Lactobacillus salivarius and application thereof in regulating flora balance of digestive tract of mutton sheep

Abstract

The invention relates to the technical field of probiotics, in particular to lactobacillus salivarius and application of the lactobacillus salivarius in regulating flora balance of digestive tracts of mutton sheep. The invention provides a lactobacillus salivarius KS1018, and the preservation number of the lactobacillus salivarius KS1018 is CCTCC (China Center for Type Culture Collection) NO: M 2025020. The lactobacillus salivarius KS1018 is a strain which is separated from dolang sheep rumen fluid and has the characteristics of high temperature resistance, acid resistance and cholate resistance, and has the advantages of low immunogenicity and high safety when being applied to mutton sheep. Due to the high-temperature-resistant characteristic, high stability can be kept in the feed mixing and processing process, and the application effect is further improved. The acid-resistant and cholate-resistant characteristics can reduce the influence of the digestive tract on the activity of the feed additive, so that the feed additive effectively plays a role in the digestive tract of the ruminant. The strain is good in palatability after being fed, and can effectively regulate digestive tract flora of ruminants, restore digestive tract flora balance and improve the disease-resistant potential of the ruminants.

Description

Technical Field

[0001] The invention relates to the technical field of probiotics, in particular to a strain of Lactobacillus salivarius and application thereof in regulating the balance of bacterial flora in the digestive tract of mutton sheep. Background Art

[0002] The mutton industry is a crucial component of animal husbandry and plays a vital role in meeting the population's demand for high-quality protein. With the rapid development of large-scale farming, high-density feeding and intensive fattening have become the norm. Improving daily weight gain, reducing feeding costs, and reducing morbidity and mortality rates have become key targets pursued by farming companies. With increasing global attention to food safety and environmental protection, restricting the use of antibiotics in feed has become a trend. Probiotics, as a potential alternative to antibiotics, have garnered widespread attention. Probiotics are a class of microorganisms that are beneficial to the host and can maintain the balance of the digestive tract's microbiome by competitively inhibiting the growth of pathogens, regulating gastrointestinal immune function, and producing beneficial metabolites. However, current probiotics have poor resistance to high temperatures, acids, and bile salts, making them ineffective in the digestive tract. Summary of the Invention

[0003] To address the above problems, the present invention provides a strain of Lactobacillus salivarius and its use in regulating the balance of the digestive tract flora of mutton sheep. The Lactobacillus salivarius provided by the present invention has the advantages of being resistant to high temperatures, acids, and bile salts, and can function in the digestive tract of ruminants, regulating the balance of the digestive tract flora of ruminants and improving their disease resistance.

[0004] In order to achieve the above object, the present invention provides the following technical solutions:

[0005] The invention provides a strain of Lactobacillus salivarius KS1018, with a preservation number of CCTCC NO: M 2025020.

[0006] The present invention provides a bacterial agent, the active ingredient of which includes the Lactobacillus salivarius KS1018 described in the above technical solution.

[0007] Preferably, the number of viable Lactobacillus salivarius KS1018 in the bacterial agent is ≥1.0×10 9 CFU / g.

[0008] The present invention provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps:

[0009] Inoculating the Lactobacillus salivarius KS1018 described in the above technical solution into a liquid culture medium and cultivating to obtain a seed liquid;

[0010] inoculating the seed liquid into a fermentation medium, performing fermentation culture, and collecting bacterial cells;

[0011] The bacterial cells are freeze-dried to obtain the bacterial agent.

[0012] Preferably, the liquid culture medium includes MRS liquid culture medium; the culture conditions of the seed liquid include: 25-80°C, 200-240r / min; the OD of the seed liquid 600 The value is 1 to 3.

[0013] Preferably, the fermentation medium includes MRS medium; the fermentation culture conditions include: temperature of 25-80°C, dissolved oxygen of 20%-40%, rotation speed of 200-600r / min, ventilation volume of 1-2VVM, and time of 14-25h.

[0014] The present invention provides the use of Lactobacillus salivarius KS1018 described in the above technical solution, or the bacterial agent described in the above technical solution, or the bacterial agent obtained by the preparation method described in the above technical solution in one or more of 1) to 3):

[0015] 1) Preparing a product for preventing and treating pathogens; the pathogens include one or more of Escherichia coli, Staphylococcus aureus, and Enterobacter cloacae;

[0016] 2) Preparation of products for improving the immune function of ruminants;

[0017] 3) Regulate the balance of ruminant digestive tract flora;

[0018] The ruminants include sheep.

[0019] Preferably, the meat sheep include Hu sheep and / or Duolang sheep.

[0020] Preferably, the product for improving the immune function of ruminants is a product for increasing the concentration of IgM and / or IgG in serum; the regulation of the balance of the digestive tract flora of ruminants includes: reducing the negative impact of opportunistic pathogens on the digestive tract flora, reducing flora disorder, and restoring the balance of the digestive tract flora; the opportunistic pathogens include Enterobacter cloacae.

[0021] The present invention provides a method for regulating the balance of ruminant digestive tract flora, comprising the following steps:

[0022] The microbial agent is mixed with feed and then fed to ruminants; the microbial agent is the microbial agent described in the above technical solution or the microbial agent obtained by the preparation method described in the above technical solution.

