Bacillus subtilis BS-1 and its application in buffalo silage

Abstract

The present application relates to the field of microbial technology, in particular to bacillus subtilis BS-1 and its application in water buffalo silage feed, the strain BS-1 of the present application has good bacteriostatic activity on escherichia coli, salmonella and streptococcus uberis, after mixing and fermenting according to a specific proportion with orange peel and cassava residue, the bacteriostatic spectrum of the obtained silage feed is significantly enhanced, which can simultaneously inhibit escherichia coli, campylobacter jejuni, salmonella, streptococcus uberis, staphylococcus aureus and streptococcus agalactiae that cause diarrhea and mastitis. In the feed, after mixing 60%-75% of orange peel and 25%-40% of cassava residue, the crude protein content of the fermentation product is significantly improved, and the fiber content is reduced. Feeding test shows that the feed can significantly improve the milk yield and milk quality of lactating water buffalo, and effectively alleviate diarrhea. The present application provides a safe, efficient and nutritious silage feed with disease prevention function.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to Bacillus subtilis BS-1 and its application in buffalo silage. Background Technology

[0002] Silage is an important component of ruminant diets, and its quality directly affects animal production performance and health. Orange peel is high in protein and rich in nutrients, while cassava residue has a large biomass and stable fiber structure. Mixing these two in silage can achieve nutritional complementarity and represents an important direction for developing high-quality roughage. However, conventional mixed silage mainly focuses on nutrient preservation and palatability; its functional role in regulating animal intestinal health and preventing specific diseases still needs further exploration.

[0003] In dairy farming, especially buffalo farming, diarrhea caused by pathogenic Escherichia coli and Salmonella, as well as mastitis caused by Streptococcus mammologica and Staphylococcus aureus, are major problems leading to production losses and increased veterinary drug costs. For a long time, the livestock industry has relied heavily on antibiotics for prevention and treatment, but this has resulted in increasingly serious risks of drug residues and bacterial resistance. Therefore, developing functional feeds that can inhibit pathogens and enhance animals' own resistance through bioregulation has become an important research direction in the industry.

[0004] Probiotic preparations, as a potential alternative to antibiotics, are widely used in feed additives. Some Bacillus strains have attracted attention due to their ability to tolerate processing and storage, colonize the gut, and inhibit pathogens. However, the antibacterial spectrum of single strains is often limited, and their function is constrained by the feed substrate and fermentation process. Combining specific probiotics with suitable plant materials through fermentation may produce synergistic effects, not only improving the nutritional value of feed but also potentially expanding its bioactivity, resulting in a feed product with both nutritional and disease prevention functions. However, currently, there is still a lack of mature and reliable technical solutions for specialized fermented silage targeting major pathogens in dairy cows, possessing both broad-spectrum antibacterial function and significant nutritional enhancement effects.

[0005] Based on the aforementioned industry demands and current technological status, developing a novel silage that uses specific probiotics to ferment forage and simultaneously improves feed nutritional quality, enhances animal production performance, and expands the antibacterial spectrum has significant application value. Summary of the Invention

[0006] In view of the above, it is necessary to develop a special fermented silage that targets the main pathogens of dairy cows, has broad-spectrum antibacterial function and significant nutritional enhancement effects. This new type of silage can simultaneously improve the nutritional quality of feed, improve animal production performance and expand the antibacterial spectrum, and has important application value.

[0007] To achieve the above objectives, this invention has screened out a new strain: Bacillus subtilis. Bacillus subtilis Strain BS-1, its classification name is: Bacillus subtilis BS-1, Chinese classification name: Bacillus subtilis BS-1, accession number GDMCC NO: 67856; this strain is deposited at Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on February 11, 2026.

[0008] The present invention also includes the Bacillus subtilis. Bacillus subtilis Inoculum of strain BS-1.

