] Biochemical identification tubes for Bacillus include catalase 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] Based on the physicochemical identification methods of the strains in the *Manual of Systematic Identification of Common Bacteria* and *Bergey's Manual of Bacteriological Identification*, the physiological and biochemical identification results of the 24 strains isolated in the experiment are shown in Tables 3 and 4. Preliminary identification indicates that PL 2, PL 6, PL 9, PL12, PL 24, PL 84, PL 86, PL 92, PL 96, PL 104, PL 106, and PL 110 are lactic acid bacteria; PB 3, PB 11, PB 27, PB 43, PB 58, PB 71, PB 76, PB 89, and PB 96 are Bacillus.

[0059] Table 3 Biochemical characteristics of lactic acid bacteria

[0060]

[0061]

[0062] Note: "+" indicates growth, and "-" indicates no growth. The same applies to the table below.

[0063] Table 4 Biochemical characteristics of Bacillus

[0064]

[0065] 1.3.3 In this application, DNA was extracted from Lactobacillus using a bacterial genomics kit. Subsequently, molecular biology techniques such as 16S rRNA gene sequence analysis and DNA hybridization were used to identify the species of the antidiarrheal probiotic. DNA extraction: First, the strain was Gram-stained and examined under a microscope and observed on agar plates to confirm that the strain had been purified. Then, single colonies were selected and inoculated into MRS liquid medium and cultured at 37°C for 24 hours. After that, the DNA of the strain was extracted and stored at -20°C.

[0066] PCR amplification: Using bacterial genomic DNA isolated from piglet intestines as a template, 16S rDNA gene fragments were amplified using universal primers: upstream 27F (5'-AGAGTTTGATCCTGGCTCAG-3') and downstream 1492R (5'-GGCTACCTTGTTACGACTT-3'). Primer synthesis and PCR product sequencing were performed by Beijing Liuhe Huada Co., Ltd.

[0067] The PCR amplification system consisted of a 25 μL reaction volume, including 1 μL each of forward and reverse primers, 2 μL of template DNA, 12.5 μL of 2×Taqplus Buffer, and 8.5 μL of sterile dEPC H2O. Sterile ultrapure water was used as a negative control. PCR amplification conditions were as follows: samples were pre-denatured at 94℃ for 5 min, denatured at 94℃ for 30 s, annealed at 55℃ for 30 s, and extended at 72℃ for 1 min 30 s. After 30 cycles, a final extension at 72℃ for 5 min was performed. After PCR amplification, 5 μL of the PCR product was extracted and electrophoresed at 110V for 30 min. The band size and intensity of all PCR products were measured using an SDS-PAGE UV gel imaging system. PCR products meeting the expected results were sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing.

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

[0069] 1.3.4 Data Analysis:

[0070] First, the sequencing results were assembled and then subjected to BLAST analysis using NCBI. Following this, relevant sequences retrieved from GenBank were used to align and compare the sequences using MEGA 11.0. A phylogenetic tree was then constructed using the neighbor-joining method. Finally, bootstrap analysis with 1000 random replicates was performed on the sequence data to evaluate the tree's topological structure.

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

[0072] BLAST comparisons were performed in GenBank to construct a genetic purification tree. PB 3 and PB 27 are in the same clade as Bacillus subtilis, with an evolutionary affinity of 92%. PB 43 and PB 96 are in the same clade as Bacillus subtilis, with an evolutionary affinity of 93%. PB 11, PB 58, and PB 76 are in the same clade as Bacillus subtilis, with an evolutionary affinity of 92%. PB 89 is in the same clade as Bacillus subtilis, with an affinity of 90%. PB 71 is related to halophilic Bacillus, with an affinity of 97%.

[0073] 2. Conclusion

[0074] Twenty-one dominant bacterial strains 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 montelukast*, and PL 84 and PL 104 were *Enterococcus faecium*. PB 3 and PB 27 belonged to *Bacillus subtilis*, PB 11, PB 43, PB 58, PB 76, PB 89 and PB 96 were *Bacillus subtilis*, and PB 71 was a halophilic bacillus.

