Weissella confusa DY12 with probiotic activity and its application

Abstract

The present invention relates to the technical field of the development and utilization of probiotics, specifically to a Weissella confusa DY12 with probiotic activity and its application. The Weissella confusa DY12 was isolated from sheep feces. The present invention identified the probiotic effect of Weissella confusa DY12 through animal experiments. The results showed that Weissella confusa DY12 could tolerate acid and bile salts and resist the gastrointestinal internal environment; its hydrophobicity was 39.47% and its auto-aggregation was 48.12%, demonstrating that the Weissella confusa DY12 of the present invention has good intestinal adhesion ability and has the potential of probiotics. The present invention verified that Weissella confusa DY12 can effectively regulate the immune function of the animal body, improve the antioxidant capacity of the body, as well as the content of lysozyme and the activity of alkaline phosphatase, and increase the daily weight gain, ultimately improving the growth performance of animals.

Description

Technical Field

[0001] The present invention relates to the technical field of the development and utilization of probiotics, and specifically to a Weissella confusa DY12 with probiotic activity and its application. Background Art

[0002] At present, antibiotics are commonly used to relieve or control clinical bacterial diseases such as Escherichia coli and Salmonella. Therefore, antibiotics are still an important means for treating bacterial infections during the breeding process. However, the repeated use, overuse, or abuse of antibiotics can lead to drug side effects. In addition, while killing pathogenic bacteria, antibiotics may also have an adverse impact on intestinal microorganisms, thereby causing irritable bowel syndrome (IBS) and various immune diseases. More importantly, the overuse and abuse of antibiotics can lead to the generation and spread of antibiotic-resistant strains, posing a serious threat to the breeding industry and thus threatening the safety and health of humans. In this context, probiotic preparations have opened up a new way for the production of natural, safe, high-quality, green, and healthy animal products. As a new type of feed additive, it has advantages in alleviating the shortage of feed resources, improving the nutritional structure, reducing feed costs, and protecting the environment, and can be widely applied to the breeding of poultry, pigs, and ruminants, which is of great significance for the healthy and sustainable development of China's breeding industry.

[0003] Lactic acid bacteria (LAB) are the most common Gram-positive bacteria among probiotics and produce various beneficial functions for the host, mainly including the following four points: 1. By producing antibacterial substances such as organic acids, bacteriocins, and hydrogen peroxide, and competitively binding to the adhesion sites of intestinal mucosal epithelial cells, the content of pathogenic bacteria such as Escherichia coli and Salmonella in the intestine is reduced, thereby maintaining the balance of the intestinal flora; 2. By stimulating the host immune system, resisting pathogen infections, and promoting the body's humoral and cellular immunity, the immunomodulatory effect is exerted; 3. By eliminating harmful substances in the body, such as free radicals and toxins, the antioxidant capacity of animals is enhanced, the fatty acid metabolism process is intervened, and the brightness and tenderness of meat products are significantly improved; 4. Produce a variety of digestive enzymes, such as cellulase and protease, to help the body better digest, promote the absorption of nutrients by the host, and thus promote the growth of the host. In addition, the feed fermented by lactic acid bacteria contains a large number of lactic acid bacteria and lactic acid on the one hand, which can inhibit the growth of spoilage bacteria and extend the storage period of the feed; on the other hand, it is also rich in organic acids and various flavor substances such as sugars, alcohols, and esters, which can improve the palatability of the feed and increase the feed intake of animals. At present, the lactic acid bacteria strains used are mostly Lactobacillus plantarum, Lactobacillus acidophilus, Pediococcus acidilactici, Enterococcus faecalis, and Enterococcus faecium. Weissella, as a kind of lactic acid bacteria, can not only produce antibacterial substances such as bacteriocins and organic acids, but also produce exopolysaccharides, and thus has attracted more attention in the pharmaceutical, food and other industries.

