Bacillus subtilis and microbial preparation and its application in silage preparation

Abstract

The present application belongs to the field of microbial application and feed processing technology, and discloses a bacillus subtilis, named bacillus subtilis YT1, which is preserved in China Center for Type Culture Collection, located at Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with a preservation number of CCTCC NO: M 20242767 and a preservation date of December 10, 2024. The present application also discloses a microbial preparation and the application of the bacillus subtilis or the microbial preparation in the processing of silage. The present application first simultaneously possesses high efficient lignocellulose degradation ability, excellent fermentation promotion characteristics, significant antioxidant activity and good probiotic characteristics in a strain of bacillus subtilis. This characteristic enables a single bacterial agent to achieve multiple goals such as fiber degradation, fermentation promotion, oxidation protection, which traditionally requires multiple compounded bacterial agents, and fundamentally overcomes the problems of poor compatibility between strains, unstable synergistic effect and high application cost of existing compounded additives.

Description

Technical Field

[0001] This invention belongs to the field of microbial application and feed preparation technology, and particularly relates to a Bacillus subtilis strain with both lignocellulose degradation and probiotic properties, its microbial preparations, and its application in silage preparation. Background Technology

[0002] Silage, as a crucial material foundation for animal husbandry, directly impacts animal nutritional balance and the economic benefits of the industry through its fermentation quality. The silage process is essentially a complex biochemical process relying on microorganisms such as lactic acid bacteria in an anaerobic environment. Its core lies in rapidly producing acid to inhibit harmful microorganisms and reduce nutrient loss. However, this process often faces multiple challenges: First, the inherent soluble sugar content in forage is often insufficient to support the full reproduction of lactic acid bacteria; second, the main component of plant cell walls—lignocellulose—has a stable structure, and conventional lactic acid bacteria lack the ability to degrade such complex polymers, resulting in low fiber utilization and limited nutrient release; furthermore, the biochemical reactions during silage and aerobic exposure upon opening the silo can easily lead to feed quality deterioration, oxidative loss of nutrients such as vitamins, and negatively impact feed value and animal health.

[0003] Additive development is an important technological approach to improving the quality of silage. For example, inoculation with Lactobacillus plantarum (… Lactiplantibacillus plantarum It can rapidly lower the pH value, and inoculation with Lactobacillus bushus ( L. buchneri Microbial additives can help improve aerobic stability, while using fiber-degrading bacteria can improve the digestibility of fibrous materials. However, these microbial additives usually have a single function and cannot simultaneously achieve multiple goals such as efficient fiber degradation, fermentation process optimization, and oxidative damage control. Although combining different microbial agents can achieve functional complementarity to some extent, the combination schemes often face problems such as poor compatibility between strains, unstable synergistic effects, and increased application costs.

[0004] Bacillus subtilis ( Bacillus subtilis Bacillus subtilis (BS), as a recognized safe probiotic for feed, has shown good application potential in silage. Studies have shown that BS, as a facultative aerobic bacterium, can consume oxygen to promote the rapid establishment of an anaerobic fermentation environment and has the potential to secrete lignin peroxidase, manganese peroxidase, laccase, or cellulase, thereby degrading lignocellulose components and improving the cellulose degradation rate of silage. However, the functions of existing Bacillus subtilis strains are often limited, usually focusing only on one aspect of fiber degradation or inhibition of harmful microorganisms. In particular, they generally lack systematic antioxidant activity, and currently no Bacillus subtilis strain has the ability to produce cellulase, lignin peroxidase, manganese peroxidase, and laccase simultaneously. Therefore, it is difficult to comprehensively meet the comprehensive requirements of high-quality silage in terms of nutritional value, fermentation quality, and antioxidant stability. Summary of the Invention

[0005] The technical problem to be solved by this invention is to overcome the shortcomings and defects mentioned in the background technology above. This invention aims to provide a new strain of Bacillus subtilis with both high efficiency in degrading lignocellulose and significant antioxidant activity, and to develop its corresponding microbial preparation. This preparation is dedicated to achieving multiple functions such as fiber degradation, fermentation promotion and oxidative protection simultaneously during silage fermentation, thereby overcoming the limitations of existing additive technologies such as single function, poor synergy and high application cost, and providing an innovative and efficient solution for the comprehensive improvement of silage quality.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A strain of Bacillus subtilis ( Bacillus subtilis The Bacillus subtilis was named Bacillus subtilis (B. subtilis). Bacillus subtilis YT1, deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO: M 20242767, and deposited on December 10, 2024.

[0008] Based on a general inventive concept, the present invention also provides a microbial preparation comprising at least one of the following: live cells, inactivated cells, culture, fermentation broth, extract or metabolite of Bacillus subtilis YT1.