[0009] The present invention also includes the Bacillus subtilis. Bacillus subtilis Application of strain BS-1 or the aforementioned inoculum in the preparation of silage from orange peel and / or cassava residue.

[0010] The present invention also includes the Bacillus subtilis. Bacillus subtilis Application of strain BS-1 in the preparation of antibacterial agents, wherein the antibacterial agent inhibits Escherichia coli (Escherichia coli). Escherichia coli Salmonella ( Salmonella spp. ) and / or Streptococcus lactis ( Streptococcus uberis ).

[0011] This invention also includes the application of the orange peel and / or cassava residue silage in inhibiting bacterial growth, wherein the bacteria are Escherichia coli (Escherichia coli). Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ),salmonella( Salmonella spp. Streptococcus lactis ( Streptococcus uberis Staphylococcus aureus Staphylococcus aureus ) and / or alactamase-free streptococci ( Streptococcus agalactiae ).

[0012] The present invention also includes the application of the orange peel and / or cassava residue silage in improving the yield and / or milk quality of dairy buffalo.

[0013] The present invention also includes the Bacillus subtilis. Bacillus subtilis Silage of strain BS-1, the method for preparing the silage is as follows: orange peel and cassava residue are crushed and mixed evenly, and then inoculated with Bacillus subtilis. Bacillus subtilis The strain BS-1 is obtained by ensiling for 30 days.

[0014] Furthermore, the orange peel accounts for 60%-75% of the total weight, and the cassava residue accounts for 25%-40% of the total weight.

[0015] Furthermore, the orange peel accounts for 75% of the total mass, and the cassava residue accounts for 25% of the total mass.

[0016] A feeding method to increase buffalo milk production, wherein the feeding method comprises: feeding lactating buffaloes with 30% by weight of the silage and 70% by weight of the basal diet, wherein the basal diet is a fermented feed.

[0017] Furthermore, the basic fermented feed consists of 16% molasses, 52% sweet corn stalks, and 32% wheat straw by weight.

[0018] The present invention has the following beneficial effects: The strain BS-1 of the present invention was isolated by the research group. Testing showed that this strain has good antibacterial activity against Escherichia coli, Salmonella, and Streptococcus lactis. Furthermore, the antibacterial spectrum of this strain was enhanced after fermentation with orange peel and cassava residue. The orange peel-cassava residue mixed silage showed antibacterial effects against Escherichia coli, Campylobacter jejuni, and Salmonella, which cause diarrhea, and against Streptococcus lactis, Staphylococcus aureus, and Streptococcus agalactiae, which cause mastitis, with an enhanced antibacterial spectrum. In addition, testing showed that when orange peel-cassava residue mixed silage was prepared with 60%-75% orange peel and 25%-40% cassava residue by weight, the protein content was significantly increased and the fiber content decreased. Feeding this silage to lactating buffalo significantly improved milk yield and quality, while also effectively alleviating diarrhea in buffalo. It is a safe, effective, and broad-spectrum antibacterial silage. Attached Figure Description

[0019] Figure 1 Plate culture medium for strain BS-1.

[0020] Figure 2 This is a microscopic image of Gram-stained strain BS-1.

[0021] Information on the preservation of biological materials

[0022] The strain information deposited in this application is: Bacillus subtilis Bacillus subtilis Strain BS-1, its classification name is: Bacillus subtilis BS-1, Chinese classification name: Bacillus subtilis BS-1, accession number GDMCC NO: 67856; this strain is deposited at Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on February 11, 2026. Detailed Implementation

[0023] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0024] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0025] Example 1

[0026] This example demonstrates the isolation, identification, and growth characteristics of the strain.

[0027] 1. Screening and isolation of strains: The strains in this application were isolated from animal feces.

[0028] 2. Morphological and molecular identification of the screened BS-1:

[0029] ① Inoculate BS-1 strain onto LB medium and observe colony morphology characteristics as follows: Figure 1 and Figure 2 As shown, it is characterized by: medium to large, grayish-white, dull colonies with round, wrinkled surfaces and irregular edges; Gram-positive bacteria that are long rod-shaped and spore-forming.