[0004] At present, there are relatively few studies and applications on the probiotic effects of Weissella in animals. However, existing studies have shown that Weissella is a potential probiotic, with great research potential. Therefore, screening out a strain of Weissella with good tolerance, auto-aggregation ability, antibacterial activity, growth promotion, and immune promotion characteristics is of great significance for the healthy and green development of the current aquaculture industry. Summary of the Invention

[0005] In response to the above problems, the present invention provides a fusant Weissella with probiotic activity, which has broad-spectrum antibacterial ability, acid tolerance, bile salt tolerance, can increase the daily weight gain of animals, and has the ability to adhere to intestinal epithelium.

[0006] To achieve the above object, the following technical solutions are provided:

[0007] The fusant Weissella of the present invention was isolated from sheep feces and named DY12. Through 16S rRNA gene sequence analysis, this strain DY12 is Weissella confusa. This strain was deposited at the China Center for Type Culture Collection (abbreviated as CCTCC) on July 22, 2024. Address: Wuhan, China. It was classified and named Weissella confusa DY12, and the deposit number is CCTCC NO: M 20241630.

[0008] The fusant Weissella confusa DY12 is a Gram-positive bacterium.

[0009] The present invention provides a bacterial agent containing the aforementioned fusant Weissella confusa DY12.

[0010] The present invention also provides the use of the fusant Weissella confusa DY12 in the preparation of feed additives or animal feeds.

[0011] Preferably, the feed additive or animal feed has broad-spectrum antibacterial activity.

[0012] Preferably, the feed additive or animal feed is a product that promotes animal growth and regulates the immune function of animals.

[0013] Preferably, the feed additive or animal feed is a product that improves antioxidant capacity.

[0014] The present invention has demonstrated through animal experiments that the above-mentioned fusant Weissella confusa DY12 can improve the growth performance of animals, enhance the antioxidant capacity of the body, and effectively regulate the immune function of animals.

[0015] In the present invention, the antibacterial spectrum of the Weissella confusa DY12 is selected from Gram-positive bacteria or Gram-negative bacteria.

[0016] In the present invention, the Gram-negative bacteria are pathogenic Escherichia coli and Salmonella enteritidis.

[0017] Salmonella is a Gram-negative, facultative anaerobic intracellular bacterium that is widely distributed in nature. Its members can infect humans and a variety of animals, and most of them are highly pathogenic. Among them, Salmonella enteritis (SE) can cause enteritis, reduced production performance, and even death in poultry and livestock, and animal products contaminated with this bacterium can further cause acute gastritis in humans, seriously endangering human health and public safety.

[0018] The present invention also provides a drug containing the aforementioned Weissella confusa DY12.

[0019] The beneficial effects of the present invention are as follows:

[0020] 1. The present invention identified the probiotic effects of Weissella confusa DY12 through animal experiments. The results showed that Weissella confusa DY12 can tolerate acid and bile salts and resist the gastrointestinal environment; its hydrophobicity is 39.47% and its auto-aggregation ability is 48.12%, proving that Weissella confusa DY12 of the present invention has good intestinal adhesion ability and has the potential of probiotics.

[0021] 2. The present invention verified that Weissella confusa DY12 can effectively regulate the immune function of animals, improve the antioxidant capacity of the body, as well as the content of lysozyme and the activity of alkaline phosphatase, and increase the daily weight gain, ultimately improving the growth performance of animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the streak plate of Weissella confusa DY12 in MRS agar medium in the present invention;

[0023] Figure 2 It is the result of staining microscopy of Weissella confusa DY12 in the present invention (10×100μm);

[0024] Figure 3 It is the phylogenetic tree in the present invention;

[0025] Figure 4 It is the antibacterial result in the embodiment of the present invention; MRS: MRS broth is used as a negative control; Figure 4 A is Salmonella; Figure 4 B is Escherichia coli;

[0026] Figure 5 The hemolysis results in the embodiments of the present invention; Figure 5 A is a positive control bacterium: Staphylococcus aureus; Figure 5 B is Weissella confusa DY12;

[0027] Figure 6 The organ index results in the embodiments of the present invention;

[0028] Figure 7 The daily weight gain results in the embodiments of the present invention;

[0029] Figure 8 The contents of IgG in the blood of mice and sIgA in the jejunum in the embodiments of the present invention; A: the content of IgG in the blood; B: the content of sIgA in the jejunum, CG represents the control group; LG represents the low-dose group; HG represents the high-dose group. Detailed implementation manners

[0030] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0031] It should be clear that the experimental methods used in the following embodiments are all conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0032] Statistical processing: Statistical analysis was performed using GraphPad Prism 8.0.2, and the significance level of difference was set to P < 0.05 or P < 0.01.

