Bacillus subtilis KY2098, compound preparation of bacillus subtilis KY2098 and grape seed extract and application of bacillus subtilis KY2098 and grape seed extract in prevention and treatment of chicken mycoplasma synoviae
A compound preparation of Bacillus subtilis KY2098 and grape seed extract has solved the treatment problem of Mycoplasma synoviae disease in chickens, achieving efficient prevention and treatment and promoting animal growth, and is suitable for the breeding industry.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUAZHONG AGRI UNIV
- Filing Date
- 2026-01-25
- Publication Date
- 2026-05-12
AI Technical Summary
Current technologies lack a unified and standardized method for treating mycoplasma synoviae infection in chickens, and drug resistance is a serious problem due to differences in drug sensitivity. There is an urgent need to explore more scientific and effective treatment strategies.
A compound preparation using Bacillus subtilis KY2098 and grape seed extract was developed. By screening out Bacillus subtilis KY2098, which has antagonistic effects against Mycoplasma synoviae in chickens, and combining it with grape seed extract, a fermentation broth or fermentation supernatant was prepared for use in the preparation of drugs and feed additives for the prevention and treatment of Mycoplasma synoviae disease in chickens.
It achieves a prevention and control effect of 90% to 100% against chicken synovitis, and promotes animal growth and enhances immunity, making it suitable for the industrialization of animal husbandry.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biological agent technology, and specifically relates to Bacillus subtilis KY2098, its compound preparation with grape seed extract, and its application in the prevention and treatment of Mycoplasma synoviae disease in chickens. Background Technology
[0002] Mycoplasma synoviae infection in chickens is a global infectious disease caused by Mycoplasma synoviae, resulting in severe economic losses to the poultry industry. The main symptoms are leg weakness, emaciation, and keel cysts, which can adversely affect the egg production performance and quality of breeding and laying hens, as well as flock growth. Improper husbandry, immunosuppression, and mixed infections with other diseases can all lead to outbreaks. With the rapid development of intensive poultry farming, the incidence of Mycoplasma synoviae infection in chickens is on the rise, posing a serious threat to the poultry industry.
[0003] Currently, there are many drugs available for treating Mycoplasma synoviae infection in chickens. However, due to varying farming environments and management levels, and individual and regional differences in the sensitivity of Mycoplasma synoviae to drugs, veterinary clinics lack a unified and standardized treatment plan for this disease. Treatment for common Mycoplasma synoviae infections often relies on the addition of antibiotics. With the increasing prevalence and spread of Mycoplasma synoviae infection, the pathogen's drug resistance is constantly increasing, and drug residue problems are becoming more serious, further limiting existing treatment options and necessitating the exploration of more scientific and effective treatment strategies.
[0004] With the arrival of the "antibiotic reduction / alternative era," the development and application of antibiotic alternatives has become a hot topic in livestock research and production, with probiotics, plant extracts, and traditional Chinese medicine products receiving significant attention. Bacillus subtilis, as a feed additive, has the potential to improve the gut microbiota of livestock and poultry, regulate metabolism, enhance immunity, and promote feed digestion and absorption. Grape seed extract (GSE), an active ingredient extracted from grape seeds, mainly contains antioxidants such as proanthocyanidins (OPCs) and polyphenols, which can improve meat quality and production performance in livestock and poultry, enhance antioxidant and immune functions, and have the potential to replace antibiotics.
[0005] This application combines probiotics and plant extracts to create a Bacillus subtilis-grape seed extract compound product. The two have a synergistic effect, providing a new strategy for the treatment of Mycoplasma synoviae disease in chickens, which is in line with the scientific concept of safe, environmentally friendly and green farming. Summary of the Invention
[0006] In view of the problems existing in the prior art, the purpose of this invention is to provide Bacillus subtilis KY2098, which has the function of preventing and treating Mycoplasma synoviae disease in chickens. The preservation number of this strain is: CCTCC NO: M 2026185.
[0007] Another object of the present invention is to provide a strain and its application in compound preparations made with grape seed extract.