[0030] ② Molecular biological identification: The above-mentioned strains were sequenced and verified: The 16S rRNA gene sequence of the strains was amplified using universal primers, and the purified PCR product was sequenced. The results are shown in SEQ ID NO.1 of the sequence listing. Comparison showed that strain BS-1 is similar to Bacillus subtilis. Bacillus subtilis With 100% similarity and based on morphological identification, this strain was named... Bacillus subtilis .

[0031] Example 2

[0032] This example describes the identification of the growth characteristics of strain BS-1, as detailed below:

[0033] 1. The growth characteristics of strain BS-1 are as follows: 0~1.5h: lag phase; 1.5~5h: logarithmic growth phase; after 5h, growth gradually slows down and enters the stationary phase; after 14h, growth decreases and enters the death phase. It exhibits rapid proliferation and high cell biomass, meeting the growth characteristics required of microorganisms as feed additives.

[0034] 2. Cellulase activity of strain BS-1: The cellulase activity of BS-1 was determined with reference to the literature of Li Riqiang et al.: the approximate values ​​of the four cellulases Cen, FPA, Cex and BG were 45 U / mL, 25 U / mL, 20 U / mL and 35 U / mL, respectively.

[0035] 3. Antimicrobial susceptibility results: The antimicrobial susceptibility results of strain BS-1 are shown in Table 1:

[0036] Table 1. Antimicrobial susceptibility results of strain BS-1

[0037]

[0038] Note: R: drug resistance; I: intermediate; S: sensitive.

[0039] As shown in Table 1, the susceptibility of strain BS-1 to 31 antibiotics across 11 classes was determined. Strain BS-1 was sensitive to amoxicillin, ampicillin, mezlocillin, cephalexin, cefaclor, cefotaxime, streptomycin, gentamicin, amikacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, acetylspiramycin, erythromycin, tylosin, azithromycin, tetracycline, minocycline, trimethoprim-sulfamethoxazole, sulfamethoxazole, vancomycin, teicoplanin, and clindamycin; resistant to penicillin, piperacillin, methoxypyrimidine, bacitracin, and rifampin; and intermediately resistant to polymyxin and lincomycin.

[0040] 4. In vivo safety test: (oral toxicity): Acute oral toxicity in mice (1×10⁻⁶) 10 In the CFU / kg test, mice in both the control and experimental groups showed normal appetite, no abnormal behavior, and good mental state within 24 hours, consistent with the control group, and no deaths occurred. There was no significant difference in average body weight before and after gavage. Dissection revealed no significant pathological changes in the major organs of the experimental group. Inoculation of cross-sections of various mouse organs onto nutrient agar plates showed no bacterial colony growth, indicating no bacterial translocation. Combined with drug sensitivity results and in vivo safety tests, this demonstrates good safety.

[0041] 5. Biological characteristics (tolerance test): The tolerance of the isolated bacteria under different pH conditions was determined, as shown in Table 2.

[0042] Table 2 Biological characteristics of strain BS-1

[0043]

[0044] As shown in Table 2, after being treated for 4 hours at pH 2.0, pH 3.0, pH 4.0 and pH 5.0, the survival rate of strain BS-1 at different pH values ​​(pH 3.0, 4.0, 5.0 and 6.0) was above 95% (95.61%, 96.82%, 98.12% and 98.64%), indicating that the isolated strain has good acid resistance.

[0045] 6. Determination of salt tolerance: The tolerance of isolated bacteria under different bile salt concentrations was determined by simulating the inhibitory effect of intestinal bile salts, as shown in Table 3.

[0046] Table 3 Salt tolerance of strain BS-1

[0047]

[0048] As shown in Table 3, the survival rate of strain BS-1 was above 80% after 4 hours of treatment in mediums with different bile salt concentrations (0.1%, 0.2%, 0.3%, 0.4%), indicating that the isolated bacteria had good tolerance to high concentrations of bile salts.