[0033] Example 1 Isolation and identification of ovine-derived Weissella confusa DY12

[0034] 1. Isolation of Weissella: Ovine-derived fecal samples were from a slaughterhouse in Sichuan Province. The collected fecal samples were serially diluted with PBS and spread on GYP solid medium, and cultured in a constant temperature anaerobic incubator at 37 °C for 24 h. Single colonies with a calcium dissolution zone were picked and purified on MRS agar medium until the colony morphology remained unchanged. The colonies of Weissella on MRS agar medium were round, slightly raised, transparent or opaque, with smooth edges Figure 1 .

[0035] Drop a drop of sterile normal saline on a clean glass slide. Use an inoculation loop to pick up a small amount of bacteria and spread it on the sterile normal saline. After drying, perform Gram staining, microscopic examination and observe the colony morphology. Select the strains with short rod-shaped cells and positive Gram staining for unified numbering. After screening tests such as resistance to artificial gastrointestinal fluid, it was found that the biological characteristics of strain DY12 were the best. Strain DY12 is a Gram-positive bacterium, usually short rod-shaped, single, paired or short-chain under the microscope, as Figure 2 shown.

[0036] 2. PCR identification

[0037] DNA extraction: Extract the DNA of the antibacterial active strain by the phenol-chloroform method, and use a ultra-micro nucleic acid and protein analyzer to detect the DNA concentration and purity, with the criteria of A260 / A280 > 1.8 and A260 / A230 > 2.0.

[0038] Using the strain DNA as a template, select the 16S rRNA universal primers

[0039] 27F(5´-AGAGTTTGATCCTGGCTCAG-3´), 1492R(5´-GGTTACCTTGTTACGACTT-3´). The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and PCR amplification was performed on the synthesized primers.

[0040] The PCR amplification system is: 2 µL of DNA template, 10 µL of 2×T5 Super PCR Mix, 1 µL of 27F, 1 µL of 1492R, filled up to 20 µL with double-distilled water (dd H2O). The PCR amplification conditions are: pre-denaturation at 95℃ for 30 min; denaturation at 94℃ for 30 s; annealing at 50.8℃ for 30 s; extension at 72℃ for 90 s, 35 cycles; and further extension at 72℃ for 5 min.

[0041] Agarose gel electrophoresis: Use a 2% agarose gel and 1×TAE buffer as the electrophoresis medium to detect the PCR amplification products by electrophoresis. Electrophoresis conditions: voltage 120 V, current 200 mA, time 26 min. The sequencing of the PCR products was completed by Sangon Biotech Co., Ltd. The sequencing results were assembled, and the sequencing results were submitted to the GenBank database of the National Center for Biotechnology Information (NCBI) in the United States for local alignment using the Basic Local Alignment Search Tool (BLAST) to determine the species. A phylogenetic tree was constructed using MEGA11, starting from Figure 3It can be seen that the isolated Weissella is Weissella confusa.

[0042] Strain preservation information:

[0043] Weissella confusa DY12 was sent to the China Center for Type Culture Collection (CCTCC) for preservation. The preservation address is Wuhan, Hubei Province. The preservation number is CCTCC NO: M 20241630. The taxonomic name is Weissella confusa DY12, and the preservation date is July 22, 2024.