[0009] Based on a general inventive concept, the present invention also provides the application of the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation in the preparation of silage.

[0010] Preferably, the above-mentioned applications are used to reduce the fiber content in silage, improve the fermentation quality of silage, and / or improve the antioxidant capacity of silage.

[0011] More preferably, the application includes at least one of the following aspects:

[0012] a) Reduce the content of neutral detergent fiber, acid detergent fiber and acid detergent lignin in silage;

[0013] b) Improve the antioxidant capacity of silage;

[0014] c) Reduce the pH and ammonia nitrogen / total nitrogen ratio of silage;

[0015] d) Increase the lactic acid content of silage;

[0016] e) Optimize the microbial community structure of silage, enrich beneficial Lactobacillus microorganisms and fiber-degrading bacteria, and inhibit harmful bacteria.

[0017] In the above-mentioned applications, preferably, the raw materials for the silage are selected from natural and advantageous forage resources or cultivated forage in southern regions, including at least one of grass forage, asteraceous forage, or leguminous forage.

[0018] In the above applications, preferably, the grass forage includes at least one of Miscanthus sinensis, Elephantgrass, or Paspalum notatum; the asteraceae forage includes at least one of Erigeron candelilla or Solidago canadensis; and the legume forage includes alfalfa.

[0019] Based on a general inventive concept, the present invention also provides a method for improving the quality of silage, comprising the following steps:

[0020] (1) Cut the silage raw materials into shorter pieces; preferably 2-3 cm;

[0021] (2) Mix the silage raw material treated in step (1) with the above-mentioned Bacillus subtilis or the above-mentioned microbial preparation evenly;

[0022] (3) Compact, seal and store the mixture obtained in step (2).

[0023] In the above-described method for improving silage quality, preferably, the amount of Bacillus subtilis or the microbial preparation added is 1×10⁻⁶. 4 cfu·g -1 -1×10 6 cfu·g -1 .

[0024] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0025] (1) The Bacillus subtilis YT1 provided by this invention is a multifunctional strain that has been artificially screened and identified. For the first time, a single Bacillus subtilis strain simultaneously possesses highly efficient lignocellulose degradation capabilities, excellent fermentation-promoting characteristics, significant antioxidant activity, and good probiotic properties. This strain can simultaneously secrete four key enzymes: cellulase, lignin peroxidase, manganese peroxidase, and laccase. It effectively breaks down the structure of plant cell walls, degrading polymers such as cellulose and hemicellulose, which are difficult to utilize, into soluble sugars, thereby providing additional nutrient substrates for fermenting microorganisms such as lactic acid bacteria. This characteristic allows a single bacterial agent to achieve multiple objectives such as "fiber degradation, fermentation promotion, and oxidative protection," which traditionally require the combination of multiple bacterial agents. This fundamentally overcomes the problems of poor inter-strain compatibility, unstable synergistic effects, and high application costs associated with existing compound additives.

[0026] (2) Applying Bacillus subtilis YT1 of the present invention to silage preparation can significantly optimize the fermentation process. Experimental results show that it can effectively reduce the pH value of silage, creating a better acidic storage environment; significantly increase lactic acid content, promoting high-quality fermentation; and generally reduce the ammonia nitrogen / total nitrogen ratio, effectively reducing protein decomposition loss. At the same time, its strong lignocellulose degradation ability can directly act on the feed fiber structure, significantly reducing the content of neutral detergent fiber (NDF), acid detergent fiber (ADF), and acid detergent lignin (ADL). This not only softens the feed texture but also fundamentally improves the nutritional value of the feed and its digestibility and utilization rate in livestock.

[0027] (3) The Bacillus subtilis YT1 of the present invention has antioxidant activity and can significantly improve the antioxidant capacity of finished silage. The key antioxidant indicators such as DPPH free radical scavenging rate, FRAP (ferric ion reducing capacity) and ABTS free radical scavenging rate of silage treated with it are significantly improved. This helps to protect easily oxidized nutrients such as vitamins and unsaturated fatty acids in the feed, prolong the shelf life of the feed, and may bring additional health benefits to the feeding animals.

[0028] (4) The Bacillus subtilis YT1 strain of the present invention is acid-resistant, bile-resistant, and tolerant of gastrointestinal fluids, exhibiting good survival ability in the gastrointestinal environment, meeting the basic requirements of probiotics. Simultaneously, it can inhibit the growth of various common pathogens (such as Staphylococcus aureus and Escherichia coli). Comprehensive safety evaluation confirms that this strain has no hemolytic activity, does not produce biological amines, and antibiotic susceptibility testing shows no risk of multidrug resistance, meeting the application standards for food-grade microorganisms, and is safe and reliable for use.