[0049] 7. Heat resistance: The growth of strain BS-1 after treatment at different temperatures is shown in Table 4:

[0050] Table 4. Heat resistance of strain BS-1

[0051]

[0052] As shown in Table 4, the survival rate of strain BS-1 was above 80% after being treated at different temperatures (60℃, 70℃, 80℃, 90℃) for 4 hours, indicating that the isolated bacteria have good tolerance to high temperatures.

[0053] Example 3

[0054] This example demonstrates an antibacterial experiment on strain BS-1.

[0055] Five indicator bacteria that commonly cause buffalo diarrhea or mastitis: Escherichia coli ( Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ),salmonella( Salmonella spp. Streptococcus lactis ( Streptococcus uberis Staphylococcus aureus Staphylococcus aureus ) and agalactococci ( Streptococcus agalactiae After activation and culture, the culture was prepared for use. The antibacterial activity of strain BS-1 against the above five indicator bacteria was determined by the perforated agar diffusion method. The results of the measured inhibition zones are shown in Table 5.

[0056] Table 5. Antibacterial effects of strain BS-1 against common pathogens.

[0057]

[0058] Note: In the table, "—" indicates no inhibition effect, different lowercase letters indicate significant differences in the same column of data (p<0.05), and the same letters indicate no significant differences in the same column (p>0.05). The same applies to the following table.

[0059] Table 5 shows that strain BS-1 is effective against Escherichia coli (E. coli). Escherichia coli Salmonella ( Salmonella spp. ) and Streptococcus lactis ( Streptococcus uberis It has antibacterial effects against Campylobacter jejuni ( ) Campylobacter jejuni Staphylococcus aureus Staphylococcus aureus ) and agalactococci ( Streptococcus agalactiaeNo antibacterial effect; Based on the inhibition zone: Escherichia coli ( Escherichia coli The inhibition zone of ) was significantly higher than that of other pathogens (p<0.05), followed by Salmonella ( Salmonella spp. Antibacterial zone and Streptococcus lactis ( Streptococcus uberis ),salmonella( Salmonella spp. Antibacterial zone and Streptococcus lactis ( Streptococcus uberis The inhibition zones of strain BS-1 showed no significant difference, indicating that strain BS-1 was not effective against Escherichia coli (E. coli). Escherichia to be cultivated The antibacterial effect of ) is the best, followed by Salmonella ( Salmonella spp. Antibacterial zone and Streptococcus lactis ( Streptococcus uberis ).

[0060] Example 4

[0061] This example demonstrates the antibacterial effect of strain BS-1 on different plant silages.

[0062] From Example 2, we know that strain BS-1 itself is effective against Campylobacter jejuni (… Campylobacter jejuni Staphylococcus aureus Staphylococcus aureus ) and agalactococci ( Streptococcus agalactiae None of the above methods showed any inhibitory effect. However, these bacteria often cause diarrhea in buffalo and easily lead to mastitis in lactating buffalo. According to existing technology reports, the metabolites of plants undergo changes after microbial fermentation. These metabolites have a wide range of pharmacological effects, including antiviral, antitumor, hepatoprotective, and antibacterial effects, as well as beneficial effects such as enhancing immunity and delaying aging. However, the effects vary depending on the plant. Fermentation of Bacillus subtilis with plants can promote the release of effective plant activity. Therefore, we considered the inhibitory effect of Bacillus subtilis fermented with several commonly used forages on common intestinal pathogens and mastitis pathogens in buffalo. The specific plan is as follows:

[0063] After sterilization, several commonly used waste residues (orange peel, pineapple peel, monk fruit residue, and cassava residue) are crushed and the moisture content is adjusted to about 45% with distilled water. An inoculum of 5% by weight (with an effective viable count of 103) is then added. 8 -10 9The BS-1 inoculant was inoculated into several sterilized forages (orange peel, pineapple peel, monk fruit residue, and cassava residue) and mixed well. The mixture was then placed in silage bags for silage, with each bag containing 200g of sample. The bags were sealed and fermented in the dark for 30 days to serve as the forage experimental group. Several commonly used waste residues in Guangxi (orange peel, pineapple peel, monk fruit residue, and cassava residue) were crushed after sterilization, and the moisture content was adjusted to about 45% with distilled water. Without inoculating any microbial agents, they were directly placed into silage bags, with each bag containing 200g of sample. After sealing, they were fermented in the dark for 30 days as a control group of forage. In the experiment, cassava residue was the waste material from cassava alcohol fermentation, and monk fruit residue was the waste material after processing monk fruit juice. The silage of the experimental group and the control group were mixed at a solid-liquid mass ratio of 2:1, boiled, and kept boiling for 15 minutes. After filtration, the filtrate was cooled, and then dripped onto filter paper. The filtrate was placed on a plate coated with pathogens and incubated for 24 hours. The size of the inhibition zone was then tested. The results are shown in Tables 6 and 7.

[0064] Table 6. Antibacterial effect of silage on pathogens in different control groups

[0065]

[0066] Table 2 shows that silage made from orange peel, pineapple peel, monk fruit residue, and cassava residue is effective against the pathogen Escherichia coli. Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ),salmonella( Salmonella spp. Streptococcus lactis ( Streptococcus uberis Staphylococcus aureus Staphylococcus aureus ) and agalactococci ( Streptococcus agalactiae None of them have antibacterial effects.

[0067] Table 7. Antimicrobial effects of silage inoculated with strain BS-1 on pathogens in different experimental groups.

[0068]

[0069] As shown in Tables 1, 2, and 3, fermented forage silage had no inhibitory effect on pathogens. After inoculation with strain BS-1, BS-1-pineapple peel silage and BS-1-monk fruit residue silage showed no inhibitory effect on Escherichia coli. Escherichia to be cultivated ),salmonella( Salmonella spp. ) and Streptococcus lactis ( Streptococcus uberis All of them have antibacterial effects, and are effective against Campylobacter jejuni ( Campylobacter jejuni Staphylococcus aureus Staphylococcus golden ) and agalactococci ( Streptococcus agalactiae It has no antibacterial effect, which is consistent with the antibacterial effect of strain BS-1 in Table 1.

[0070] The antibacterial activity of BS-1 strain changed after orange peel and cassava residue were inoculated with them. BS-1-orange peel silage showed improved antibacterial activity against Escherichia coli (E. coli). Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ),salmonella( Salmonella spp. Streptococcus lactis ( Streptococcus uberis ) and Staphylococcus aureus ( Staphylococcus aureus All of them have antibacterial properties, but only against Streptococcus agalactiae (Streptococcus agalactiae). Streptococcus agalactiae ) has no antibacterial effect; BS-1-cassava residue silage has no effect on Escherichia coli ( Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ),salmonella( Salmonella spp. Streptococcus lactis ( Streptococcus you are pregnant ) and agalactococcus ( Streptococcus agalactiae All of them have antibacterial properties, but only against Staphylococcus aureus ( Staphylococcus aureus The BS-1 strain showed no antibacterial activity. This indicates that the antibacterial spectrum of BS-1 inoculated with orange peel and cassava residue was broader than that of strain BS-1 in Table 1. This suggests that the antibacterial effect of strain BS-1 was enhanced after inoculation with orange peel and cassava residue, which could inhibit the growth of more bacteria and better prevent and treat animal diarrhea and mastitis. Therefore, we chose orange peel and cassava residue for the next experiment.