[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solutions:
[0009] The applicant screened a Bacillus subtilis strain that antagonizes Mycoplasma synoviae from multiple strains. This bacterium was deposited at the China Center for Type Culture Collection on January 22, 2026, with the classification name: Bacillus subtilis KY2098, accession number: CCTCC NO: M 2026185, address: Wuhan University, Wuhan, China.
[0010] The scope of protection of this invention includes:
[0011] Fermentation broth or fermentation supernatant of Bacillus subtilis KY2098, wherein the fermentation broth contains live Bacillus subtilis KY2098 bacteria.
[0012] A compound preparation containing Bacillus subtilis KY2098, its fermentation broth, or fermentation supernatant.
[0013] Preferably, the compound preparation described above also contains grape seed extract.
[0014] In the above-mentioned compound preparation, preferably, the grape seed extract is a water extract of grape seeds.
[0015] The above-mentioned compound preparation, preferably, the preparation method of the grape seed extract includes: extracting crushed and sieved grape seeds multiple times at a material-to-water ratio of 1:5-20, combining the filtrates, heating and refluxing, ultrafiltration, concentrating, and then spray drying to obtain the final product.
[0016] In the above-described compound preparation, preferably, the mass ratio of Bacillus subtilis KY2098 bacterial powder to grape seed extract is 1:0.005-0.02, and the effective bacterial concentration of Bacillus subtilis KY2098 bacterial powder is 5.0 × 10⁻⁶. 9 CFU / g.
[0017] Application of Bacillus subtilis KY2098, fermentation broth of Bacillus subtilis KY2098, fermentation supernatant of Bacillus subtilis KY2098, or compound preparations in the preparation of drugs for the treatment or prevention of Mycoplasma synoviae disease in chickens.
[0018] Application of Bacillus subtilis KY2098, fermentation broth of Bacillus subtilis KY2098, fermentation supernatant of Bacillus subtilis KY2098, or compound preparations in the preparation of drugs for the treatment or prevention of Mycoplasma synoviae infection in chickens.
[0019] Application of Bacillus subtilis KY2098, fermentation broth of Bacillus subtilis KY2098, fermentation supernatant of Bacillus subtilis KY2098, or compound preparations in the preparation of chicken feed additives.
[0020] The above-described applications refer to pharmaceutically acceptable dosage forms, including but not limited to tablets, capsules, granules, injections, powders, or drops.
[0021] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0022] The applicant screened a strain of Bacillus subtilis from multiple strains that antagonizes Mycoplasma synoviae. This strain can regulate metabolism, enhance immunity, and promote feed digestion and absorption, while also exhibiting good inhibitory effects against Mycoplasma synoviae. Grape seed extract improves meat quality and production performance in livestock and poultry, enhances antioxidant and immune functions, and inhibits the proliferation of Mycoplasma synoviae. These two components were prepared into a Bacillus subtilis-grape seed extract compound preparation, which exhibits a synergistic effect. Experiments have confirmed that the compound preparation described in this invention achieves a 90%–100% prevention and treatment effect against Mycoplasma synoviae disease in chickens, and also promotes growth and enhances immunity in animals, which is of great significance for the industrialization of antibiotic-free animal husbandry. Attached Figure Description
[0023] Figure 1 The colony morphology, Gram staining microscopy, and scanning electron microscopy results of strain KY2098 are shown.
[0024] Wherein: A: Macroscopic morphology of strain KY2098 on NA culture medium; B: Microscopic examination results of strain KY2098 under an optical microscope (10×100); C: Microscopic examination results of strain KY2098 under an electron microscope.
[0025] Figure 2 Phylogenetic tree of the 16S rDNA sequence of strain KY2098.
[0026] Figure 3 The results show the stress resistance of strain KY2098;
[0027] Where: A: acid resistance result of KY2098; B: bile salt resistance result of KY2098.
[0028] Figure 4 Concentration detection of MS from different sources.
[0029] Figure 5 The minimum inhibitory concentration (MIC) for strain KY2098 was determined.