[0029] In summary, the Bacillus subtilis YT1 provided by this invention can serve as a highly efficient, safe, and multifunctional microbial additive. It can effectively solve problems such as insufficient soluble sugar and low fiber utilization during silage production, comprehensively improving the fermentation quality, nutritional value, and antioxidant properties of silage. This has significant value in promoting cost reduction, efficiency improvement, and sustainable development in animal husbandry. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0031] Figure 1The image shows the Congo red staining effect of Bacillus subtilis YT1 on CMC-Na medium: A: Blank control; B: Bacillus subtilis YT1; C: Control bacteria, Bacillus subtilis 168.

[0032] Figure 2 The colony morphology (A) and Gram staining results of Bacillus subtilis YT1 are shown in Figure B.

[0033] Figure 3 It is the phylogenetic tree of Bacillus subtilis YT1.

[0034] Figure 4 It is the size of the inhibition zone of Bacillus subtilis YT1 against four pathogens: Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Pichia pastoris.

[0035] Figure 5 It is the tolerance of Bacillus subtilis YT1 to pH (A), bile salts (B), gastric juice (C), and intestinal juice (D).

[0036] Figure 6 It refers to the ability of Bacillus subtilis YT1 to produce biofilms.

[0037] Figure 7 Evaluation of hemolytic activity of Bacillus subtilis YT1: A: Staphylococcus aureus; B: Bacillus subtilis YT1.

[0038] Figure 8 The results of biogenic amine production by Bacillus subtilis YT1 are as follows: A: Positive control for amino acid decarboxylase; B: Bacillus subtilis YT1.

[0039] Figure 9 These are SEM images of each treatment group in Example 6.

[0040] Figure 10 This refers to the OTU analysis of each treatment group in Example 6.

[0041] Figure 11 This refers to the relative abundance of each treatment group at the genus level in Example 6.

[0042] Figure 12 This is a bubble chart showing the species distribution of each treatment group at the genus level (top 20) in Example 6.

[0043] Figure 13 This is a bubble chart showing the species distribution of each treatment group at the microbial species level (top 10) in Example 6.

[0044] Figure 14 This refers to the linear discriminant analysis of each treatment group in Example 6.

[0045] Figure 15 This refers to the RDA dual-sequence graph analysis in Example 6.

[0046] Figure 16 This is the correlation analysis between the microbial community and various indicators in Example 6. Detailed Implementation

[0047] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0048] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0049] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0050] Example 1: Isolation, screening and identification of Bacillus subtilis YT1

[0051] 1. Source of strain isolation

[0052] A mixture of alfalfa, rice straw, and wheat bran in a mass ratio of 8:1:1 was used as the separation source. Testing of this silage revealed a significant decrease in neutral detergent fiber (NDF) content and a significant increase in relative feed value (RFV). Furthermore, bacterial diversity analysis showed an increase in the abundance of cellulose-decomposing bacteria within the bacterial community of the added mixed silage.

[0053] 2. Initial screening

[0054] Alkali-free lignin agar and carboxymethyl cellulose sodium (CMC-Na) medium were used to selectively screen microorganisms from the above-mentioned isolated sources using a dilution-spreading method, targeting strains with the ability to degrade lignocellulose.

[0055] After purifying the initially isolated colonies, the enzyme production capacity of the strains was qualitatively evaluated using Congo red staining and aniline blue decolorization tests. Finally, 18 bacterial isolates with lignocellulose degradation potential were preliminarily screened out (the qualitative test results are shown in Table 1).

[0056] Table 1. Colony morphology and enzyme production qualitative detection results of 118 bacterial strains

[0057]

[0058] Note: (1) The meaning of the numbers is as follows: C, screening on CMC-Na medium; M, screening on lignin medium; MM, repeated twice on lignin medium; U2 and U3 represent the isolation sources, which are 3.0 and 4.5 g·kg, respectively. -1 FW urea in alfalfa mixed silage; the suffix number indicates the single colony number picked from the plate; e.g., CU3-4 indicates the addition of 4.5 g·kg. -1 FW urea-containing alfalfa mixed silage strain No. 4 was obtained by screening on CMC-Na medium. (2) "+" indicates fading effect, and the number of "+" indicates the degree of fading; "-" indicates no fading effect; (3) CK is the amount of 500 μ·mL -1 The diameter of the decolorization zone of Congo red obtained from cellulase standard solution.

[0059] 3. Secondary screening

[0060] The enzyme activity of the above 18 strains was quantitatively determined, and the results are shown in Table 2. Among them, MU3-13-11-1-1 showed the following result: lignocellulose degradation enzyme activity: 33.49 U·mL. -1 Lignin peroxidase activity: 32.38 U·mL -1 Manganese peroxidase activity: 1.19 U·mL -1 Laccase activity: 0.25 U·mL -1 It has the highest overall ability. Therefore, this strain was identified as the target strain for subsequent research.