[0023] Beneficial effects:

[0024] The present invention provides a strain of Lactobacillus salivarius KS1018, with a deposit number of CCTCC NO: M2025020. The Lactobacillus salivarius KS1018 described in the present invention is a strain of probiotic bacteria from Duolang sheep isolated from the rumen fluid of Duolang sheep, and has the characteristics of high temperature resistance (37°C to 80°C), acid resistance (pH value 3 to 7) and bile salt resistance (bile salt concentration tolerated is 0.3% to 0.5%). When applied to meat sheep, it has the advantages of low immunogenicity and high safety, and can avoid safety issues such as immune response when used as a feed additive. In addition, a high temperature resistance step is added in the screening process, so that the screened Lactobacillus salivarius KS1018 can maintain a high stability during feed mixing and processing, further improving the application effect. The acid resistance and bile salt resistance steps are added in the screening process, so that the screened Lactobacillus salivarius KS1018 can effectively play a role in the digestive tract of ruminants, reducing the influence of the digestive tract on its activity. This strain has good palatability after feeding, can effectively regulate the digestive tract flora of ruminants, reduce the negative impact of common opportunistic pathogens in farms such as Enterobacter cloacae on the digestive tract flora, help alleviate flora disorders, restore the balance of digestive tract flora, and enhance the disease resistance potential of meat sheep. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0026] Figure 1 The relative abundance of dominant bacterial phyla in the rumen fluid of Hu sheep and Duolang sheep;

[0027] Figure 2 is the colony morphology of strain KS1018;

[0028] Figure 3 The results of microscopic examination after Gram staining of KS1018 strain;

[0029] Figure 4 This is the result of α-diversity analysis of rumen microbiota;

[0030] Figure 5 This is the PCoA analysis result of rumen flora;

[0031] Figure 6 This is a histogram of community composition at the phylum level of rumen bacteria;

[0032] Figure 7 The results of Lefse multi-level species difference discriminant analysis of rumen flora;

[0033] Figure 8 This is the result of fecal microbiome α diversity analysis;

[0034] Figure 9 This is the PCoA analysis result of fecal microbiota;

[0035] Figure 10 The bar graph shows the community composition of fecal flora at the phylum level;

[0036] Figure 11 These are the results of Lefse's multi-level species discrimination analysis of fecal flora.

[0037] Biological Deposit Description

[0038] Lactobacillus salivarius KS1018, classified as Ligilactobacillus salivarius KS1018, was deposited in the China Center for Type Culture Collection on January 6, 2025. The deposit address is China Center for Type Culture Collection, Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, and the deposit number is CCTCC NO: M 2025020. DETAILED DESCRIPTION

[0039] The invention provides a Lactobacillus salivarius KS1018, with a preservation number of CCTCC NO: M2025020.

[0040] The Lactobacillus salivarius KS1018 provided by the present invention is isolated from a Duolang sheep that is tolerant to roughage, has strong stress resistance, and has good growth performance. It is a target strain that maintains a high survival rate in a temperature range of 37°C to 80°C, a pH value of 3 to 7, and a bile salt concentration of 0.3% to 0.5% (w / v). Due to the homology between the source of the strain and the species of the feeding animal, it has the excellent effect of low rejection and high safety when feeding meat sheep. The single colony of the strain is a round, medium-sized, raised, neatly edged, smooth-surfaced, opaque milky white strain, is non-hemolytic, and has the ability to produce acid ( Figure 2 Gram staining of the strain revealed a bluish-purple color (Gram-positive), with a short rod-like shape, existing singly or in pairs, and without spores or flagella ( Figure 3). Lactobacillus salivarius KS1018 has an inhibitory effect on the growth of Staphylococcus aureus and Escherichia coli, with the diameters of the inhibition zone being 5.23±0.08mm and 6.91±0.06mm, respectively. It has an inhibitory effect on the growth of Enterobacter cloacae, with a diameter of the inhibition zone being 16.95±0.67mm. It is sensitive to penicillin and moderately sensitive to cephalothin, tetracycline, erythromycin and clindamycin. It can be seen that when conventional antibiotics are used in aquaculture, the function of the Lactobacillus salivarius KS1018 provided by the present invention will not be significantly affected by the interference of antibiotics, and its role in regulating the digestive tract flora can be guaranteed. Lactobacillus salivarius KS1018 has good palatability, has no effect on the feeding preferences of the test sheep, can significantly improve the serum immune indicators of Hu sheep, and to a certain extent, improve the growth performance of meat sheep and improve the rumen and fecal microbial flora, which helps to alleviate flora disorders, restore the balance of the digestive tract flora, and enhance the disease resistance potential of meat sheep.

[0041] Based on the above advantages, the present invention provides a bacterial agent, the active ingredient of which includes the Lactobacillus salivarius KS1018 described in the above technical solution.

[0042] As an embodiment, the number of viable bacteria of Lactobacillus salivarius KS1018 in the bacterial agent is ≥1.0×10 9 CFU / g.

[0043] The present invention provides a method for preparing the bacterial agent described in the above technical solution, comprising the following steps:

[0044] Inoculating the Lactobacillus salivarius KS1018 described in the above technical solution into a liquid culture medium and cultivating to obtain a seed liquid;

[0045] inoculating the seed liquid into a fermentation medium, performing fermentation culture, and collecting bacterial cells;

[0046] The bacterial cells are freeze-dried to obtain the bacterial agent.

[0047] As an embodiment, the liquid culture medium includes MRS liquid culture medium; the culture conditions of the seed liquid include: 25-80°C, 200-240r / min; the OD of the seed liquid 600 As another embodiment, the culture conditions of the seed solution include: 25-40°C, 210-230r / min; the OD of the seed solution 600 As another embodiment, the culture conditions of the seed solution include: 37°C, 220r / min; the OD of the seed solution 600 The value is 1 to 3.

[0048] As one embodiment, the fermentation medium includes MRS medium; the fermentation culture conditions include: temperature of 25-80°C, dissolved oxygen of 20%-40%, rotation speed of 200-600 r / min, ventilation of 1-2 VVM, and time of 14-25 hours. As another embodiment, the fermentation culture conditions include: temperature of 25-40°C, dissolved oxygen of 25%-35%, rotation speed of 300-550 r / min, ventilation of 1-1.2 VVM, and time of 15-20 hours. As another embodiment, the fermentation culture conditions include: temperature of 37°C, dissolved oxygen of 30%, dissolved oxygen of 30%, rotation speed of 500 r / min, ventilation of 1 VVM, and time of 18 hours.