[0071] Example 5

[0072] Preparation and quality assessment of silage:

[0073] In Example 3, we found that orange peel and cassava residue fermented with strain BS-1 had an inhibitory effect on a variety of pathogens. In order to improve the antibacterial effect of silage, we considered mixing orange peel and cassava residue for silage to prepare mixed silage. Specifically, orange peel and cassava residue were mixed according to the mass ratio in Table 8 and then inoculated with strain BS-1 for 30 days to obtain the corresponding silage.

[0074] Table 8. Percentage of orange peel and cassava residue by mass in different experimental groups

[0075]

[0076] The nutritional components of the silage in the above experimental groups were tested, including the contents of dry matter (DM), crude protein (CP), neutral detergent fiber (NDF), and acid detergent fiber (ADF). The results are shown in Table 9.

[0077] Table 9. Nutritional composition of silage from different experimental groups

[0078]

[0079] As shown in Table 5, the dry matter (DM) of silage prepared from the mixture of orange peel and cassava residue fermented by strain BS-1 was significantly higher in group 5 than in groups 1-4 (p<0.05), and in group 4 significantly higher than in groups 1-3 (p<0.05). This indicates that the overall dry matter content increased with the increase of the proportion of cassava residue. This may be because the dry matter content of cassava residue itself is higher than that of orange peel, resulting in a higher proportion of cassava residue in the mixture and a higher overall DM.

[0080] Regarding crude protein (CP), there was no significant difference between groups 1 and 2 (p>0.05), but the CP was significantly higher than that of groups 3-5 (p<0.05). There was no significant difference between groups 3-5 (p>0.05), indicating that the crude protein content of orange peel is higher than that of cassava residue. To produce high-protein silage, a high proportion of orange peel must be ensured.

[0081] Regarding the content of neutral detergent fiber (NDF), group 5 > group 4 > group 3 > group 2 > group 1. Group 5 was significantly higher than groups 1-4 (p<0.05), group 4 was significantly higher than groups 1-3 (p<0.05), group 3 was significantly higher than groups 1-2 (p<0.05), and there was no significant difference between groups 1 and 2 (p>0.05). Regarding the content of acid detergent fiber (ADF), group 5 > group 4 > group 3 > group 2 > group 1. There was no significant difference between groups 3 and 5 (p>0.05), but there was a significant difference between groups 3-5 and groups 1-2 (p<0.05), and a significant difference between groups 1 and 2 (p<0.05). This indicates that the content of NDF and ADF increases continuously and significantly with the increase of the proportion of cassava residue, clearly indicating that cassava residue is rich in structural fiber. High fiber content can affect feed palatability and animal digestibility.

[0082] The sensory evaluation criteria are shown in Table 10:

[0083] Table 10 Sensory Evaluation Criteria

[0084]

[0085] The evaluation results based on the evaluation criteria in Table 10 are shown in Table 11:

[0086] Table 11 Sensory Evaluation Results

[0087]

[0088] As shown in Table 11, in terms of total score and grade, the total sensory evaluation score gradually decreased from 20 points in Group 1 to 15 points in Group 5. Correspondingly, the grades for Groups 1 to 4 were all "Excellent" (Grade I), while Group 5 dropped to "Fair" (Grade II). This indicates that the higher the proportion of orange peel, the better the overall sensory quality of the mixed silage. However, when the proportion of orange peel falls below a certain threshold (1:3 in group 5), the quality declines significantly, dropping below the "excellent" level. In terms of aroma, structure, and color, group 1 received the highest score of 14, while group 5 only scored 10. This indicates that a high proportion of orange peel helps produce aromatic substances and inhibits the production of undesirable fermentation odors (such as butyric acid), which aligns with the chemical properties of orange peel, which is rich in soluble carbohydrates and conducive to lactic acid fermentation. In terms of structure, groups 1-5 all maintained good structure. Regarding color, group 5 was the only group to lose points in color, while groups 1-4, with a high proportion of orange peel, maintained optimal color. This again demonstrates the positive role of orange peel in maintaining the good color of silage.