[0061] Table 2 Results of quantitative determination of enzyme activity of 18 bacterial strains

[0062]

[0063] Note: "ND" indicates not detected.

[0064] 4. Strain identification

[0065] 16S rRNA sequencing was performed on MU3-13-11-1-1, and the resulting sequence (as shown in SEQ ID No: 1) was compared with the NCBIBLAST database. Sequences from 19 standard strains most closely related to strain MU3-13-11-1-1 were downloaded, and a phylogenetic tree was constructed using MEGA X software based on the Maximum Likelihood method, as shown below. Figure 3 As shown (the accession number of the standard strains used for comparison is listed before the strain name; the percentage of branch positions represents the coverage of internal node data; the scale bar length is 0.001 nucleotide substitution rate). Combined with morphology ( Figure 2 Based on the sugar source fermentation characteristics (Table 3), etc., strain MU3-13-11-1-1 was preliminarily identified as belonging to the genus Bacillus subtilis. Bacillus subtilis The strain was named Bacillus subtilis (B. subtilis) Bacillus subtilis )YT1.

[0066] > Bacillus subtilis YT1

[0067]

[0068] Table 3. Glucose source utilization, gas production, and catalase activity.

[0069]

[0070] 5. Strain preservation

[0071] This strain was deposited at the China Center for Type Culture Collection (CCTCC) on December 10, 2024, with accession number CCTCC NO: M 20242767, at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China.

[0072] Example 2: Comparison of enzyme production capacity between Bacillus subtilis YT1 and existing Bacillus subtilis technologies

[0073] To further clarify the cellulase production effect of Bacillus subtilis YT1 under simulated silage conditions, model strains Bacillus subtilis 168 and Bacillus subtilis YT1 were cultured in carboxymethyl cellulose sodium (CMC-Na) medium (37℃, anaerobic) for 2 days. A Congo red staining comparison experiment was then conducted, and the cellulase production capacity of the two strains was compared by the size of the bleaching zone. Figure 1 It can be seen that the diameter of the bleaching zone of Bacillus subtilis YT1 (32.50 mm) is significantly larger than that of Bacillus subtilis 168 (24.50 mm). P (Value < 0.05).

[0074] Example 3: Evaluation of the antibacterial, antioxidant and probiotic properties of Bacillus subtilis YT1

[0075] 1. Antibacterial ability test

[0076] The antibacterial activity of BS YT1 was determined using the double-layer agar diffusion method, and the results are as follows: Figure 4 As shown.

[0077] The results showed that BSYT1 exhibited varying degrees of inhibitory effects against Staphylococcus aureus, Escherichia coli, Listeria monocytogenes, and Pichia pastoris, but the antibacterial effects differed. The strongest inhibitory effect was observed against Staphylococcus aureus, with an inhibition zone diameter of 38.33 mm, while the weakest inhibitory effect was observed against Escherichia coli, with an inhibition zone diameter of 8.67 mm. This indicates that BSYT1 can produce antibacterial secondary metabolites and possesses antibacterial activity.

[0078] 2. Evaluation of antioxidant activity

[0079] The in vitro antioxidant capacity of BS YT was evaluated by measuring the BS YT content, and the results are shown in Table 4.

[0080] Table 4 Antioxidant capacity of Bacillus subtilis YT1

[0081]

[0082] As can be seen from Table 4, the ABTS free radical scavenging rate, hydroxyl free radical scavenging ability, and DPPH free radical scavenging rate reached 42.97%, 26.64%, and 7.46% respectively.

[0083] 3. Analysis of probiotic characteristics

[0084] By measuring the tolerance of BS YT1 to pH, bile salts, and gastrointestinal fluids, its tolerance to the gastrointestinal environment was judged, as Figure 5 shown.

[0085] From Figure 5 it can be seen that BS YT1 has significant acid tolerance. As the pH value increases, the survival rate shows an upward trend. At pH 2, pH 3, and pH 4, the survival rates of BS YT1 are 37.92%, 77.36%, and 76.37% respectively ( Figure 5 A). On the contrary, as the bile salt concentration increases, the survival rate of BS YT1 shows an obvious downward trend. At bile salt concentrations of 0.01%, 0.3%, and 0.5%, the survival rates are 82.22%, 77.93%, and 62.88% respectively ( Figure 5 B). Under the conditions of artificial intestinal fluid and gastric juice, the survival rate of BS YT1 at 20 h is significantly higher than that at 4 h, and the overall survival rate ranges from 27.41% to 62.33%. It is worth noting that the survival rate in artificial gastric juice at 20 h is the highest, reaching 62.33% ( Figure 5 C). In addition, BS YT1 has the characteristic of adhering to the intestine. The autoaggregation and hydrophobicity of BS YT1 at 20 h are significantly greater than those of BS YT1 at 4 h. Among them, the autoaggregation and hydrophobicity at 20 h are 39.88% and 37.49% respectively ( Figure 5 D).