[0049] In one embodiment, when freeze-drying the bacterial cells, the collected bacterial cells are freeze-dried at -80 to -20°C for 72 to 120 hours. In another embodiment, when freeze-drying the bacterial cells, the collected bacterial cells are freeze-dried at -80 to -60°C for 120 hours.

[0050] As an embodiment, after the bacteria are freeze-dried, the process further includes crushing the freeze-dried bacteria to 80-100 mesh, sieving, and adding maltodextrin as an auxiliary material to obtain the bacterial agent; the mass ratio of the sieved freeze-dried bacteria to maltodextrin is 1:39.

[0051] Based on the above advantages, the present invention provides the use of Lactobacillus salivarius KS1018 described in the above technical solution, or the bacterial agent described in the above technical solution, or the bacterial agent obtained by the preparation method described in the above technical solution in one or more of 1) to 3):

[0052] 1) Preparing a product for preventing and treating pathogens; the pathogens include one or more of Escherichia coli, Staphylococcus aureus, and Enterobacter cloacae;

[0053] 2) Preparation of products for improving the immune function of ruminants;

[0054] 3) Regulate the balance of ruminant digestive tract flora;

[0055] The ruminants include sheep.

[0056] As an embodiment, the meat sheep include Hu sheep and / or Duolang sheep.

[0057] As an embodiment, the product for improving the immune function of ruminants is a product for increasing the concentration of IgM and / or IgG in serum.

[0058] As an embodiment, the regulating the balance of the digestive tract flora of ruminants includes: reducing the negative impact of opportunistic pathogens on the digestive tract flora, reducing flora disorder, and restoring the balance of the digestive tract flora; the opportunistic pathogens include Enterobacter cloacae.

[0059] The present invention provides a method for regulating the balance of ruminant digestive tract flora, comprising the following steps:

[0060] The microbial agent is mixed with feed and then fed to ruminants; the microbial agent is the microbial agent described in the above technical solution or the microbial agent obtained by the preparation method described in the above technical solution.

[0061] As an embodiment, the ruminant includes mutton sheep; the mutton sheep include Hu sheep and / or Duolang sheep, and the amount of the bacterial agent consumed by each sheep per day is ≥1×10 9 CFU.

[0062] As an embodiment, the regulating the balance of the digestive tract flora of ruminants includes: reducing the negative impact of opportunistic pathogens on the digestive tract flora, reducing flora disorder, and restoring the balance of the digestive tract flora; the opportunistic pathogens include Enterobacter cloacae.

[0063] To further illustrate the present invention, a strain of Lactobacillus salivarius provided by the present invention and its application in regulating the balance of the digestive tract flora of mutton sheep are described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0064] Preparation Example

[0065] Enterobacter cloacae is an important opportunistic pathogen of the Enterobacteriaceae family and is widely present in feed, water sources and livestock house environments on farms. The Enterobacter cloacae used in the present invention was isolated and extracted from the rumen fluid of Duolang sheep and was identified as Enterobacter cloacae.

[0066] Example 1

[0067] 1. Determine the source of the strain:

[0068] Since Duolang sheep have the characteristics of tolerance to roughage, good stress resistance and excellent growth performance, the rumen fluid of Duolang sheep was selected as the source of bacteria by aseptic puncture sampling. Rumen fluid samples of 12 Hu sheep and 12 Duolang sheep were selected for 16S rRNA sequencing. The level of Firmicutes in Duolang sheep was significantly higher than that in Hu sheep (P<0.01), and the levels of Bacteroidetes, Proteobacteria and Candidate Radials were significantly lower than those in Hu sheep (P<0.05). Three phyla of bacteria with a relative abundance of >1% were identified in the rumen fluid of Duolang sheep, namely Firmicutes (56.45%), Bacteroidetes (39.62%) and Actinobacteria (1.07%) ( Figure 1 Firmicutes are the primary rumen microbiome that promotes the breakdown of insoluble carbohydrates in feed. These results suggest that Duolang sheep have greater potential for feed efficiency. Proteobacteria, composed of many pathogenic bacteria, have been associated with increased abundance in the rumen and high-concentrate diets, subacute ruminal acidosis, and ruminal dysbiosis. The significantly lower abundance of Proteobacteria in the rumen of Duolang sheep may be related to the stability of their rumen microbial environment and their strong adaptability to adverse conditions.

[0069] Lactic acid bacteria in the Firmicutes have many important characteristics. Lactic acid bacteria are safe to be developed as potential probiotic species, and can enhance the immune function of the intestinal mucosa. They can prevent the growth and reproduction of pathogenic microorganisms by promoting the expression and secretion of β-defensins. Combined with the comparison results of the rumen flora of Duolang sheep and Hu sheep, it was decided to screen lactic acid bacteria of the Firmicutes as potential probiotic species.

[0070] 2. Isolation, screening and identification of bacterial strains:

[0071] (1) Twelve healthy, nulliparous, and medically healthy Duolang sheep aged 18 to 24 months were selected from Kashgar, Xinjiang. Rumen fluid was collected aseptically using rumen puncture and filtered through four layers of gauze.

[0072] (2) Rumen fluid samples were inoculated into MRS solid selective culture medium containing 2% CaCO3 under anaerobic conditions and cultured for 48 h. The strains that produced obvious calcium-dissolving circles around the colonies, i.e., those with acid-producing ability, were selected.

[0073] (3) During the initial screening of lactic acid bacteria, catalase test and hydrogen sulfide production test were used. The catalase test was positive and the hydrogen sulfide production test was negative. During the rescreening, hemolysis test was performed: the bacteria were inoculated on blood agar medium containing 5% sheep blood and cultured at 37°C overnight to screen for safe strains without hemolysis. Colonies with morphological characteristics consistent with lactic acid bacteria were selected and subjected to tolerance tests based on the rumen environment. The survival rates of the strains were tested under different temperatures, acidic conditions and bile salt concentrations. Some of the results are shown in Table 1. A target strain was screened out that maintained a high survival rate in the temperature range of 37°C to 80°C, pH values ​​of 3 to 7, and bile salt concentrations of 0.3% to 0.5% (w / v). It was numbered KS1018. The survival rate of this strain under different conditions was significantly higher than that of the previously screened Lactobacillus salivarius strain 1018 (see Chinese Patent CN119020218A) and was used for subsequent tests.