[0075] Example 2: Biological characteristics of strains in the dominant gut microbiota 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, PL 106 and PL 110, Enterococcus lactis PL 24, Enterococcus montelukastii 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, and Bacillus halophilus PB 71 were isolated from the intestines of healthy piglets.

[0079] Broths at different pH values: Weigh out MRS liquid culture medium and NA broth separately, and dilute both types of broth to 1000 mL with double-distilled water. Divide the broth into 5 Erlenmeyer flasks and adjust the pH of the broths with 2 mol hydrochloric acid to pH 3.0, pH 3.5, pH 4.0, pH 4.5 and pH 5.0 respectively. Then, autoclave the Erlenmeyer flasks (115℃, 30 min) and place them in a clean bench to cool for later use.

[0080] Broth with different bile salt concentrations: Weigh MRS liquid culture medium and NA broth separately, and dilute them to 200 mL in Erlenmeyer flasks with double-distilled water. Add 0, 0.10, 0.20, and 0.30 g of ox bile salts to the dispensed culture media, respectively, so that the bile salt concentrations in the MRS and NA liquid culture media are 0%, 0.2%, 0.4%, and 0.6%. Then, autoclave the Erlenmeyer flasks (115℃, 20 min) and place them in a clean bench to cool for later use.

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

[0082] 1.3. Data Processing:

[0083] This experiment first used Excel 2021 to calculate and organize the experimental data, then used SPSS 23.0 statistical software to perform analysis of variance, and used Tukey's method to conduct multiple comparisons. The significance level of the differences was P<0.05. Finally, Graphpad Prism 8 was used to plot and present the experimental results.

[0084] 1.4. Experimental methods and results:

[0085] 1.4.1 Effect of different acid concentrations on the growth of the strain:

[0086] In this study, the pH of the liquid culture medium was adjusted using hydrochloric acid to five levels: pH 3.0, pH 3.5, pH 4.0, pH 4.5, and pH 5. The prepared culture medium was then dispensed into 5 mL portions per test tube and sterilized at 121°C under high pressure for 15 minutes. The culture medium of the test strain was added to the sterilized culture medium at the same dosage of 0.1 mL. After shaking at 160 rpm for 24 hours at 37°C, the acid and salt tolerance of the test strains was observed. The OD values ​​of each group of bacterial solutions were measured, with three replicates per group, and the average value was taken.

[0087] Effects of different acid concentrations on bacterial growth: Analysis of OD values ​​of 12 Lactobacillus strains cultured in MRS liquid at pH 5.8 for 24 hours and inoculated into MRS liquid media at pH 3–5 for 24 hours was performed. The results are as follows: Figure 5-1 Three strains of lactic acid bacteria were screened that showed relatively high tolerance to pH 3 and pH 3.5 in vitro. Their survival in MRS liquid culture medium is shown below. Figure 5-1In MRS liquid medium at pH 3, PL 96 showed a significantly higher survival rate than other strains (P < 0.05), but no significant difference compared to PL 86. At pH 3.5, PL 86 showed a significantly higher survival rate than other strains (P < 0.05), but no significant difference compared to PL 96. At pH 4, PL 86, PL 92, PL 96, and PL 104 showed significantly higher survival rates than other strains (P < 0.05), indicating good acid tolerance.

[0088] Depend on Figure 5-2 It can be seen that the nine Bacillus strains have poor tolerance to acidity in the pH range of 3 to 4.5. Among them, PB 43 has a higher survival rate at pH 4.5, which is significantly higher than that of other strains (P<0.05). At pH 5, PB 43 and PB 89 have higher survival rates and are significantly more tolerant to acid than other strains (P<0.05).