[0089] Based on the nutritional composition analysis results in Table 5, we conclude that the experimental groups with high orange peel content (Groups 1 and 2) are both nutritionally and sensorily excellent. These two groups not only have the highest crude protein (CP) content but also achieved "excellent" sensory evaluations, with Group 1 receiving a perfect score. This is the ideal feed formulation, meeting the nutritional needs of animals while also providing good palatability and appearance. In contrast, the medium-proportion groups (Groups 3 and 4) represent a trade-off between nutrition and sensory evaluation. While the CP content decreased significantly with increasing cassava residue proportions, the sensory scores (especially for Group 4) remained at the "excellent" level. This indicates that if the production goal is not to pursue the highest protein content but rather to focus on feed structure and cost, these proportions represent an acceptable balance.

[0090] Example 6

[0091] This example demonstrates an experiment using silage on dairy buffalo, as detailed below:

[0092] Sixty mid-lactation buffaloes with similar lactation days, milk yield, parity, and body condition were selected and randomly divided into 6 groups of 10 each. Each group was fed the following different experimental feeds:

[0093] CK: Basal diet is based on fermented feed.

[0094] Group 1: 50% by weight of silage from Group 1 of Example 5 + 50% by weight of basic fermented feed.

[0095] Group 2: 50% by weight of silage from Group 2 of Example 5 + 50% by weight of basic fermented feed.

[0096] Group 3: 50% by weight of silage from Group 3 of Example 5 + 50% by weight of basic fermented feed.

[0097] Group 4: 50% by weight of silage from Group 4 of Example 5 + 50% by weight of basic fermented feed.

[0098] Group 5: 50% by weight of silage from Group 5 of Example 5 + 50% by weight of basic fermented feed.

[0099] In this embodiment, the basic fermented feed consists of 16% molasses, 52% sweet corn stalks, and 32% wheat straw by weight.

[0100] The experiment lasted for 6 weeks, including a 2-week pre-trial period and a 4-week formal trial period. During the experiment, the buffalo were fed twice a day (06:00 and 14:00) and milked twice a day (06:00 and 14:00). The daily milk yield was recorded, and routine management was carried out according to the farm's prescribed procedures. Milk samples were collected on days 7, 21, and 42 of the experiment to determine the content of milk fat, milk protein, and lactose. The number of times the buffalo experienced diarrhea during the experiment was recorded: one episode of diarrhea was recorded as 1, and two episodes of diarrhea were recorded as 2, as shown in Table 12.

[0101] Table 12 Effects of silage on buffalo milk composition

[0102]

[0103] Table 12 shows that, in terms of milk production: there was no significant difference between groups 1 and 2, but groups 1 and 2 were significantly higher than groups 3-5. Considering the nutritional conditions of the silage, we believe that high-protein, low-fiber silage can significantly increase the milk production of dairy buffalo. The milk production of groups 1-5 was significantly higher than the control group (p<0.05), which may be due to the silage's effect on the bacteria causing mastitis, Streptococcus lactis, after inoculation with strain BS-1. Streptococcus uberis ), agalactococcus ( Streptococcus agalactiae ) and Staphylococcus aureus ( Staphylococcus aureus Both have antibacterial properties, reducing the probability of mastitis in dairy cows and ensuring milk production.

[0104] In terms of milk quality, there was no significant difference in milk fat content between groups 1 and 2 (p>0.05), but the milk fat content of groups 1-5 was significantly higher than that of groups 3-5 and the control group (p<0.05). The milk fat content of groups 3-5 was higher than that of the control group, but the difference was not significant (p>0.05). This may be related to the fact that easily digestible carbohydrates and appropriate fiber levels in the feed promote rumen acetic acid synthesis. There was no significant difference in milk protein content between groups 1 and 2 (p>0.05), but the milk protein content of groups 1-2 was significantly higher than that of groups 3-5 and the control group (p<0.05). This indicates that the higher the crude protein in the feed, the higher the milk protein content may be, directly benefiting from the highest crude protein supply in the feed. In terms of lactose content, there was no significant difference between groups 1-5 and the control group (p>0.05), indicating that silage has little effect on lactose content.