[0044] Example 2 Determination of the antibacterial ability of the supernatant of Weissella confusa from sheep

[0045] The bacterial solution was inoculated into MRS liquid medium in proportion. After culturing at 37 °C for 24 h, a seed solution was prepared. The fermentation supernatant was obtained by centrifuging at 10,000 rpm for 15 min at 4 °C using a high-speed refrigerated centrifuge. After removing the bacteria and other impurities in the supernatant with a 0.22 µm disposable sterile filter, it was stored at -20 °C for later use.

[0046] The Oxford cup method antibacterial test was used to measure the antibacterial ability of the fermentation supernatant. The quality control strains were Escherichia coli ATCC25922 and Salmonella ATCC14028 respectively. The detection plate was selected as the nutrient agar medium LB. The indicator bacteria were adjusted to a turbidity of 0.5 with an ultraviolet spectrophotometer OD 600 nm as the turbidity of the indicator bacteria suspension. 0.3 mL of the bacterial solution was pipetted onto the LB medium and evenly spread on the surface of the medium with a disposable spreading rod. The Oxford cups were placed in the center of the petri dish. Approximately 0.2 mL of the fermentation supernatant was slowly added to the Oxford cups, and a control was set. Subsequently, it was placed upright in a 4 °C refrigerator for 8 h. After the supernatant diffused, it was placed in a 37 °C incubator for 12 h, and then the antibacterial zone was observed and recorded. (The results are shown in Table 1, Figure 4 )

[0047] Table 1 Antibacterial effect

[0048]

[0049] Note: "-" indicates that the antibacterial zone < 8 mm has no antibacterial activity; +: the diameter of the antibacterial zone is 8 - 11 mm, ++: the diameter of the antibacterial zone is 12 - 17 mm, +++: the diameter of the antibacterial zone is 18 - 23 mm.

[0050] Table 1, Figure 4 The results showed that Weissella confusa DY12 had good inhibitory effects on Escherichia coli and Salmonella enteritidis.

[0051] Example 3 Biological characteristics of Weissella confusa DY12 from sheep

[0052] 1. Growth curve of Weissella confusa DY12

[0053] Inoculate Weissella confusa DY12 into 50 mL of MRS broth at a ratio of 2%, and culture it at a constant temperature of 37°C. Starting from 0 h, take 3 mL every two hours to measure the OD 600 nm value, record the data, and repeat three parallel controls, using the blank MRS medium as a control. The results show that Weissella confusa DY12 follows the laws of the lag phase, logarithmic phase, stationary phase, and decline phase. The strain is in the lag phase in the first 2 h, the logarithmic growth phase from 2 - 10 h, and enters the plateau phase after 10 h. After entering the stationary phase and decline phase, the viable bacteria will be inhibited by their own metabolites. It can be determined that the optimal harvesting time of this bacterium is 10 h.

[0054] 2. Experiment on the gastrointestinal fluid tolerance of ovine-derived Weissella confusa DY12

[0055] Preparation of gastrointestinal fluid: Add 0.35 g of pepsin to 100 mL of 0.2% sterile physiological saline to make gastric juice. Adjust the pH of the solution to 2.5 with 1 mol / L HCl, and store it for later use through a 0.22 μm filter. Prepare intestinal fluid by adding 0.1 g of trypsin and 1.8 g of bovine bile salt to 100 mL of distilled water containing 1.1 g of NaHCO3 and 0.2 g of NaCl. Adjust the pH of the solution to 8.0 with 1 mol / L NaOH, and store the solution after filtering through a 0.22 μm filter for later use. Inoculate the bacterial solution (10 8 ~10 9 CFU / mL) into the simulated gastric juice at an inoculation amount of 10%, and perform plate counting at 0 h and 3 h (intestinal fluid at 4 h) respectively to measure the viable bacteria count. Calculate the survival rate (%) of the strain according to the following formula:

[0056] Survival rate (%) = C1 / C2 × 100%

[0057] Where: C1 is the viable bacteria count (CFU / mL) measured after gastrointestinal fluid treatment; C2 is the viable bacteria count (CFU / mL) measured in the gastrointestinal fluid at 0 h.