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

[0090] The salt concentration (w / v%) of the culture medium was adjusted to three levels: 0.2, 0.4, and 0.6 (w / v%) using analytical grade ox bile salt. The prepared culture medium was then dispensed into 5 mL portions per test tube. After sterilization at 121°C for 15 min under high pressure, 0.1 mL of the culture medium of the test strain was added to the sterilized culture medium. The culture was then shaken at 160 rpm for 24 h at 37°C. The acid and salt tolerance of the test strains was observed, and the OD values ​​of each group of bacterial solutions were measured, with three replicates per group. The average value was then taken.

[0091] Effects of different bile salt concentrations on bacterial growth: Lactic acid bacteria: Experimental results are from... Figure 6-1 As shown, the growth of *Lactobacillus* was inhibited to some extent at three different bile salt concentrations. However, strain PL 24 showed significantly higher survival rates than other strains at a bile salt concentration of 0.2% (P<0.05), while the difference between PL 12 and PL 96 was not significant. PL 24 and PL 84 showed significantly higher survival rates than other strains at bile salt concentrations of 0.4% and 0.6% (P<0.05). The fact that strains PL 12, PL 24, and PL 84 could grow and reproduce normally indicates that these strains have relatively good bile salt tolerance.

[0092] Bacillus: Experimental results by Figure 6-2As shown, the growth of Bacillus was inhibited to some extent at three different bile salt levels, and the viability of Bacillus decreased with increasing bile salt concentration in the culture medium. At a bile salt concentration of 0.2%, strain PB96 had a significantly higher survival rate than other strains (P<0.05), but no significant difference compared to PB58. At 0.4%, PB76 showed higher tolerance to bile salt concentration than other strains (P<0.05), while PB58 showed significantly higher tolerance to bile salt concentration than PB27 at a concentration of 0.6% (P<0.05).

[0093] 1.4.3 Sensitivity test of strains to different temperatures:

[0094] The temperature was adjusted to four levels: 45℃, 50℃, 55℃, and 60℃. After treatment for 30 minutes, the temperature sensitivity of the test strains was observed. The bacterial cultures were shaken at 160 rpm for 12 hours at 37℃ (with the temperature group set to stand for 6 hours). The OD values ​​of each group of bacterial cultures were measured, with three replicates per group, and the average value was taken.

[0095] Effects of different temperature levels on bacterial growth: Lactic acid bacteria: There were no significant differences in the growth and reproduction of bacterial strains at different temperatures. As the temperature increased within the range of 45–50℃, the strains exhibited relatively high sensitivity, with their growth and reproduction capacity gradually decreasing, although they could still grow. Specifically, at 45℃, the survival rate of PL 12 was significantly higher than that of PL 24 and PL 6 (P<0.05), and at 50℃, the growth and reproduction rates of PL 2, PL 9, and PL 86 were significantly higher than other strains (P<0.05). At 55–60℃, their reproductive capacity decreased, making it unsuitable for the growth and reproduction of lactic acid bacteria. Strain PL 24 showed relatively high temperature sensitivity (P<0.05), and its growth was not significant after treatment. Other temperatures had no effect on the growth and reproduction of the strains. Specific data can be found in [link to data]. Figure 7 .

[0096] Bacillus: When screening strains, they were subjected to a 75℃ water bath for 15 minutes. The strains were not sensitive to temperature, and their growth did not change significantly.

[0097] 1.4.4 Determination of the beneficial effects of dominant bacterial strains:

[0098] After centrifuging the activated strain at 6000×g for 5 min, the supernatant was filtered through a 0.22 μm sterile filter to remove bacteria. The bacterial culture was collected using Oxford cups, and the bacterial protease production capacity was determined using the casein hydrolysis test, while the amylase production capacity was detected using the starch hydrolysis test. Four wells (7 mm in diameter) were then evenly punched in the center of a culture plate. 150 μL of cell-free supernatant was added to each well, and uninoculated culture medium was added as a control. After incubation at 37℃ for 12 h, the diameter of the hydrolysis zone was measured.