[0030] Figure 6 Anatomical diagrams for animal experiments;
[0031] Among them: A: Blank control group; B: Virus challenge control group; C: KY2098 group; D: Grape seed extract group; E: Compound preparation prevention group; F: Compound preparation treatment group.
[0032] Figure 7 A graph showing the viral load in throat swabs used in animal experiments. Detailed Implementation
[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to embodiments. It should be understood that the embodiments described herein are for illustrative purposes only and are not intended to limit the invention.
[0034] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0035] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0036] Example 1: Strain Screening and Identification
[0037] 1.1 Isolation and culture of strain KY2098
[0038] Fecal, milk, and intestinal samples were collected from farms in multiple provinces across China. 1g or 1ml of each sample was dissolved in 100mL of sterile physiological saline, serially diluted, and then spread onto NB solid medium (10g peptone, 3g beef extract, 5g sodium chloride, 15g agar, 1000ml distilled water, autoclaved at 121℃ for 15min). The medium was incubated at 37℃ for 24h. Based on morphological characteristics such as colony size, color, transparency, and edge, 104 strains of Bacillus subtilis were isolated and purified. Using Mycoplasma synoviae HB03 as an indicator bacterium, initial screening and multiple rescreenings were performed, ultimately yielding one bacterial strain with strong antibacterial effect, named KY2098. The results are shown in Tables 1 and 2.
[0039] Table 1. Preliminary screening of Bacillus subtilis strains that inhibit Mycoplasma synoviae HB03.
[0040]
[0041] Note: "-" indicates invalid, and "+" indicates valid.
[0042] Table 2. Secondary screening of Bacillus subtilis strains that inhibit Mycoplasma synoviae HB03.
[0043]
[0044] Note: The numbers in the table indicate that the supernatant of the strain's fermentation broth has an inhibitory effect on HB03 at that dilution factor. The larger the number, the stronger the inhibitory effect.
[0045] 1.2 Identification of strain KY2098
[0046] Based on comprehensive colony characteristics, physiological and biochemical properties, and molecular biological detection results, all bacteria were identified as *Bacillus subtilis*. The specific technical solution is as follows:
[0047] (1) Colony characteristics and bacterial cell morphology
[0048] In NB solid medium, colonies are milky white or slightly yellowish-white, initially appearing as concave or round shapes with a moist, opaque surface and neat edges. Later growth results in rough, irregularly edged, and opaque colonies. In NB liquid medium, a thin film forms on the surface, and the liquid is uniformly turbid. Gram staining and microscopic examination reveal Gram-positive bacteria; the bacteria are short rod-shaped, encapsulated in mucus, and can produce spores, with plump, terminal spores (e.g., ...). Figure 1 and Figure 2 ).
[0049] (2) Identification of physiological and biochemical characteristics
[0050] Single colonies were picked and inoculated into bacterial micro-test tubes for the determination of physiological and biochemical indicators. Classification and determination were performed according to Bergey's Manual of Bacteriological Identification and the Manual of Systematic Identification of Common Bacteria. The results are shown in Table 3. Strain KY2098 was preliminarily identified as belonging to the genus Bacillus.
[0051] Table 3. Physiological and biochemical test results of strain KY2098
[0052]
[0053] Note: + indicates positive, – indicates negative.
[0054] (3) Phylogenetic tree of strain KY2098
[0055] BLAST analysis was performed on 16S rDNA of strain KY2098, and a phylogenetic tree was constructed using MEGA 7.0 with high homology and the Neighbor-Joining method (e.g.,...). Figure 2 ).
[0056] Based on the phylogenetic tree and physiological and biochemical characteristics analysis of 16S rDNA, strain KY2098 was identified as Bacillus subtilis. This bacterium was deposited at the China Center for Type Culture Collection (CCTCC) on January 22, 2026, with the classification name: Bacillus subtilis KY2098, accession number: CCTCC NO: M2026185, address: Wuhan University, Wuhan, China.