[0086] After incubating BS YT1 at 37 °C for 48 h and standing still, the formation of biofilm was observed and measured, as Figure 6 shown. From Figure 6 it can be seen that BS YT1 has strong biofilm formation ability and belongs to a strain with strong biofilm formation ability. Note: Biofilm judgment standard: The limit value is 2 times the negative value (Dc). According to the D value, the strains can be divided into strong biofilm-forming strains (D > 2Dc), weak biofilm-forming strains (Dc < D < 2Dc), and non-biofilm-forming strains (D < Dc).

[0087] Based on the above experimental results, Bacillus subtilis YT1 has excellent tolerance to the gastrointestinal environment, adhesion, and film-forming ability. These characteristics meet the basic standards of probiotics, indicating its great potential for application as a probiotic.

[0088] Example 4: Safety evaluation of Bacillus subtilis YT1

[0089] A comprehensive in vitro safety assessment of the Bacillus subtilis YT1 strain of the present invention was conducted, including hemolytic activity, biogenic amine production potential, and antibiotic susceptibility testing.

[0090] 1. Hemolytic activity assay

[0091] Based on their ability to induce hemolysis in sheep blood cells, bacteria can be classified into complete hemolysis (β-hemolysis, exhibiting a clear hemolytic halo), partial hemolysis (α-hemolysis, exhibiting a translucent, grass-green hemolytic halo), and non-hemolytic (γ-hemolysis). Some strains, when cultured on Columbia blood agar plates, will form a distinct hemolytic zone around the colony. Most bacteria with hemolytic activity are typically highly pathogenic.

[0092] The hemolytic activity of the strain was determined using Columbia blood agar plates. Figure 7 As shown, the positive control strain (Staphylococcus aureus) produced a clear hemolytic zone around its colonies, while no hemolytic zone appeared around the colonies of Bacillus subtilis YT1. This result indicates that strain YT1 does not exhibit hemolytic activity in vitro, preliminarily demonstrating its lack of potential cytotoxicity and high safety profile, consistent with the basic characteristics of a safe strain.

[0093] 2. Determination of biogenic amine production potential

[0094] Biogenic amines are alkaloids. In liquid culture media containing bromocresol purple indicator (color range: yellow at pH 5.2, purple at pH 6.8), a bluish-purple hue indicates the production of alkaline substances, suggesting the potential for biogenic amine production.

[0095] The determination was performed using a decarboxylase culture medium supplemented with bromocresol purple indicator. Figure 8 As shown, the culture medium in the positive control tube (A) turned blue-purple, indicating alkali production (i.e., potential biogenic amine production); while the culture medium (B) inoculated with YT1 turned yellow due to acid production from glucose fermentation by the strain, and the color did not revert to blue-purple throughout the entire culture process. This result indicates that Bacillus subtilis YT1 does not produce amino acid decarboxylase and therefore does not have the potential to generate biogenic amines, eliminating the risk of foodborne biogenic amine poisoning caused by it.

[0096] 3. Antibiotic susceptibility testing

[0097] Susceptibility testing of Bacillus subtilis strain YT1 to 30 antibiotics was conducted using antimicrobial disks (Bikman Biotechnology, catalog number 110705004) (Table 5). The results showed that Bacillus subtilis YT1 remained sensitive or moderately sensitive to 17 commonly used clinical antimicrobial agents (including key therapeutic agents such as vancomycin and imipenem) (penicillins, cephalosporins, aminoglycosides, tetracyclines, macrolides, sulfonamides, quinolones, lincomycins, glycopeptides, carbapenems, and others), with no multidrug resistance risk detected. It exhibited inherent resistance to only five antibiotics: penicillin, cephalexin, streptomycin, lincomycin, and polymyxin B. This is related to the natural β-lactamase activity of Bacillus spp., consistent with the typical characteristics of this genus.

[0098] Table 5. Sensitivity of Bacillus subtilis YT1 to 30 antibiotics.

[0099]

[0100] It is worth noting that Bacillus subtilis YT1 completely lacks the ability to synthesize biogenic amines, eliminating the associated risk of foodborne poisoning. Combining the characteristics of Bacillus subtilis as a Generally Recognized as Safe (GRAS) species with the above experimental results, Bacillus subtilis YT1 demonstrates good application safety, meeting the biosafety standards for food-grade microorganisms and probiotic preparations.