[0099] The results of the 21 dominant strains hydrolyzing protein and starch are shown in Table 5. PL 86 showed significantly higher protease and amylase production capabilities than 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 all produce protease and amylase.

[0100] Table 5. Casein hydrolysis and starch hydrolysis tests for each strain

[0101]

[0102] Note: "-" indicates that no hydrolysis reaction occurred.

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

[0104] The strain was cultured for 48 hours, with 5 mL samples taken every 4 hours. The supernatant was collected by centrifugation, and its pH was measured. 200 μL of each supernatant was then used for antibacterial testing. A 100-fold diluted *E. coli* bacterial suspension was evenly spread on LB agar plates. Two 6 mm inner diameter Oxford cups were placed at equal intervals on each plate, and 200 μL of anti-diarrheal probiotic supernatant was added to each cup. The plates were incubated at 37°C for 24 hours, and the diameter of the inhibition zone in the fermentation supernatant was measured using calipers at each time point, with three replicates for each sample.

[0105] Results of antibacterial activity detection of strains against pathogenic Escherichia coli ATCC 25922: Twenty-one dominant strains were cultured for 48 hours, and the changes in the inhibition diameter of the supernatant after fermentation 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 inhibition diameter of PL 9 against pathogenic Escherichia coli ATCC25922 was significantly larger than that of other lactic acid bacteria strains (P<0.05), reaching 26 mm, which was not significantly different from PL 96 (P>0.05). However, the pH difference between PL 96 and PL 86 after 48 hours of culture was not significant (P>0.05). In contrast, the inhibition diameter of PB 96 in the Bacillus group was significantly larger than that of other Bacillus strains (P<0.05), reaching 11 mm, which was significantly different from that of PB 89 (P<0.05).

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

[0107] The dominant bacterial strain and Escherichia coli culture were diluted separately with sterile water to approximately 1.0 × 10⁻⁶. -8 CFU / ml, 2% of each was inoculated into MRS liquid medium and cultured at 37℃ with shaking at 150 rpm. 5 mL of bacterial culture was collected at 0h, 2h, 4h, 6h, 8h, 10h, 12h, 14h, and 16h, and the pH was measured. The culture was then serially diluted, and 200 μL of each diluted solution was evenly spread onto EMB plates and incubated at 37℃ for 24h. The number of viable E. coli remaining was counted, and the survival rate at each treatment time point was calculated.

[0108] Results of in vitro co-culture experiments between strains and pathogenic Escherichia coli ATCC 25922: The pH results of co-culturing 21 strains with pathogenic Escherichia coli ATCC 25922C are shown in Table 6. After co-culturing, the pH value initially decreased from 0 to 24 h, increased at 48 h, and then decreased again at 72 h. The pH of the co-cultured Bacillus group decreased slowly with time and then reached equilibrium. At 8 h, PB3 and PB9 were significantly lower than other strains (P<0.05). At 12 h, PB3, PB11, and PB27 were significantly lower than other strains (P<0.05). From 24 to 72 h, the pH of the PB3 co-cultured solution was significantly lower than other strains (P<0.05). The pH of the co-cultured Lactic Acid Bacteria group gradually decreased with time and then leveled off. At 12 h, PL86 and PL86 showed significantly lower pH values. The acid production capacity of PL86 mixed bacterial solution was significantly lower than that of other strains (P<0.05) at 24-48h, and significantly higher than that of other strains at 72h (P<0.05).

[0109] Table 6 pH values ​​of mixed culture

[0110]

[0111]

[0112] The residual colony counts of 21 Bacillus strains co-cultured with pathogenic Escherichia coli ATCC 25922C are shown in Table 7. The log value of the residual viable E. coli count initially increased with increasing culture time, reached an equilibrium at 4-8 hours, and then decreased sharply. After 24 hours of culture, no viable E. coli remained in the EMB culture medium. In the Bacillus group, strains PB 3 and PB 11 showed significantly lower values ​​at 8 hours of culture compared to other strains (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 those of strains cultured alone. This indicates that these dominant strains generally have strong acid-producing capabilities, although their acid production rate is slow, it is largely unaffected by the co-cultured E. coli in the medium.