[0057] (4) Shake flask experiment of strain KY2098
[0058] Take 10 μL of KY2098 from a glycerol tube and incubate it on NB solid medium at 37°C for 12 h. Pick a single colony and add it to 3 mL of NB liquid medium. Incubate at 37°C with shaking at 200 rpm for 4 h to obtain activated bacterial culture. Inoculate the above bacterial culture at a 1% inoculum into 100 mL of NB liquid medium and incubate at 37°C with shaking at 200 rpm for 24 h to obtain fermentation broth. At this time, the viable cell count of the fermentation broth is approximately 5 × 10⁻⁶. 8 CFU / mL, used in the following examples.
[0059] The fermentation broth was centrifuged at 5000 rpm for 10 min to obtain the supernatant.
[0060] 1.3 Enzyme production capacity of Bacillus subtilis KY2098
[0061] (1) Protease activity
[0062] Two μL of KY2098 fermentation broth was vertically dropped onto NB solid medium containing 10% skim milk powder. After 10 min, the cultures were incubated at 37℃ for 1, 2, and 3 days, respectively. The presence of a distinct proteolytic zone around the strain was observed, and the diameter of the zone was measured to assess the strain's protease production capacity. The results showed that the diameters (D) of the proteolytic zones of KY2098 were 13.50±2.86 mm, 20.38±2.87 mm, and 25.00±2.94 mm, respectively, indicating that Bacillus subtilis KY2098 can produce protease with a prominent protease production characteristic. As a feed additive, it can significantly improve the utilization rate of feed protein.
[0063] (2) Amylase activity
[0064] Two μL of KY2098 fermentation broth was vertically dropped onto NB solid medium containing 1% soluble starch. After static incubation at 37℃ for 1, 2, and 3 days, 1 mL of iodine solution was added. The presence of a clear starch-dissolving zone around the strain was observed within 1 minute, and the diameter of the dissolving zone was measured to assess the strain's amylase production capacity. The measured diameters (D) of the KY2098 dissolving zone were 18.00±0.00 mm, 23.25±3.89 mm, and 35.75±1.77 mm, respectively, indicating that Bacillus subtilis KY2098 can produce amylase.
[0065] 1.4 Stress resistance characteristics of Bacillus belyssus KY2098
[0066] (1) Acid resistance test
[0067] Strain strain KY2098 was cultured on NB solid medium for 3 days. Colonies were washed off with 6 mL of physiological saline and thoroughly mixed. The bacterial suspension was then inoculated at a 10% inoculum into NB liquid medium at pH values of 2, 3, 4, 5, 6, and 7 (initial bacterial concentration was 5 × 10⁻⁶). 8 (CFU / mL), with 3 replicates per group. After incubation at 37℃ with shaking for 3 hours, observe the growth and count the colonies.
[0068] The results show that ( Figure 3 In section A), within the pH range of 2–7, the viable count of strain KY2098 was consistently 2 × 10⁻⁶. 8 The presence of CFU / mL or higher indicates that the Bacillus subtilis KY2098 in this invention has a high survival rate in a low-pH gastric acid environment, allowing a sufficient number of live bacteria to pass through the stomach and reach the intestines to exert their effects.
[0069] (2) Bile salt tolerance test
[0070] Strain strain KY2098 was cultured on NB solid medium for 3 days. Colonies were washed off with 6 mL of physiological saline and thoroughly mixed. The bacterial suspension was then inoculated at 10% in NB liquid medium at concentrations of 0.0%, 0.1%, 0.2%, 0.3%, and 0.4% (initial bacterial concentration was 5 × 10⁻⁶). 8 (CFU / mL), with 3 replicates per group. After incubation at 37℃ with shaking for 3 hours, observe the growth and count the colonies.
[0071] The results show that ( Figure 3 In sample B), after tolerating for 3 hours in a liquid with a bile salt concentration of 0.1%-0.4% (mass percentage), the viable count of strain KY2098 and the zero-bile-salt group (bile salt concentration of 0.0%) were both above 1×10⁻⁶. 8 A CFU / mL level above indicates that Bacillus subtilis KY2098 can tolerate a high bile salt environment.