[0074] Table 1 Survival rate of KS1018 under different conditions (%)

[0075] strain Bile salt concentration 0.3% (w / v) 6h pH 3.024h 37℃10min 80℃5min KS1080 68.72 95.80 100 1.9

[0076] (4) Morphological observation of the colony morphology, size, color and other characteristics, the results show that the single colony of the strain is round, medium size, convex, regular edge, smooth surface, opaque, white strain, non-hemolytic, with acid-producing ability Figure 2 ). Gram staining of the strain showed blue-violet after staining (Gram-positive), short rod-shaped, single or paired, no spores or flagella Figure 3

[0077] (5) After amplifying the 16S rRNA gene, the amplified fragment was sequenced, and BLAST homology comparison was performed combined with NCBI database. After sequence similarity analysis, the strain KS1018 was identified as Ligilactobacillus salivarius (recorded as Ligilactobacillus salivarius KS1018), and the strain was preserved in China Center for Type Culture Collection, with the preservation number of CCTCC NO: M 2025020.

[0078] Example 2 Growth characteristics, pathogenic bacteria antagonistic ability and common antibiotic resistance test of Ligilactobacillus salivarius KS1018

[0079] 1. Strain growth characteristics

[0080] The bacterial liquid of Ligilactobacillus salivarius KS1018 screened in Example 1 was inoculated in MRS liquid medium at a concentration of 2% (v / v) and cultured at 37°C, 220 r / min. The absorbance OD 600 value and pH value of the bacterial liquid were measured every 2h, and the growth curve was drawn.

[0081] The results are as follows: when the culture temperature is 37°C, Ligilactobacillus salivarius KS1018 enters the logarithmic growth phase after 4h of inoculation in MRS liquid medium, and enters the slow growth phase after 12h. The pH of the culture medium decreases rapidly to 4.6 within 2-8h after inoculation of Ligilactobacillus salivarius KS1018 in MRS liquid medium, and slowly decreases to be constant after 12h of inoculation. The strain grows rapidly under the optimal growth conditions (37°C, pH 6.5), and the pH decreases rapidly to 4.6 within 6h due to acid production, indicating that it has high fermentation activity and can rapidly proliferate in the digestive tract and adjust the pH after feeding.

[0082] 2. Pathogenic bacteria inhibition test of the strain

[0083] ​Lactobacillus salivarius KS1018 was used for pathogen inhibition tests. The pathogens were Escherichia coli (ATCC700928) and Staphylococcus aureus (ATCC25923), purchased from the American Type Culture Collection. Using the Oxford cup method, a sterilized Oxford cup was placed vertically on LB solid medium coated with pathogen liquid, and 1×10 7 200 μL of Lactobacillus salivarius KS1018 bacterial solution with a CFU / mL was cultured at 37°C for 24 h and the diameter of the inhibition zone was recorded.

[0084] The results showed that Lactobacillus salivarius KS1018 inhibited the growth of both Staphylococcus aureus and Escherichia coli, with inhibition zone diameters of 5.23±0.08mm and 6.91±0.06mm, respectively. This suggests that it possesses some anti-pathogen activity and is significantly more effective than the previously screened Lactobacillus salivarius strain 1018 (see Chinese Patent CN119020218A).

[0085] 3. Strain Competition Inhibition Test

[0086] A strain competition inhibition test was conducted using Lactobacillus salivarius KS1018 and Enterobacter cloacae. KS1018 and Enterobacter cloacae were added to 8 mL of MRS liquid medium at a 1% (v / v) addition rate to prepare the fermentation supernatant. The concentration of each was 9.0×10 6 Use a sterile pipette to dispense 120 μL of an indicator bacterial solution with a CFU / mL concentration onto an indicator plate and spread evenly using a sterile spreading rod. After the indicator plate has dried, place three sterile, room-temperature-dried Oxford cups onto the indicator plate and add 200 μL of the treated test bacteria fermentation supernatant. Diffusion is allowed at 4°C for 6 hours, followed by incubation at 37°C for 24 hours. Measure the diameter of the inhibition zone.

[0087] Competitive inhibition tests showed that the supernatant of Lactobacillus salivarius inhibited the growth of Enterobacter cloacae, with an inhibition zone diameter of 16.95 ± 0.67 mm. The supernatant of Enterobacter cloacae had no inhibitory effect on the growth of Lactobacillus salivarius. This suggests that Lactobacillus salivarius has the ability to inhibit the growth of Enterobacter cloacae, but its growth is not affected by the inhibitory effect.

[0088] 4. Common antibiotic resistance test of strains

[0089] The susceptibility of Lactobacillus salivarius KS1018 to common antibiotics used in mutton farming was evaluated using the disc agar diffusion method. The test bacteria were evenly spread onto MRS agar and LB solid medium, respectively. Sensitivity discs were evenly placed on the agar medium. The culture was incubated inverted at 37°C for 24 hours, and the diameter of the inhibition zone was recorded.

[0090] The results showed that Lactobacillus salivarius KS1018 was sensitive to penicillin and cefazolin, and moderately sensitive to gentamicin, tetracycline, and erythromycin. This suggests that the function of this strain is not significantly affected by conventional antibiotics used in mutton sheep farming, ensuring its role in regulating the digestive tract flora.

[0091] Example 3

[0092] 1. Preparation of Bacterial Powder

[0093] (1) Small test (100L)

[0094] Strain Activation: Use Lactobacillus salivarius KS1018 strain to culture on MRS solid plates to obtain monoclonal colonies. Pick a monoclonal colony from the MRS solid plate and inoculate it into 100 mL of MRS liquid medium. Incubate for 18 hours to prepare the strain activation solution to achieve the desired fermentation activity.