[0101] Example 5: Application and effect evaluation of Bacillus subtilis YT1 in silage of grass and Asteraceae forage

[0102] This embodiment aims to illustrate the specific methods and effects of applying Bacillus subtilis YT1 to silage of grasses (such as Miscanthus sinensis, Elephantgrass, and Paspalum distichum) and Asteraceae (such as Erigeron canadensis and Solidago canadensis) to improve their silage quality.

[0103] 1. Methods for preparing silage

[0104] The specific operational methods for improving the quality of silage are as follows:

[0105] (1) Cut the forage to be silage into 2-3 cm pieces to facilitate compaction and fermentation.

[0106] (2) The bacterial suspension addition amount was set at 4 gradients: BS0 group (control group): no bacterial agent added; BS4 group: 1×10 4 cfu·g -1 BS5 group: Add 1×10 5 cfu·g -1BS6 group: Add 1×10 6 cfu·g -1 Spray the corresponding Bacillus subtilis YT1 onto the chopped forage and mix thoroughly.

[0107] (3) Filling and sealing: Weigh 500 g of the mixed material and quickly fill it into a polyethylene silage bag. Use a vacuum pump to remove the air from the bag and then heat-seal it to create an anaerobic fermentation environment.

[0108] (4) Fermentation and storage: Place the sealed silage bags in the ambient temperature (25-30℃) and store them in the dark for 45 days.

[0109] 2. Effect Evaluation

[0110] After 45 days of silage fermentation, samples were taken from the sealed containers to determine the fermentation quality and fiber quality of the silage in each treatment group, in order to evaluate the effect of adding Bacillus subtilis YT1.

[0111] 2.1 Effects of different concentrations of Bacillus subtilis YT1 on the fermentation quality of silage from gramineous forages

[0112] As shown in Table 6, the addition of Bacillus subtilis YT1 can effectively improve the fermentation quality of gramineous forage grasses (Miscanthus sinensis, Elephantgrass, and Paspalum distichum), such as reducing pH, increasing lactic acid, and reducing ammonia nitrogen.

[0113] Table 6. Effects of different concentrations of Bacillus subtilis YT1 on the fermentation quality of gramineous forage.

[0114]

[0115] Note: NH3-N / TN, ammonia nitrogen / total nitrogen; LA, lactic acid; AA, acetic acid; PA, propionic acid; BA, butyric acid. Different lowercase letters in the same column indicate significant differences between different addition amounts of the same species, Bacillus subtilis YT1. P <0.05); the same applies below.

[0116] 2.2 Effects of different concentrations of Bacillus subtilis YT1 on the silage fiber composition of Gramineae and Asteraceae forages

[0117] Table 7 shows that adding Bacillus subtilis YT1 for silage significantly reduced the fiber content (NDF, ADF, and CL) of the five forage varieties. P <0.05).

[0118] Table 7. Effects of different concentrations of Bacillus subtilis YT1 on the fiber composition of grass and asteraceous forage grasses.

[0119]

[0120] Note: BS0, BS4, BS5, and BS6 have 0 and 10 added respectively. 4 10 5 10 6 cfu·g -1 Bacillus subtilis YT1; DM, dry matter; NDF, neutral detergent fiber; ADF, acid detergent fiber; ADL, acid detergent lignin; HC, hemicellulose; CL, cellulose.

[0121] 2.4 Effects of Bacillus subtilis YT1 addition on the antioxidant capacity of silage from grasses and asteraceae forages

[0122] Based on the aforementioned effects of adding Bacillus subtilis YT1 on fermentation and fiber quality, antioxidant capacity was measured in treatment groups with the optimal Bacillus subtilis YT1 concentration for each forage and in treatment groups without Bacillus subtilis YT1 (Table 8). A T-test was performed on the antioxidant capacity of each forage group with and without Bacillus subtilis YT1. The results showed that the antioxidant capacity of all forage silages generally improved after adding Bacillus subtilis YT1. The FRAP iron ion reducing capacity of all forage groups treated with Bacillus subtilis YT1 was significantly higher than that of the untreated group (…). P The values ​​were all <0.05, indicating that Bacillus subtilis YT1 treatment had a consistent effect on improving the iron ion reduction capacity of various forages; the DPPH free radical scavenging capacity of all forages treated with Bacillus subtilis YT1 was higher than that of the untreated groups.

[0123] Table 8. Effects of Bacillus subtilis YT1 supplementation on the antioxidant capacity of gramineous and asteraceous forage grasses.

[0124]

[0125] Example 6: Application and Effect Evaluation of Bacillus subtilis YT1 in Alfalfa Silage

[0126] Alfalfa is known as the "king of forages." Given that leguminous forages have low carbohydrate content and high crude protein content, this example uses 10... 6 cfu·g -1 Bacillus subtilis YT1 was added to alfalfa (low dry matter content: 25.81±0.04% FW) for silage alone or mixed with soybean meal and DDGS (corn distillers grains and solubles) (high dry matter content: 38.81±0.08% FW) for silage to illustrate the effect of BSYT1 on silage raw materials with different moisture contents.