[0058] The results show that the survival rate of Weissella confusa DY12 in gastric juice at 3 h is 72.35%, and the survival rate of the bacterial solution in intestinal fluid at 4 h is 48.49%. Weissella confusa DY12 has good acid and bile salt tolerance characteristics and has the potential to become a probiotic.

[0059] 3. Determination of autoaggregation and hydrophobicity of ovine-derived Weissella confusa DY12

[0060] (1)Auto-aggregation: Inoculate the seed liquid into the sterilized liquid MRS medium at 2% respectively, and culture at 37 °C for 24 h. Centrifuge the culture solution at 4 °C and 10,000 r / min for 10 min, discard the supernatant, wash twice with phosphate buffer solution at pH 7.2, and then resuspend in PBS. Adjust the concentration of the bacterial suspension to 10 7 ~10 8 CFU / mL. Take 3 mL of the bacterial suspension and measure the absorbance at a wavelength of 600 nm, denoted as A0. After the bacterial suspension stands at 37 °C for 5 h, take 3 mL of the supernatant into another test tube and measure the absorbance at 600 nm, denoted as At. Repeat the independent experiment 3 times and take the average value.

[0061]

[0062] At represents the OD value at t = 5 h; A0 is the OD value at t = 0 h. 600 value; A0 is the OD value at t = 0 h 600 value.

[0063] (2)Hydrophobicity: Prepare the bacterial suspension according to (1), then add an equal volume of xylene. Vortex-mix the two-phase system for 3 min, let it stand at 37 °C for 1 h, take out the supernatant, and measure the absorbance of the aqueous phase at 600 nm, denoted as A. Calculate the surface hydrophobicity of the strain according to the formula. Repeat the independent experiment 3 times and take the average value.

[0064]

[0065] In the formula: A0 and A represent the absorbances of the Weissella confusa suspension and the aqueous phase after mixing the two phases respectively.

[0066] Weissella confusa needs to have the ability to colonize in the host intestine and prevent pathogenic bacteria from colonizing in the intestine. The auto-aggregation ability is an important index for evaluating its ability to adhere to intestinal cells in vitro. The auto-aggregation ability is generally divided into the following three categories: weak (16% - 35%), medium (35% - 50%), and strong (higher than 50%). The auto-aggregation and hydrophobicity of Weissella confusa DY12 determined in the present invention are 48.12% and 39.47% respectively, indicating that the strain has good intestinal adhesion ability.

[0067] 4. Determination of the antioxidant capacity of Weissella confusa DY12 from sheep

[0068] (1)Determination of H2O2 tolerance: Add H2O2 solution to the sterilized MRS liquid medium respectively to make the initial H2O2 concentrations in the medium be 0, 1.0, 2.0, and 3.0 mmol / L respectively. The cell concentration is 1×10 8The Weissella confusa bacterial solution with a concentration of CFU / mL was inoculated into a liquid medium containing different concentrations of H2O2 at an inoculation amount of 1%, and cultured in a constant temperature incubator at 37°C. After 8 h, the OD value at 600 nm of the Weissella confusa bacterial solution grown at different H2O2 concentrations was measured using a spectrophotometer.

[0069] (2) DPPH free radical scavenging activity: Take 1 mL of the sample, add 2 mL of a 0.2 mmol / L DPPH anhydrous ethanol solution, mix well, and react in the dark at room temperature for 30 min. Centrifuge at 8000 g for 10 min at 4°C, take the supernatant, measure the absorbance of the supernatant at 517 nm, zero with deionized water, and perform each experiment in parallel three times.

[0070] DPPH scavenging rate (%) = ×100%

[0071] Where: A i is the absorbance of the sample group; A j is the absorbance of the blank group; A0 is the absorbance of the control group.

[0072] (3) Hydroxyl radical scavenging activity: Pipette 0.5 ml of the sample and 1 ml of o-phenanthroline (concentration 0.1%), add 1 mL of PBS, 1 mL of 2.5 mmol / L FeSO4, and 1 mL of 20 mmol / L H2O2. After incubating in a water bath at 37°C for 1.5 h, measure the absorbance at 536 nm. Calculate the scavenging rate of hydroxyl radicals using the following formula, and perform each experiment in parallel three times.