[0095] First-stage seed liquid culture: transfer 20 mL of the activated bacterial culture liquid to five 1 L MRS culture media, for a total of 5 L. Culture for 8.5 h until the seed liquid (OD 600 value reaches 2).

[0096] Fermentation of 100 L of culture medium: Inoculate 5 L of primary seed culture medium into a 100 L fermentor. Add 15 L of MRS medium during fermentation. The fermentor volume is 60 L. Adjust the dissolved oxygen to 30%, the rotation speed to 500 rpm, and the ventilation rate to 1 vvm, which is linked to the dissolved oxygen control.

[0097] (2) Expanded experiment (1000L)

[0098] Seed tank culture: Use 100 L of MRS medium for seed tank culture.

[0099] Fermentation: Inoculate the culture medium from the seed tank into a 1-ton fermenter at a volume ratio of 1:20. Add 150 L of feed medium during fermentation. The working volume of the fermenter is 600 L. Adjust the dissolved oxygen to 30%, set the maximum speed, and set the ventilation rate to 1 vvm, which is linked to the dissolved oxygen control.

[0100] (3) Extraction

[0101] Bacteria collection: Use a disc centrifuge for centrifugation at a speed of 5000r / min to collect bacteria in the fermentation broth.

[0102] (4) Refining

[0103] Freeze-drying: The collected cells were freeze-dried at -45°C for 120 h.

[0104] The equipment's water consumption is ≥220 L / min, with a pressure of ≥0.5 MPa; cooling water consumption is ≥16 T / h, with a temperature of 0.15 ≤ P ≤ 0.2 MPa and a temperature of t ≤ 25°C; and compressed air flow is ≥40 L / min, with a temperature of 0.5 ≤ P ≤ 0.8 MPa. During sterilization and defrosting, the steam flow rate is ≥150 kg / h, with a temperature of 0.2 ≤ P ≤ 0.3 MPa. During the freeze-drying process, the cold trap temperature is set to -45°C, and the product temperature is controlled by switching the plate cooling valve and condensing valve. The chamber is evacuated, maintaining the cold trap temperature at -45°C. The vacuum pump and related valves are operated to reach the pre-vacuum value before proceeding to the next stage.

[0105] (5) Crushing

[0106] Crushing treatment: crush the dried bacteria into 100 mesh. The preparation of Lactobacillus salivarius inoculum is completed. After sieving, maltodextrin is added as an auxiliary material to obtain bacterial powder (the number of viable bacteria is 7.6×10 9 CFU / g); the mass ratio of sieved freeze-dried bacteria to maltodextrin is 1:39. Store in a frozen environment below -20°C.

[0107] 2. Palatability test

[0108] To investigate the effects of feeding Lactobacillus salivarius KS1018 on the feeding behavior and palatability of experimental Hu sheep, a pilot study was conducted before the formal feeding experiment began. Thirty-two fattening male Hu sheep, approximately three months old and in good condition and weighing approximately 30 ± 4 kg, were randomly divided into four groups of eight each. The control group (Con) was fed a basal diet. The three Lactobacillus salivarius treatment groups (LA, MA, and HA) each received 0.53 g (LA), 1.05 g (MA), and 1.58 g (HA) of the bacterial powder prepared in step 1, respectively, added to the basal diet. Feed was prepared daily.

[0109] Each group of lambs was fed a sufficient basal diet. After 24 hours, the amount of feed leftover was measured, and behavioral changes in the lambs were monitored during the 24-hour feeding period. The results are shown in Table 2. The results showed no significant differences in average daily feed intake among the experimental groups, demonstrating good feeding motivation. Palatability tests showed that the addition of Lactobacillus salivarius did not affect the palatability of the experimental lambs, and the lambs were able to consume the feed normally.

[0110] Table 2 Palatability test results

[0111] Group Concentrated feed + grass feed / kg Residue / kg Feed intake per group / kg Average feed intake of sheep / kg Con 14 4.7 9.3 1.1625 LA 13.5 5.1 8.4 1.05 MA 14.5 4.6 9.9 1.2375 HA 12.7 3.2 9.5 1.1875

[0112] Example 4 Effect of feeding Lactobacillus salivarius strains on serum immunoglobulins (IgG, IgM)

[0113] 1. Experimental Animals and Design

[0114] Thirty-two fattening male Hu sheep of approximately three months of age with similar body weight (30 ± 4 kg) and good physical condition were randomly divided into four groups of eight sheep each. The control group (Con) was fed a basal diet, while the three Lactobacillus salivarius treatment groups (LA, MA, and HA) each received 0.53 g, 1.05 g, and 1.58 g of the bacterial powder prepared in step 1 of Example 3, respectively, added to the basal diet. Feed was prepared daily.

[0115] Before the experiment began, the sheep shed was disinfected, the test sheep were dewormed, and numbered. Roughage was fed once daily at 9:00 AM and 5:00 PM, and concentrate feed was fed once daily at 10:00 AM and 6:00 PM. Feed and water were available ad libitum, ensuring a small amount of leftover feed was present. Lactobacillus salivarius KS1018 powder was mixed evenly with the first morning concentrate feed and fed (referred to as the main feeding period). The health of the sheep was observed and recorded daily during the feeding period.

[0116] 2. Collect blood samples to measure serum immunoglobulin concentration

[0117] On day 56 of the feeding period, 10 mL of venous blood was collected from the experimental Hu sheep. After serum separation, serum immunoglobulin IgM and IgG concentrations were measured using ELISA kits. IgM and IgG are important indicators of humoral immune function and play a key role in the primary and secondary immune responses, respectively. By measuring serum IgM and IgG concentrations, we can assess the role of Lactobacillus salivarius in regulating the humoral immune function of meat sheep, enhancing disease resistance, and promoting immune health. The results are shown in Table 3.