[0113] Table 7. Number of residual colonies 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 bacterial flora of healthy piglets. Among them, 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 porcine Enterococcus montelukastii PL 86 on growth performance and diarrhea in mice:

[0118] 1. Materials and Methods:

[0119] 1.1 Test Materials:

[0120] Twenty-four 8-week-old SPF-grade male Bal b / c mice, weighing 20–22 g, were purchased from Changsheng Biotechnology Co., Ltd. They were kept under 12-hour light per day at a temperature of 21±2℃ and a relative humidity of 45%±10%. They were isolated and acclimatized for 7 days, during which time they had free access to food and water. Enterococcus montelukastii PL 86 and pathogenic Escherichia coli ATCC 25922 were preserved at the Key Laboratory of High-Efficiency Utilization and Nutritional Regulation of Cold Region Feed Resources at Heilongjiang Bayi Agricultural Reclamation University.

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

[0122] Table 8 Main Instruments and Equipment

[0123]

[0124] 1.3 Experimental Methods and Results

[0125] 1.3.1 Effects of Enterococcus montelukast PL 86 on body weight and feed intake in mice:

[0126] Twenty-four 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 consisted of two phases: the first phase was the construction of the probiotic prevention barrier, and the second phase was the challenge with pathogenic Escherichia coli ATCC 25922.

[0127] The first phase lasted 21 days, during which the PL 86 group and the PL 86+EcN group received 200 μL of 1.0 × 10⁻⁶ solution via gavage daily. 8 Enterococcus mongolicus PL 86 (CFU / mL) was administered to both the CON and EcN groups via gavage daily at the same dose of MRS liquid culture medium.

[0128] The second phase began on day 22 of the experiment and continued until all mice in the EcN group developed diarrhea. During this period, the PL 86+EcN group and the EcN group were administered 200 μL of 1.0 × 10⁻⁶ solution by gavage daily. 10 Pathogenic Escherichia coli ATCC 25922 (CFU / mL) was administered to both the CON and PL86 groups via gavage at the same dose of LB liquid culture medium daily.

[0129] In the first phase, mouse weight and food intake were measured every 3 days. In the second phase, mouse weight and food intake were measured daily.

[0130] Effects of Enterococcus montelukast PL 86 on body weight and feed intake in mice:

[0131] As shown in Table 9, the body weight of mice in all groups showed an increasing trend from 0 to 21 days. The body weight of mice gavaged with PL 86 did not decrease, and the mice in all groups were in good spirits. From 21 to 24 days, the body weight of mice in the EcN group showed a decreasing trend, while the body weight of the PL 86+EcN group did not change significantly. Furthermore, at 24 days, the body 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 food intake of all other groups showed a decreasing trend from 0 to 6 days. From 21 to 24 days after challenge, the food intake of the EcN group decreased significantly, while the food intake of the other groups remained unchanged.

[0132] Table 9. Changes in mouse body weight

[0133]

[0134] Note: Superscript letters indicate significant differences between groups; the same applies to the table below.

[0135] As shown in Table 10, except for the EcN group, the food intake of all other groups showed a decreasing trend from 0 to 6 days. After challenge with the virus at 21 to 24 days, the food intake of the EcN group decreased significantly. At 21 and 22 days, there was no significant difference in food intake between the EcN group and the other groups (P<0.05), while the food intake of the other groups did not change.