[0105] Regarding the frequency of diarrhea, the control group experienced 10 episodes of diarrhea, while groups 1-5 experienced 0 episodes. This indicates that inoculating the mixed silage of orange peel and cassava residue with strain BS-1 effectively inhibits Escherichia coli, a bacteria associated with intestinal diarrhea. Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ) and Salmonella ( Salmonella spp. This growth has had a remarkable effect on improving the intestinal health of buffalo.

[0106] In summary, the antibacterial activity of strain BS-1, which was selected by the applicant of this invention, was enhanced after fermenting orange peel and cassava residue, and it showed increased activity against Escherichia coli (E. coli). Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ),salmonella( Salmonella spp. Streptococcus lactis ( Streptococcus uberis ), agalactococcus ( Streptococcus agalactia ) and Staphylococcus aureus ( Staphylococcus aureus Both of these have antibacterial effects. After fermentation, the two mixed forages can significantly reduce the incidence of mastitis and diarrhea in dairy buffalo, and improve the milk yield and quality of dairy buffalo. They are a good buffalo silage, especially suitable for feeding lactating buffalo.

[0107] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. Bacillus subtilis Bacillus subtilis Strain BS-1, characterized in that, the bacillus subtilis Bacillus subtilis The strain BS-1 has the accession number GDMCC NO: 67856.

2. A microbial agent comprising the Bacillus subtilis strain BS-1 of claim 1. Bacillus subtilis The microbial agent comprising the Bacillus subtilis strain BS-1 of claim 1.

3. The Bacillus subtilis strain BS-1 or inoculant of claim 1. Bacillus subtilis Use of the Bacillus subtilis strain BS-1 or inoculant of claim 2 in the preparation of orange peel and / or cassava pulp silage.

4. The Bacillus subtilis strain of claim 1 Bacillus subtilis Use of the Bacillus subtilis strain BS-1 for the preparation of a bacteriostatic agent, characterized in that, The pathogenic bacteria inhibited by the bacteriostatic agent are Escherichia coli (E. coli) Escherichia coli ), Salmonella (Salmonella) Salmonella spp. ) and / or Streptococcus uberis (S. uberis) Streptococcus uberis ).

5. Use of orange peel and / or cassava pulp silage feed according to claim 3 for inhibiting the growth of bacteria, characterized in that, The bacteria in question is Escherichia coli (E. coli) Escherichia coli Campylobacter jejuni ( Campylobacter jejuni ),salmonella( Salmonella spp. Streptococcus lactis ( Streptococcus uberis Staphylococcus aureus Staphylococcus aureus ) and / or alactamase-free streptococci ( Streptococcus agalactiae ).

6. The application of the orange peel and / or cassava residue silage as described in claim 3 in improving the yield and / or milk quality of dairy buffalo.

7. A silage comprising the Bacillus subtilis strain BS-1 of claim 1. Bacillus subtilis The silage feed comprising the Bacillus subtilis strain BS-1, characterized in that, The preparation method of the silage is as follows: the orange peel and cassava residue are crushed and uniformly mixed and stirred, then Bacillus subtilis is inoculated Bacillus subtilis The silage of strain BS-1 is obtained after 30 days.

8. The silage according to claim 7, characterized in that, The orange peel comprises 60%-75% by weight, and the cassava residue comprises 25%-40% by weight.

9. The silage according to claim 8, characterized in that, The orange peel comprises 75% by weight and the cassava residue comprises 25% by weight.

10. A feeding method for increasing milk production in buffaloes, characterized in that, The feeding method is as follows: 30% by weight of the silage as described in claim 9 is mixed with 70% of the basal diet and fed to lactating buffalo, wherein the basal diet is a fermented feed.

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