[0118] Table 3 Serum immunoglobulin concentrations of Hu sheep

[0119] Indicators / Groupings Con LA MA HA P-value IgM (μg / mL) 8.53±1.49 9.70±1.18 15.26±8.67 11.91±4.78 0.0758 IgG (μg / mL) 189.70±27.59 227.62±26.85 334.51±140.30 289.47±67.03 0.0088

[0120] Results showed that serum IgM concentrations in the Hu sheep were nearly significantly different across groups (P = 0.0758). The serum IgM concentrations of the Hu sheep fed different concentrations of Lactobacillus salivarius increased compared to the control group. Serum IgG concentrations were significantly different across groups (P = 0.0088), with the MA group showing a significant difference compared to the Con group (P = 0.0097). These results suggest that feeding Lactobacillus salivarius can help improve serum immune indicators and enhance the disease resistance of meat sheep.

[0121] Example 5 Regulation of the Digestive Tract Flora of Sheep by Lactobacillus salivarius

[0122] 1. Experimental Animals and Design

[0123] Enterobacter cloacae is an important opportunistic pathogen of the Enterobacteriaceae family and is widely present in the feed, water sources and livestock house environment of farms. It is transmitted through contact and easily infects mutton sheep with low immunity, thereby causing diseases such as pleuropneumonia and sepsis. At the same time, it causes an imbalance in the digestive tract flora, significantly affecting the feed utilization, growth and health of mutton sheep. Therefore, in this experiment, Enterobacter cloacae was selected as a pathogen model, and its pathogenic effects in an actual breeding environment were simulated by adding it to the feed, and the effect of Lactobacillus salivarius on improving the digestive tract flora of mutton sheep and combating the negative effects of pathogens was evaluated. A similar palatability test was used to determine that the addition of Enterobacter cloacae would not affect the palatability of the feed of the experimental Hu sheep.

[0124] Thirty-two fattening male Hu sheep of about 105 days of age with similar body weight (30 ± 4 kg) and good physical condition were randomly divided into four groups, with 8 sheep in each group. The control group (Con) and the medium-dose Lactobacillus salivarius KS1018 treatment group (MA) were as described in Example 4 (the number of viable Lactobacillus salivarius KS1018 used per sheep per day was 1×10 9 CFU); Enterobacter cloacae treatment group (MB) was treated with 1×10 viable bacteria per sheep per day. 9 To calculate the CFU count, 1.05g of Enterobacter cloacae powder was added. For the mixed treatment group (AB), 1.05g of Lactobacillus salivarius KS1018 powder and 1.05g of Enterobacter cloacae powder were added to the daily feed, with the feed prepared fresh daily. The preparation method for the Enterobacter cloacae powder was similar to that for the Lactobacillus salivarius KS1018 powder in Example 3, except that LB medium was added as the feed medium during fermentation, and the final mass ratio of freeze-dried bacteria to maltodextrin was 7:13.

[0125] Before the experiment began, the sheep shed was disinfected, the test sheep were dewormed, and numbered. Roughage was fed once daily at 9:00 AM and 5:00 PM, and concentrate feed was fed once daily at 10:00 AM and 6:00 PM. Feed and water were freely available, ensuring that there was a small amount of leftover feed after each meal. Lactobacillus salivarius and Enterobacter cloacae powder were mixed evenly with the first morning concentrate feed and fed to the sheep. The health of the sheep was observed and recorded daily during the feeding period.

[0126] 2. Lactobacillus salivarius regulates the digestive tract flora after being added to the feed, improving the disease resistance potential of mutton sheep

[0127] 2.1 Effect of Lactobacillus salivarius on growth performance

[0128] The fasting weight of each sheep was weighed before morning feeding on the 1st and 56th day of the normal feeding period, and the individuals with poor health status were culled after the normal feeding period, and the final average daily gain of each group was calculated to evaluate the ability of Lactobacillus salivarius KS1018 to improve the growth rate of mutton sheep and antagonize the negative effects of Enterobacter cloacae. The results, as shown in Table 4, show that the average daily gain of the MA group is higher than that of the Con group, while the average daily gain of the AB and MB groups is lower than that of the Con group, and the average daily gain of the AB group is less than that of the MB group. This shows that Lactobacillus salivarius KS1018 can improve the growth performance of mutton sheep, while Enterobacter cloacae has the opposite effect, and the addition of Lactobacillus salivarius KS1018 in the AB group alleviates the negative effects of Enterobacter cloacae on growth performance.

[0129] Table 4 Body weight indicators of Hu sheep

[0130] Indicator / Group Con MA AB MB Average daily weight gain during the entire period / g 289.80±68.49 296.68±52.25 254.64±36.53 242.35±64.79

[0131] 2.2 Effect of Lactobacillus salivarius on rumen flora

[0132] On the 56th day of the normal feeding period, rumen fluid was collected aseptically and sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for microbial community diversity analysis.

[0133] 2.2.1 Analysis of rumen flora a diversity

[0134] The rumen flora a diversity parameters (Chao, Shannon and Simpson indices) are shown in Table 5, and there was no significant difference in Chao richness, Shannon diversity and Simpson index among the groups. Figure 4 The Chao richness index is used to evaluate the total number of species in the microbial community, reflecting the potential number of species in the flora; the Shannon diversity index combines the richness and evenness of the community, measuring the distribution of different species in the community; and the Simpson index focuses more on the distribution proportion of dominant species, reflecting the uniformity of the community and whether it is dominated by a few species. Under the current experimental conditions, the addition of Lactobacillus salivarius KS1018 did not significantly change the species richness and diversity distribution of the rumen microbial community of the test sheep, and had certain stability in maintaining the overall composition and function of the rumen flora.