[0136] Table 10 Changes in food intake of mice

[0137]

[0138] 1.3.2 Effects of Enterococcus montelukast PL 86 on the number of lactic acid bacteria and Escherichia coli in the mouse intestine:

[0139] In the first stage, every 3 days, 1g of feces was collected from the tray 5 hours after the mice were gavaged and placed in MRS liquid culture medium. Then, the MRS liquid culture medium was placed in a shaker at 160 rpm and incubated at 35°C for 24 hours. After the culture was completed, the medium was serially diluted and plated. The number of lactic acid bacteria was counted after anaerobic in MRS culture dishes for 24 hours. Each group was replicated 3 times.

[0140] In the second stage, the number of lactic acid bacteria was measured daily. At the same time, 1g of feces was collected from the tray 5 hours after the mice were gavaged and placed in LB liquid medium. The LB liquid medium was then placed in a shaker at 160 rpm and incubated at 35°C for 24 hours. After the incubation was completed, the medium was serially diluted and plated. The number of Escherichia coli was calculated after 24 hours of anaerobic incubation in LB culture dishes. Each group had 3 replicates.

[0141] Results of the effects of Enterococcus montelukast PL 86 on the number of lactic acid bacteria and Escherichia coli in the mouse intestine:

[0142] Table 11 shows the lactic acid bacteria in the mouse intestines. The lactic acid bacteria content in the feces of mice in the PL 86 and PL 86+EcN groups showed an increasing trend from 0 to 3 days. Throughout the 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 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 Lactic acid bacteria content in mouse feces

[0144]

[0145] Note: Superscript letters indicate significant differences between groups; the same applies to the table below.

[0146] The levels of E. coli in the feces of mice after challenge are shown in Table 12. The levels of E. coli in the feces of the EcN group continued to rise, and the average levels of E. coli from 21 to 24 days were significantly higher than those of the PL 86+EcN group (P<0.05). The levels of E. coli in the feces of the PL 86+EcN group decreased on day 23, at which point the levels of fecal E. coli were significantly lower than those of other groups (P<0.05). The levels rebounded on day 24, but were still significantly different from those of the EcN group (P<0.05).

[0147] Table 12 Escherichia coli content in mouse feces

[0148]

[0149] 1.3.3 Effects of Enterococcus montelukast PL 86 on organ indices and diarrhea indices in mice:

[0150] In the second phase of the experiment, the number of mouse feces and the amount of loose stool were recorded daily, and the loose stool was graded as follows: Figure 9 As shown, the diarrhea index was calculated. During the experimental period, all mice in the EcN group experienced diarrhea. All mice were sacrificed on the same day, and the colon length was measured. The weights of the liver, kidneys, and spleen were measured, and the indices of each organ were calculated. The specific calculation formulas are 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 per animal

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

[0154] Average loose stool grade = Total number of 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 montelukastii PL 86 on organ indices and diarrhea indices in mice:

[0158] On day 24 of the experiment, all mice in the EcN group experienced diarrhea, while mice in the other groups did not experience diarrhea and remained in good condition throughout the experiment. Organ and diarrhea indices are shown in Table 13. The diarrhea index of the EcN group was significantly higher than that of the other groups (P<0.001). After challenge, the colon length in the EcN group was significantly shortened, showing a significant difference compared to the PL 86 and PL 86+EcN groups (P<0.05). After feeding with PL 86, the liver index of the mice significantly increased, and was higher than that of the EcN group, with significant differences between groups (P<0.05). The kidney index did not change significantly. The spleen index was significantly higher in the EcN group, showing differences between all groups (P<0.05).

[0159] Table 13 Organ Index and Diarrhea Index of Mice

[0160]

[0161] 1.4 Data Processing

[0162] This experiment first used Excel 2021 to calculate and organize the experimental data, then used SPSS 23.0 statistical software to perform analysis of variance, and used Tukey's method to conduct multiple comparisons. The significance level of the differences was P<0.05. Finally, Graphpad Prism 8 was used to plot and present the experimental results.

[0163] 2. Conclusion: Feeding mice with Enterococcus mundtii PL 86 did not cause lethargy or weight loss, and it could increase the number of lactic acid bacteria in the intestines to a certain extent, inhibiting the invasion of bacterial pathogens into the animal intestines and effectively preventing the occurrence of diarrhea in animals.