[0072] 1.5 Determination of the minimum inhibitory concentration (MIC) of fermentation supernatant of Bacillus subtilis KY2098 against different MS concentrations
[0073] Strains of KY2098 were cultured on NB solid medium for 12 h. Single colonies were picked and added to 3 mL of NB liquid medium, and cultured at 37°C with shaking for 4 h to obtain activated bacterial solution. This activated bacterial solution was then inoculated into 100 mL of NB liquid medium at a 1% inoculum size and cultured at 37°C with shaking for 24 h to obtain fermentation broth. The fermentation broth was centrifuged at 5000 rpm for 10 min to obtain the supernatant for later use. For MS analysis, five strains with high virulence from different regions were selected: HB03 (Hubei), 6s (Hebei), MS-1 (Henan), MS-2 (Shandong), and MS-3 (Jiangxi), with concentrations of 10... 8 10 8 10 7 10 6 10 8 CCU / mL ( Figure 4 ).
[0074] The fermentation broth supernatant at different dilutions was cultured with MS at 37°C for 2 days using the two-fold dilution method. The minimum inhibitory concentration (MIC) of strain KY2098 was determined by the color change of the culture medium. Figure 5 The results showed that the supernatant of the fermentation broth of strain KY2098 still had an inhibitory effect on HB03 (Hubei), 6s (Hebei), MS-1 (Henan), MS-2 (Shandong) and MS-3 (Jiangxi) at dilution ratios of 96, 48, 48, 48 and 96 times, respectively.
[0075] Example 2:
[0076] Fermentation process of Bacillus subtilis KY2098
[0077] After activating Bacillus subtilis KY2098, single colonies were picked and inoculated into NB liquid medium. The culture was then incubated at 37℃ with shaking at 220 rpm for 12 hours to obtain the seed culture. The seed culture was then inoculated into fermentation medium (25g corn starch, 10g peptone, 2g soybean meal, 3g calcium chloride, 2g sodium chloride, 2g dipotassium hydrogen phosphate, 0.5g magnesium sulfate, 0.3g manganese sulfate, 0.002g antifoaming agent, 1000ml distilled water, pH adjusted to 7.0 with sodium hydroxide, autoclaved at 121℃ for 15 minutes) at a constant temperature of 37℃ with a pH of 6.5±0.2. The initial stirring speed was 75 rpm, and the stirring speed was gradually increased as the dissolved oxygen level decreased, eventually reaching 200 rpm. The initial aeration rate was 1 m³ / min. 3As fermentation time increases and fermentation density rises, dissolved oxygen decreases, the aeration rate is gradually increased, controlling dissolved oxygen (DO) at 5%-10%, and the aeration rate reaches 8 m³ / h. 3 / h, after culturing under these conditions for 24 hours, the mixture is transferred to a container. Once the transgenic rate reaches 90%, the Bacillus subtilis KY2098 fermentation broth is obtained. The effective bacterial count of KY2098 can reach 5.0 × 10⁶. 9 CFU / mL.
[0078] Example 3:
[0079] Preparation of a compound formulation containing Bacillus subtilis and grape seed extract, and other control groups:
[0080] Compound preparation: Bacillus subtilis KY2098 powder and grape seed extract are mixed at a mass ratio of 1:0.01.
[0081] The preparation method of the Bacillus subtilis KY2098 powder is as follows: Bacillus subtilis KY2098 fermentation broth is spray-dried to obtain Bacillus subtilis KY2098 (the effective bacterial concentration after spray drying is 5.0 × 10⁻⁶). 9 CFU / g), also used in the following examples.
[0082] Grape seed extract is an aqueous extract of grape seeds, which is obtained by drying, pulverizing and sieving grape seeds, extracting twice at a ratio of 1:10 between the material and the liquid (i.e., pure water), combining the filtrates, heating and refluxing, ultrafiltration to obtain the extract, concentrating the obtained extract and spray drying it, and is also used in the following examples.