[0127] 1. Methods for preparing silage

[0128] (1) Experimental design

[0129] A single-factor completely randomized design was used, with 4 treatment groups and 5 replicates per treatment. The treatment design is shown in Table 9.

[0130] Table 9 Experimental Groups

[0131]

[0132] (2) Silage operation

[0133] After finely crushing alfalfa during its initial flowering stage, add microbial agents according to the above design, mix evenly with all raw materials, pack into laboratory silage bags, compact and vacuum seal, and anaerobic ferment at ambient temperature for 45 days.

[0134] 2. Evaluation of silage effect

[0135] 2.1 Effects of adding Bacillus subtilis YT1 on alfalfa fiber composition, fermentation quality, and antioxidant capacity

[0136] As shown in Table 10, the addition of Bacillus subtilis YT1 significantly optimized the fermentation quality and nutritional value of alfalfa silage. Specifically, in high-moisture silage, it significantly reduced the content of dry matter, acid detergent fiber, acid detergent lignin, and pH value, while significantly increasing the content of crude protein and lactic acid. In low-moisture silage, it significantly reduced the content of neutral detergent fiber, acid detergent fiber, acid detergent lignin, and cellulose, as well as the ammonia nitrogen / total nitrogen ratio, while significantly increasing the lactic acid content. Furthermore, the antioxidant capacity of the silage was significantly improved under both treatments.

[0137] This indicates that adding Bacillus subtilis YT1 can significantly increase the lactic acid yield of alfalfa silage, inhibit protein degradation, and reduce lignin cellulose content, demonstrating good fermentation promotion and fiber degradation functions.

[0138] Table 10 Effects of Bacillus subtilis YT1 addition on alfalfa silage quality

[0139]

[0140] Note: * indicates P <0.05, ** indicates P <0.01; ND indicates not detected.

[0141] 2.2 Effect of Bacillus subtilis YT1 addition on alfalfa fiber morphology

[0142] The alfalfa silage was observed using a scanning electron microscope (SEM). Figure 9The images, from top to bottom, show the macroscopic morphology of BS0M, BS1M, BS0MM, and BS1MM at different magnifications. Analysis of the images reveals that while the fiber structure of alfalfa in the BS0M and BS0MM groups shows some damage, it still maintains a relatively continuous elongated shape with clear surface texture and no obvious signs of breakage or degradation. In contrast, the fibers in the BS1M and BS1MM groups exhibit obvious breakage and fragmentation, with a rough surface accompanied by numerous cracks and peeling marks. Some areas show characteristics of "porous and flocculent disintegration" degradation. This indicates that the addition of Bacillus subtilis YT1 has a significant microbial decomposition effect on the fiber structure of alfalfa during silage, and this effect is more pronounced in the low-moisture silage group (BS1MM), where the degree of fiber breakage and porosity is more significant.

[0143] It is noteworthy that in high-moisture alfalfa silage (BS1M), the strictly anaerobic environment is unfavorable for the large-scale proliferation of facultative anaerobic Bacillus subtilis YT1, yet it still exhibits significant fiber degradation and fermentation-promoting effects. This indicates that, in addition to the action of live bacteria, the enzyme system (such as cellulase and lignin peroxidase) and its metabolites produced by YT1 during fermentation play a continuous and crucial role in the silage process.

[0144] 2.3 Adding Bacillus subtilis YT1 to optimize and regulate the microbial community in silage

[0145] High-throughput sequencing technology was used to analyze the microbial community structure of silage in each treatment group to illustrate the optimized regulation of silage microecology by Bacillus subtilis YT1.

[0146] OTU analysis for each treatment group as follows Figure 10 As shown in Figure 11, the relative abundance of microorganisms in each treatment group was analyzed. At the genus level, the dominant genera in the BS0M treatment group were dispersed, with *Bacillus cereus* being the most abundant. Ochrobactrum Lactobacillus pentosophila (S) ecundilactobacillus Environmentally adapted or basal metabolic bacteria, such as lactic acid bacteria, dominate. Lactobacillus The abundance of *Lactobacillus* spp. was low, and the bacterial community structure leaned towards a "natural fermentation mixed bacteria type." In contrast, the BS1M-treated group showed a higher abundance of *Lactobacillus* spp. (…). Lactobacillus The abundance of *Lactobacillus* significantly increased, becoming the core dominant genus; the abundance of other genera such as *Ailuropoda* decreased, and the microbial community shifted towards a "fermentation-type dominated by lactic acid bacteria"; in the BS0MM treatment group, *Lactobacillus* became the dominant genus, but its abundance was limited, and a small number of environmental bacteria genera (such as *Pseudomonas*) remained. Pseudomonas In the BS1MM treatment group, the abundance of *Lactobacillus* surged to absolute dominance, while other genera (such as *Pseudomonas*) were almost eliminated, indicating a highly specialized microbial community structure for lactic acid fermentation. Furthermore, the species distribution bubble diagram at the genus level in the treatment group (…) Figure 12It can be seen that after adding Bacillus subtilis YT1, regardless of whether alfalfa is used alone or mixed with other alfalfa in silage, the microbial community structure containing microorganisms closely related to the degradation or conversion of lignocellulose, such as Bacillus (…), is significantly improved. Bacillus ), fibrous microbacteria ( Cellulosimicrobium Significant enrichment in substances such as )