[0073] Hydroxyl radical scavenging rate (%) = ×100%

[0074] Where: A0 is the absorbance of the blank group; A1 is the absorbance of the control group; A2 is the absorbance of the sample group.

[0075] (4) Determination of O 2 - radical scavenging ability: The mixed reaction solution includes: 2.8 mL of Tris-HCl (0.05 mol / L, pH 8.2), 0.1 mL of pyrogallol (0.05 mol / L), and 0.1 mL of the sample. Mix the mixed reaction solution well and react at 25°C in the dark for 4 min. After the reaction is completed, add 1 mL of 8 mol / L HCl to terminate the reaction, and detect the absorbance at 320 nm.

[0076] Superoxide anion scavenging ability (%) = ×100%

[0077] Where: A0 is the blank control without adding the sample, and A1 is the absorbance of the sample added.

[0078] (5)Determination of the total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) of Weissella confusa DY12: The T-AOC and T-SOD activities of the supernatant of Weissella confusa DY12 were determined using a kit, and the specific method was carried out strictly according to the kit instructions.

[0079] The results showed that when the H2O2 concentrations of Weissella confusa were 0, 1.0, 2.0, and 3.0 mmol / L respectively, the OD 600 values were 1.351, 1.040, 0.527, and 0.510 respectively, indicating that it could tolerate a relatively high concentration of H2O2; the DPPH radical scavenging rate of Weissella confusa was 15.32%; the hydroxyl radical scavenging rate was 6.58%; the O 2- radical scavenging rate was 6.58%; the total antioxidant capacity (T-AOC) was 1.36 U / mL; the superoxide dismutase (T-SOD) was 106.032 U / mL. It was shown that Weissella confusa DY12 had certain antioxidant characteristics and could improve the antioxidant capacity of the animal body.

[0080] Example 4 Safety of the ovine-derived Weissella confusa DY12 strain

[0081] (1)Hemolytic experiment of ovine-derived Weissella confusa DY12: The centrifuged Weissella confusa was inoculated on a blood agar plate in a streaking form and cultured at 37 °C for 48 h. The hemolysis situation was observed. Strains that produced a green zone (α-hemolysis) around the colony or did not produce any hemolysis (γ-hemolysis) on the blood agar plate were considered non-hemolytic. Strains with a hemolytic zone (transparent zone) around the colony were classified as microorganisms with hemolytic (β-hemolysis) characteristics. The appearance of a transparent β-hemolytic ring was a positive hemolytic phenotype. Staphylococcus aureus was used as a positive control strain.

[0082] The results showed that the hemolysis result of Weissella confusa DY12 was negative and its safety was relatively high, as shown in Figure 5 .

[0083] (2)Antibiotic sensitivity test of ovine-derived Weissella confusa DY12: The K-B disk diffusion method was used to detect the sensitivity of the screened strain to common antibiotics. 200 μL of the seed solution was pipetted and inoculated into 8 mL of MRS broth, cultured at 37 °C for 24 h, and the bacterial solution was adjusted to a concentration of 10 8 CFU / mL. A disposable sterile cotton swab was dipped in the bacterial solution and evenly spread on the MH medium. The drug sensitivity disks were taken out with sterile forceps and placed on the surface of the medium in turn. The drug sensitivity disks were gently pressed with forceps to make them fit tightly with the medium. Four drug sensitivity disks were placed on each petri dish. It was cultured at 37 °C for 16 h, and the diameter of the inhibition zone was measured with a ruler and the results were recorded.

[0084] Table 2 Drug sensitivity results

[0085]

[0086] According to the test standards of the Clinical and Laboratory Standards Institute (CLSI) of the United States, antibiotic resistance was evaluated by the Kirby-Bauer disk diffusion method. The drug sensitivity test results were divided into the following three levels: Resistant, Intermediate, and Susceptible. As shown in Table 2, the Weissella confusa DY12 was sensitive to most antibiotics, had no drug resistance, and had relatively high safety.