[0164] Example 4: Effects of porcine Enterococcus montelukastii PL 86 on diarrhea in piglets:

[0165] 1. Materials and Methods:

[0166] 1.1 Test Materials:

[0167] Enterococcus montelukast PL 86 was preserved in the Key Laboratory of High-Efficiency Utilization and Nutritional Regulation of Cold Region Feed Resources at Heilongjiang Bayi Agricultural Reclamation 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 trials were conducted at the following three pig farms: Lvkang Livestock Farm in Baicheng City, Jilin Province; Dabeinong Qian'an Farm in Songyuan City, Jilin Province; and Qian'an Farm in Zhaozhou County, Daqing City, Heilongjiang Province.

[0172] 1.3.2 Grouping of experimental animals

[0173] In each of the aforementioned pig farms, male and female piglets of similar age and weight were selected. Each farm had 12 healthy piglets (6 males and 6 females) and 36 piglets with diarrhea (18 males and 18 females). The experimental treatments were divided into four groups of 12 piglets each (6 males and 6 females): a control group (6 healthy males and 6 females), a diarrhea group (6 males and 6 females with diarrhea, not fed any probiotics or medications), a probiotic group (6 males and 6 females with diarrhea, fed Enterococcus montelukast PL86), and an antibiotic group (6 males and 6 females with diarrhea, fed the diarrhea treatment drug enrofloxacin). Enterococcus montelukast PL86 was cultured at a concentration of 1.0 × 10¹⁰ CFU / mL, and 0.2% Enterococcus montelukast PL86 was added to the diet. Enrofloxacin was administered according to the instructions. Each group had free access to food and water and was managed according to the farm's standard feeding methods for 7 days.

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

[0175] Diarrhea Index = Loose Stool Rate × Average Loose Stool Grade

[0176] Loose stool rate = number of loose stools per piglet ÷ total number of stools per piglet.

[0177] The loose stool grade indicates the degree of loose stool in each piglet. The scoring standard is based on the appearance of the feces and is divided into 4 grades: Grade 1 is strip-shaped or granular and relatively hard; Grade 2 is normal shape and soft; Grade 3 is thick, partially formed, and without water separation; and Grade 4 is unformed, liquid, and with fecal-water separation.

[0178] Average loose stool grade = Total number of 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 experiment used SPSS 23.0 statistical software to perform analysis of variance and Tukey's method to conduct multiple comparisons. The significance level of the differences was P<0.05.

[0182] 2. Results and Analysis

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

[0184] Table 14 Diarrhea Index in Piglets

[0185]

[0186] 3. Conclusion: Feeding piglets with Enterococcus mundtii PL 86 can effectively inhibit the occurrence of diarrhea in piglets, and its therapeutic and control effects on piglet diarrhea are comparable to those of enrofloxacin.

Claims

1. The application of a strain of bacteria that prevents diarrhea in weaned piglets in the preparation of drugs for preventing diarrhea in weaned piglets, characterized in that: It is Enterococcus montelukastii ( Enterococcus mundtii PL86 is deposited at the China Center for Type Culture Collection, located at Luojia Mountain, Bayi Road, Wuchang District, Wu'an City, Hubei Province, on July 10, 2023, with accession number CCTCCNO: M 20231246. The aforementioned Enterococcus montelukast ( Enterococcus mundtii PL86 can inhibit the number of diarrhea-causing bacteria in the intestines and prevent diarrhea in weaned piglets. The aforementioned prevention of diarrhea in weaned piglets refers to feeding them Enterococcus montelukastii (Enterococcus mongolicus). Enterococcus mundtii PL86 can reduce the content of E. coli in the intestines, increase the content of lactic acid bacteria, and prevent diarrhea. The diarrheagenic bacteria in the gut is pathogenic E. coli ATCC 25922.

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