[0135] 2.2.2 Analysis of rumen flora β diversity

[0136] The first two axes of the principal coordinate analysis (PCoA) of the rumen flora show the distribution of microbial communities in different treatment groups (Con, MA, AB, MB) (Figure 2). Figure 5). PC1 and PC2 explained 24.23% and 11.65% of the variation, respectively, and a total of 35.88% of the microbial community differences. The inter-group differences were tested by ANOSIM analysis (R=0.0171, P=0.325). The results showed that the microbial community differences between the Con group, MA group, AB group and MB group were not significant. There was a certain degree of overlap in the community composition of each group, and no obvious inter-group differentiation trend was shown. The concentrated distribution trend of the microbial community in the MA group suggests that Lactobacillus salivarius KS1018 has optimized the composition structure of rumen microorganisms to a certain extent. In contrast, the microbial community distribution of the MB group and the AB group was more dispersed, indicating that the pathogenic effect of Enterobacter cloacae interfered with the rumen flora, but the interference effect in the AB group was partially alleviated by Lactobacillus salivarius.

[0137] 2.2.3 Analysis of species composition of rumen flora

[0138] The composition of rumen microbiota in mutton sheep was analyzed in different feeding groups. The results showed that there were some differences in the relative abundance of microbiota at the phylum level among the groups ( Figure 6 At the phylum level, Firmicutes and Bacteroidota were the dominant bacterial groups in each group, accounting for more than 80% of the rumen microbiome. The detailed analysis is as follows:

[0139] In the Con group, Firmicutes (red) dominated, with an abundance of approximately 60%. Bacteroidetes (blue-green) was second, accounting for approximately 30%. Other phyla, such as Actinobacteriota and Proteobacteria, had relatively low abundances. Compared with the control group, the relative abundance of Firmicutes in the MA group decreased slightly, while the abundance of Bacteroidetes increased. Bacteroidetes is one of the important microbial groups in the rumen of mutton sheep that promotes the decomposition of insoluble carbohydrates in feed. This indicates that feeding Lactobacillus salivarius KS1018 promoted the increase in the abundance of Bacteroidetes, which helped improve the digestion and utilization of feed in the rumen.

[0140] The relative abundance of Firmicutes in the MB group was similar to that in the control group, but the abundance of Bacteroidetes decreased slightly, while the abundance of other bacterial groups, such as Proteobacteria and Cyanobacteria, increased slightly. This may be related to the dysbiosis caused by Enterobacter cloacae. The abundance of Firmicutes in the AB group was higher, similar to that in the control group, and the abundance of Bacteroidetes was between the MA and MB groups. This suggests that co-feeding with Lactobacillus salivarius KS1018 alleviated the dysbiosis caused by Enterobacter cloacae to some extent and adjusted some of the bacterial structure.

[0141] 2.2.4 Lefse multi-level species discrimination analysis of rumen microbiota

[0142] Linear discriminant analysis (LDA) combined with effect size measurement (LEfSe) determined the taxa with significant differences in abundance Figure 7 ). Only taxa with LDA scores greater than 3.5 were shown. The results showed that the abundance of norank_f__Lachnospiraceae was significantly increased in Con group, suggesting that this taxon was dominant in the control group. In MA, the abundance of Negativicutes and Veillonellales-Selenomonadales was significantly increased, indicating that the feeding of Lactobacillus salivarius KS1018 promoted the growth of these flora. Bacteria in the Negativicutes class are commonly found in anaerobic digestive environments, and some species are associated with carbohydrate metabolism and short-chain fatty acid (SCFA) production. The Veillonellales-Selenomonadales order belongs to the Negativicute class and has the functions of metabolizing lactic acid and carbohydrates and producing short-chain fatty acids, which has an important influence on the host's digestive flora. These significantly changed flora are related to the regulatory effect of Lactobacillus salivarius KS1018 on rumen flora structure, especially the increase in Negativicutes and Veillonellales-Selenomonadales, which helps to maintain the homeostasis of the digestive microbiota and improve the health of the digestive tract.

[0143] 2.3 Effect of Lactobacillus salivarius on fecal flora

[0144] On day 56 of the positive feeding period, feces were collected aseptically and sent to Shanghai Meiji Biomedicine Technology Co., Ltd. for microbiome diversity analysis.

[0145] 2.3.1 Analysis of fecal flora alpha diversity

[0146] The alpha diversity parameters (Chao, Shannon, and Simpson indices) of the fecal flora were as shown in Table 2.3.1. Figure 8 There was no statistically significant difference in Chao richness, Shannon diversity, and Simpson index. Under the current experimental conditions, the addition of Lactobacillus salivarius KS1018 did not significantly change the species richness and diversity distribution of the fecal flora of the test sheep.

[0147] 2.3.2 Analysis of fecal flora beta diversity

[0148] The OTU level principal coordinate analysis (PCoA) of the fecal flora was as shown in Figure 2.3.2. Figure 9As shown. The first two axes explained 17.57% and 11.86% of the microbial community variation, respectively. By ANOSIM analysis (R=0.0103, P=0.364), there was no statistically significant difference between the groups. Judging from the distribution of the confidence interval ellipses, the microbial community variability in the MB group was the largest, which suggests that the treatment of Enterobacter cloacae may have a certain effect on the stability of the fecal microbial community structure; while the variability in the MA group was smaller, indicating that the intervention of Lactobacillus salivarius KS1018 relatively stabilized the microbial community structure. The variability of the AB group was between the MA group and the MB group, and was significantly lower than that of the MB group, indicating that Lactobacillus salivarius KS1018 improved the potential impact of Enterobacter cloacae on the microbial community structure to a certain extent. Overall, the intervention of Lactobacillus salivarius KS1018 has a certain regulatory and restorative effect on the changes in fecal microbial communities caused by Enterobacter cloacae.

[0149] 2.3.3 Analysis of fecal microbial species composition

[0150] By analyzing the phylum-level composition of fecal flora, the relative abundance distribution of flora in each group can be observed ( Figure 10 ).