[0087] (2)Acute oral toxicity experiment in mice

[0088] Twelve male SPF-grade KM mice were selected, with 6 in each of the experimental group and the control group. After one week of adaptive feeding, the experiment was carried out. In the experimental group, the mice were intragastrically administered with the bacterial solution of Weissella confusa at 1×10 10 CFU / mL, and the blank control group was intragastrically administered with the same volume of MRS broth. The administration was continued for 21 days. During this period, the mental state, diet, feces, etc. of the mice were observed. After 21 days, the mice were dissected, and the heart, lungs, liver, spleen, and kidneys of the mice were removed to observe any obvious pathological changes, and the organ activity index was measured.

[0089] The results showed that during the intragastric administration of the mice, the mental state and diet were good, and there were no cases of diarrhea or death. After dissection, it was found that there were no obvious differences and pathological changes in the weights of the heart, liver, spleen, and lungs of the mice in the blank control group compared with those in the experimental group. By measuring the organ weights of the mice, it was found that there was no significant difference in the organ indices between the experimental group and the blank control group (P>0.05). See Figure 6 . From the results of the safety experiment, it can be seen that Weissella confusa DY12 as an animal feed additive does not have a high risk.

[0090] Example 5 Effects of sheep-derived Weissella confusa DY12 on the growth performance, antioxidant capacity, and immunity of mice

[0091] The Weissella confusa was streaked on a plate and cultured at 37°C for 24 h. Single colonies were picked and cultured in MRS liquid medium for 24 h. After being washed twice with PBS and resuspended, they were serially diluted. After plate counting, the final concentrations were determined to be 1×10 8 CFU / mL and 1×10 10 CFU / mL. They were aliquoted into 10 mL centrifuge tubes and stored at -20°C in a refrigerator.

[0092] Male SPF-grade KM mice at 4 weeks of age, weighing 20 ± 2 g, were selected. Sixty mice were randomly divided into three groups: a control group, a low-dose group, and a high-dose group. Control group: The mice were not treated and were normally raised. Low-dose group: The mice were intragastrically administered 0.2 mL of Weissella confusa bacterial solution at 10 8 CFU / mL every day. High-dose group: The mice were intragastrically administered 0.2 mL of Weissella confusa bacterial solution at 10 10 CFU / mL every day. The experiment was conducted for 21 days. During the experiment, the mice had free access to food and water, and the physiological conditions of the mice were observed.

[0093] (1) Determination of daily weight gain of mice: Sixty male SPF-grade KM mice weighing 20 ± 2 g were selected and divided into three groups of 20 mice each. Mice in the low-dose group were intragastrically administered 0.2 mL of Weissella confusa DY12 bacterial solution at 10 8 CFU / mL every day. Mice in the high-dose group were intragastrically administered 0.2 mL of Weissella confusa DY12 bacterial solution at 10 10 CFU / mL every day. Mice in the control group were intragastrically administered 0.2 mL of normal saline for 21 consecutive days. Starting from the beginning of intragastric administration, the mice in each group were weighed every 7 days, and the results were recorded.

[0094] The results showed that there was a significant difference in the daily weight gain of the mice in the low-dose group compared with the control group after 7 days of intragastric administration (P < 0.05); after 14 days, the body weight of the mice in the low-dose group increased significantly (P < 0.01); after 21 days, both the high-dose group and the low-dose group could increase the body weight of the mice, but the difference in the low-dose group was more significant (P < 0.01), as shown in Figure 7 . The results indicated that Weissella confusa DY12 had a promoting effect on the weight gain of mice.

[0095] (2) Effect of feeding Weissella confusa DY12 from sheep on the antioxidant capacity of mice: On the basis of the experiment in (1), 5 mice were randomly selected from the experimental group and the control group after 21 days, and blood was collected from the orbital cavity. The blood was stored at 37°C for 2 h, centrifuged at 3,500 rpm at 4°C for 15 min, the supernatant was aspirated, and stored at 4°C. SOD detection kit (Lanjieke Technology Co., Ltd.), AKP kit, and LZM kit (Nanjing Jiancheng Bioengineering Institute) were used to detect the activities of SOD and AKP and the content of LZM in the serum.