[0083] Comparative Example 1:
[0084] KY2098 formulation: Only effective bacterial concentration is 5.0 × 10⁻⁶. 9 Bacillus subtilis KY2098 powder, CFU / g.
[0085] Comparative Example 2:
[0086] Grape seed extract preparations: Grape seed extract only.
[0087] Example 4:
[0088] Preparation of animal models and animal experiments
[0089] 1.1 Experimental animals: 21-day-old Leghorn white laying hens were randomly divided into 6 groups of 10 each. All hens were housed in the same environmentally controlled facility (clean cages equipped with fiberglass feeders and plastic mesh floors), with free access to food and water. They were provided with the same photoperiod (16 hours of light: 8 hours of darkness), relative humidity (60%-70% for days 1-7; 50%-60% for days 8-42), and room temperature (33℃±2℃ for days 1-7; gradually reduced to 24℃ for days 8-16; 24℃ for days 17-42) for one week of acclimatization.
[0090] 1.2 The experiment included a blank control group, a challenge control group, a KY2098 group, a grape seed extract group, a compound preparation prevention group, and a compound preparation treatment group.
[0091] Blank control group and challenge control group (CON): fed a corn-soybean based diet that meets the nutritional requirements of laying hens for pelleted feed.
[0092] Compound preparation prevention group: Feed containing compound preparation was fed for seven days before the challenge until the end of the experiment. The amount of compound preparation added was 10,000 g / T of feed.
[0093] Compound preparation treatment group: Feed containing compound preparation was given from the day of the challenge until the end of the experiment. The amount of compound preparation added was 10,000g / T of feed.
[0094] KY2098 group: Feed containing KY2098 was added for seven days before the challenge until the end of the experiment. The amount of KY2098 added was 10,000 g / T of feed.
[0095] Grape seed extract group: Feed containing grape seed extract was fed for seven days before the challenge until the end of the experiment. The amount of grape seed extract added was 1000g / T of feed.
[0096] At 28 days of age, each group of chickens was challenged with Mycoplasma synoviae strain HB03 in the larynx and trachea. Ten chickens were challenged in each group, and each chicken was challenged with 1×10 strains. 7 In the CCU, after challenge, all groups continued to be fed according to the above method for 28 days, and necropsy was performed at 56 days of age. During the experiment, the mental state of the experimental animals was observed at all times, and the incidence and mortality were recorded; after the experiment, growth performance and other relevant indicators were calculated and obtained as shown in Table 4.
[0097] 1.3 Measurement Indicators
[0098] (1) Clinical symptoms and pathological autopsy
[0099] The results showed that the control group exhibited typical symptoms of Mycoplasma synoviae infection, such as lethargy, ruffled feathers, and swollen joints, with a morbidity rate of 80% after challenge. The KY2098 group and grape seed extract group showed slight improvement in symptoms compared to the challenge group after challenge, with a morbidity rate of 50% in both groups. The compound preparation prevention group showed significant improvement in symptoms compared to the challenge group after challenge, consistent with the control group, with a morbidity rate of 0%. The compound preparation treatment group showed improvement in symptoms compared to the challenge group after challenge, with a morbidity rate of 10%. No chicken deaths occurred in this animal experiment.
[0100] Post-mortem examination revealed that half of the chickens in the challenged control group exhibited severe airsacculitis and peritonitis symptoms after dissection; approximately 30% of the chickens in the KY2098 group and grape seed extract group showed airsacculitis symptoms, with slight improvement compared to the challenged group; all chickens in the compound preparation prevention group showed normal dissection results, essentially reaching the dissection level of the blank control group, with significant improvement compared to the challenged control group; 10% of the chickens in the compound preparation treatment group showed airsacculitis symptoms, with improvement compared to the challenged control group (see...). Figure 6 ).
[0101] Experimental results confirmed that, with the same dosage of the formulation, the combination of Bacillus subtilis and grape seed extract was more effective than the intervention of a single substance, and had a synergistic effect.