[0151] In the MA group, the relative abundance of Firmicutes decreased compared with the Con group, while the abundance of Bacteroidota increased relatively. This change is consistent with the trend of improvement in the balance of digestive tract microbial flora, indicating that Lactobacillus salivarius KS1018 has the effect of regulating the digestive tract flora of mutton sheep, promoting the growth of beneficial bacteria (such as Bacteroidetes), and inhibiting the overgrowth of potential pathogenic bacteria. In the MB group, the relative abundance of Firmicutes was high, and the abundance of Bacteroidetes decreased significantly, indicating that the modeling of pathogenic bacteria led to the imbalance of digestive tract microbial flora, which may affect digestive tract health. In the AB group, the abundance of Bacteroidetes rebounded compared with the MB group, suggesting that Lactobacillus salivarius KS1018 reversed the digestive tract flora imbalance caused by Enterobacter cloacae to a certain extent.

[0152] Feeding Lactobacillus salivarius KS1018 modulated the digestive tract microbiota composition of mutton sheep, increasing the abundance of Bacteroidetes, decreasing the proportion of Firmicutes, and promoting a balanced digestive tract microbiota. Even under conditions of pathogenic bacteria intervention (A and B groups), Lactobacillus salivarius KS1018 still demonstrated some potential to regulate and reverse digestive tract dysbiosis. This suggests that Lactobacillus salivarius KS1018 exerts a prebiotic effect by improving digestive tract microbiota composition and promoting digestive tract health.

[0153] 2.3.4 Lefse multi-level species discrimination analysis of fecal microbiota

[0154] Linear discriminant analysis (LDA) combined with effect size measurement (LEfSe) identified characteristic taxa with significant differences in abundance ( Figure 11). Only taxa with LDA scores greater than 2.5 are shown. The results showed that the abundance of the genera Prevotellaceae and Blautia increased significantly in the MA group. This indicates that feeding Lactobacillus salivarius KS1018 promoted the growth of these probiotic bacteria. Prevotellaceae belongs to the Bacteroidetes phylum, and its bacteria often participate in the decomposition and metabolism of carbohydrates in the intestinal environment. They can produce short-chain fatty acids (SCFAs), such as acetic acid and propionic acid, to maintain the pH stability of the digestive tract and promote digestive tract health. The genus Blautia is an important member of the Firmicutes phylum, and some species are also closely related to the production of SCFA, which contributes to the host's energy supply and anti-inflammatory response. SCFA is the main source of energy for ruminants. The increase in its production reflects the increase in feed utilization efficiency, indicating that the addition of Lactobacillus salivarius KS1018 has the effect of promoting the digestion of meat sheep. The abundance of the classes Negativicutes and Campylobacteria increased significantly in the AB group, suggesting that Lactobacillus salivarius KS1018 has regulated the microbial structure under the intervention of pathogens to a certain extent. The increase in these bacterial communities may be related to anaerobic metabolism and the production of short-chain fatty acids, which helps restore digestive tract homeostasis and resist the effects of pathogenic bacteria.

[0155] In summary, feeding Lactobacillus salivarius KS1018 significantly impacted the digestive tract microbiome of mutton sheep, promoting the increase of Prevotellaceae and Blautia genera, which are associated with carbohydrate metabolism and SCFA production. This helps maintain digestive tract health and promotes digestion. Even in the presence of pathogenic bacteria, Lactobacillus salivarius KS1018 still exhibited a modulatory effect, demonstrating its potential to stabilize the digestive tract microbiome. These significant changes in the microbiome further validate the potential of Lactobacillus salivarius KS1018 as a probiotic, particularly in promoting digestive tract metabolic balance and improving host health.

[0156] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A strain of Lactobacillus salivarius KS1018, characterized in that: The deposit number is CCTCC NO: M 2025020.

2. A bacterial agent, characterized in that The active ingredient comprises the Lactobacillus salivarius KS1018 according to claim 1.

3. The microbial agent according to claim 2, characterized in that The number of viable bacteria of Lactobacillus salivarius KS1018 in the bacterial agent is ≥1.0×10 9 CFU / g.

4. The method for preparing the bacterial agent according to claim 2 or 3, characterized in that: The following steps are involved: inoculating the Lactobacillus salivarius KS1018 according to claim 1 into a liquid culture medium and culturing to obtain a seed liquid; inoculating the seed liquid into a fermentation medium, performing fermentation culture, and collecting bacterial cells; The bacterial cells are freeze-dried to obtain the bacterial agent.

5. The preparation method according to claim 4, characterized in that The liquid culture medium includes MRS liquid culture medium; the culture conditions of the seed liquid include: 25-80°C, 200-240r / min; the OD of the seed liquid 600 The value is 1 to 3.

6. The preparation method according to claim 4, characterized in that The fermentation medium includes MRS medium; the fermentation culture conditions include: temperature of 25-80°C, dissolved oxygen of 20%-40%, rotation speed of 200-600r / min, ventilation volume of 1-2VVM, and time of 14-25h.

7. Use of the Lactobacillus salivarius KS1018 according to claim 1, the bacterial agent according to claim 2 or 3, or the bacterial agent obtained by the preparation method according to any one of claims 4 to 6 in one or more of 1) to 3): 1) Preparation of products for preventing and treating pathogens; the pathogens include: One or more of Escherichia coli, Staphylococcus aureus, and Enterobacter cloacae; 2) Preparation of products for improving the immune function of ruminants; 3) Regulate the balance of ruminant digestive tract flora; The ruminants include sheep.

8. The use according to claim 7, characterized in that The meat sheep include Hu sheep and / or Duolang sheep.

9. The use according to claim 7, characterized in that The product for improving the immune function of ruminants is a product for increasing the concentration of IgM and / or IgG in serum; the regulation of the balance of the digestive tract flora of ruminants includes: reducing the negative impact of opportunistic pathogens on the digestive tract flora, reducing flora disorder, and restoring the balance of the digestive tract flora; the opportunistic pathogens include Enterobacter cloacae.

10. A method for regulating the balance of ruminant digestive tract flora, characterized in that: The following steps are involved: The bacterial agent is mixed with feed and then fed to ruminants; the bacterial agent is the bacterial agent according to claim 2 or 3 or the bacterial agent obtained by the preparation method according to any one of claims 4 to 6.

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