[0096] Table 3 Antioxidant results

[0097]

[0098] As can be seen from Table 3, the content of SOD in the serum of the low-dose group of the three groups of mice was significantly increased compared with that of the control group (P<0.05); the content of AKP in the serum of the low-dose and high-dose groups was extremely significantly increased compared with that of the control group (P<0.01), and the content of AKP in the low-dose group was extremely significantly higher than that in the high-dose group (P<0.01); the content of LZM in the low-dose group was significantly increased compared with that of the control group (P<0.05). These results indicate that feeding the fusion Weissella confusa DY12 bacterial solution with different doses can increase the contents of superoxide dismutase, alkaline phosphatase and lysozyme in the serum, suggesting that gavage of different doses of fusion Weissella confusa DY12 to mice can improve the antioxidant activity to varying degrees, among which 1×10 8 CFU / mL has a better antioxidant effect.

[0099] (3)Effect of feeding sheep-derived fusion Weissella confusa DY12 on mouse blood IgG: On the basis of experiment (1), on the 21st day, 5 mice were randomly selected from the experimental group and the control group, blood was collected from the orbital cavity, stored at 37°C for 2 h, centrifuged at 3,500 rpm for 15 min at 4°C, the supernatant was aspirated and stored at 4°C. The content of IgG in the blood was detected using an ELISA kit (Wuhan Boster Biological Engineering Co., Ltd.).

[0100] After gavage for 21 d, the content of IgG in the serum of the low-dose group was extremely significantly increased compared with that of the control group (P<0.01), and was significantly increased compared with that of the high-dose group (P<0.01). It shows that 1×10 8 CFU / mL can significantly increase the secretion of IgG in the blood of mice, as shown in Figure 8 A.

[0101] (4)Effect of feeding sheep-derived fusion Weissella confusa DY12 on mouse intestinal mucosal sIgA: On the basis of experiment (2), the mice were sacrificed by dislocation, the jejunum of the same length was taken, the contents were rinsed with sterile normal saline, homogenized on ice, stored at -20°C overnight, repeatedly frozen and thawed 2 times, centrifuged at 5000×g for 5 min at 4°C, and the supernatant was taken. Referring to the instructions of the ELISA kit, the content of IgA in the mouse intestine was measured (Wuhan Huamei Biological Engineering Co., Ltd.).

[0102] After gavage for 21 d, there was a significant difference in the secretion amount of sIgA between the high-dose group and the control group (P<0.05), and the content of sIgA in the low-dose group was extremely significantly increased compared with that of the control group (P<0.01). It shows that 1×10 8 CFU / mL can significantly increase the secretion of sIgA in the jejunum of mice, as shown in Figure 8 B.

[0103] It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the present invention. Therefore, from any point of view, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be embraced within the present invention. Any reference signs in the claims should not be construed as limiting the claims involved.

Claims

1. Fusion of Weissella weissella confusa )DY12, characterized by: It is isolated from sheep feces, its deposit number is CCTCC NO: M 20241630, and its rDNA sequence is shown in SEQ ID NO:

1.

2. Use of the fused Weissella DY12 according to claim 1 in the preparation of feed additives or animal feed.

3. Use of the fused Weissella DY12 described in claim 1 in promoting animal growth and regulating animal immunity.

4. Use of the fused Weissella DY12 described in claim 1 in improving the antioxidant capacity of animals.

5. A feed additive for promoting animal growth, regulating animal immunity, and improving animal antioxidant capacity, characterized in that: It includes the fused Weissella DY12 described in claim 1.

6. An animal feed for promoting animal growth, regulating animal immunity, and improving animal antioxidant capacity, characterized in that: It comprises the fused Weissella DY12 as described in claim 1 and common animal feed nutritional components or carriers.

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