[0102] (2) Growth performance
[0103] All laying hens were weighed on an empty stomach at 21, 28 and 56 days of age, and their feed intake was recorded. Average body weight (BW), average daily gain (ADG), average daily feed intake (ADFI) and feed conversion ratio (F / G) were calculated.
[0104] Average body weight (BW) = Body weight at the end of the experiment / Number of chickens in each group;
[0105] Average daily weight gain (ADG) = (body weight at the end of the experiment - body weight at the beginning of the experiment) / (number of days in the experiment × number of chickens per group);
[0106] Average daily feed intake (ADFI) = Feed intake during the trial period / (Number of trial days × Number of chickens per group)
[0107] Feed conversion ratio (F / G) = Average daily feed intake / Average daily weight gain
[0108] Table 4 Effects on broiler growth performance
[0109]
[0110] Note: * indicates a significant difference compared to the challenge control group (p < 0.05), and no annotation indicates no significant difference (p > 0.05).
[0111] As shown in Table 4, at the end of the experiment, compared with other groups, the average body weight (BW), average daily weight gain (ADG), and average daily feed intake (ADFI) of the challenged group all showed a decreasing trend (P>0.05). Among them, compared with the challenged group, the average body weight (BW) and average daily weight gain (ADG) of the hens in the compound preparation prevention group showed a significant increasing trend (P=0.099, P=0.095).
[0112] (6) Detection of viral load in pharyngeal swabs
[0113] Pharyngeal swabs were collected from each group of chicks on days 3, 7, 11, 14, 18, and 25 post-infection. DNA was extracted, and the CT value was determined by quantitative real-time PCR for viral load detection. Results showed that from day 11 post-infection, the MS viral load in the combination therapy prevention and treatment groups showed a significant decreasing trend compared to the challenge control group. Bacillus subtilis KY2098 and grape seed extract alone showed no significant beneficial effect compared to the combination therapy (see [link to study]). Figure 7 ).
[0114] Experimental results confirmed that the combination of Bacillus subtilis and grape seed extract can exert a probiotic effect by inhibiting the proliferation of MS in chicks. Its effect is better than that of intervention by a single substance, and it has a synergistic effect.
[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.
Claims
1. An isolated strain of Bacillus subtilis KY2098, with accession number CCTCC NO: M 2026185.
2. The fermentation broth or fermentation supernatant of Bacillus subtilis according to claim 1, wherein the fermentation broth contains live Bacillus subtilis KY2098 bacteria.
3. A compound preparation comprising Bacillus subtilis KY2098 as described in claim 1, its fermentation broth or fermentation supernatant.
4. The compound preparation according to claim 3, characterized in that: The compound preparation contains grape seed extract.
5. The compound preparation according to claim 4, characterized in that: The grape seed extract mentioned above is a water extract of grape seeds.
6. The compound preparation according to claim 5, characterized in that: The method for preparing the grape seed extract includes: extracting crushed and sieved grape seeds multiple times at a material-to-water ratio of 1:5-20, combining the filtrates, heating and refluxing, ultrafiltration, concentrating, and then spray drying to obtain the extract.
7. The compound preparation according to claim 4, characterized in that: The mass ratio of Bacillus subtilis KY2098 bacterial powder to grape seed extract is 1:0.005-0.02, and the effective bacterial concentration of Bacillus subtilis KY2098 bacterial powder is 5.0 × 10⁻⁶. 9 CFU / g.
8. Any of the following applications of the Bacillus subtilis KY2098 of claim 1, the fermentation broth or fermentation supernatant of Bacillus subtilis KY2098 of claim 2, or the compound preparation of claim 3: 1) Its application in the preparation of drugs for the treatment or prevention of mycoplasma synoviae infection in chickens; 2) Its application in the preparation of drugs for the treatment or prevention of Mycoplasma synoviae infection in chickens; 3) Application in the preparation of chicken feed additives.
9. The application according to claim 8, wherein the dosage form of the drug is any pharmaceutically acceptable dosage form.