[0147] Bubble chart of species distribution at the species level for each treatment group ( Figure 13 It can be seen that the addition of Bacillus subtilis YT1 can increase the number of lactobacilli-like bacteria in the silage of the BS1M treatment group. Lactobacillus similis ), Enterobacter sieboldii ( Weissella paramesenteroides Lactobacillus hamsii ( Levilactobacillus hammesii The relative abundance of beneficial lactic acid bacteria such as ) and Brucella intermedia ( Brucella intermedia ), Brucella pseudoglynnosis ( Brucella pseudogrignonensis The relative abundance of harmful bacteria such as Bacillus subtilis YT1 was reduced; although the addition of Bacillus subtilis YT1 had no significant effect on the relative abundance of lactic acid bacteria in the BS1MM treatment group, it reduced the relative abundance of Bacillus cytogenes (Bacillus subtilis). Cytobacillus kochii ) and Brucella pseudoglynnii ( Brucella pseudogrignonensis The relative abundance of harmful bacteria such as ).

[0148] Microbial species with significant differences in abundance between groups were screened using linear discriminant analysis (LDA). Figure 14 It can be seen that there are significant differences in microbial species among the BS0M, BS0MM, and BS1M treatment groups. The BS1M treatment group showed a higher concentration of Lactobacillus species. Lactobacillus Multiple species in ) Lactobacillus similies, Lactobacillus concavus, Lactobacillus vaccinostercus Brucella spp. is the most critical differentially expressed bacterium. In the BS0M treatment group, Brucella spp. ( Brucella The presence of differentially expressed bacteria is often associated with "environmental adaptation" or "low-quality fermentation," indicating that when alfalfa is directly ensiled, it is easy for residual bacteria to remain, resulting in low fermentation quality.

[0149] Analysis using RDA bi-sequence graphs ( Figure 15 The correlation between microbial community distribution (scatter plots) and silage indicators (arrows) was revealed. ADL (acid-washed lignin) and Ash (ash) were positively correlated with the BS1 group microbial community. The scatter plots of the BS1M / BS1MM group (with added Bacillus subtilis YT1) separated from the BS0 group and were closer to the ADL and Ash arrows. Correlation heatmap analysis ( Figure 16 ), genus *Pseudomonas* Ochrobactrum The abundance of *Lactobacillus* showed a strong positive correlation with Ash, NDF, and ADF, and a strong negative correlation with CP, indicating that higher abundance of this genus may correlate with higher ash and fiber content, and potentially lower crude protein content. SecundilactobacillusThe abundance of this genus showed a strong negative correlation with Ash, NDF, and ADF, and a strong positive correlation with CP. Higher abundance of this genus was associated with lower fiber content and better crude protein retention. This suggests that *Ailuropoda* spp. may be related to fiber accumulation and protein loss in silage, while *Lactobacillus* spp. may inhibit fiber accumulation and promote protein preservation, making them potential markers of high-quality silage. Therefore, *Bacillus subtilis* YT1 may influence fermentation by regulating bacteria that decompose lignin and enrich minerals.

[0150] In summary, the addition of Bacillus subtilis YT1 to alfalfa silage provides beneficial lactic acid bacteria, including similar lactobacilli (…). Lactobacillus similis ), Enterobacter sieboldii ( Weissella paramesenteroides Lactobacillus hamsii ( Levilactobacillus hammesii The relative abundance of bacteria such as Brucella (Brucella) increased significantly, and the abundance of harmful bacterial groups among them also increased. Brucella intermedia ), Brucella pseudoglynnosis ( Brucella pseudogrignonensis ) and others were effectively suppressed; in addition, microbial community correlation analysis showed that the genus *Pseudomonas* ( Ochrobactrum ) and Lactobacillus spp. Secundilactobacillus The relative abundance of bacteria such as Bacillus subtilis and Bacillus thuringiensis was significantly correlated with key indicators such as fiber content, suggesting that Bacillus subtilis YT1 may affect the fermentation process by regulating these bacteria related to fiber degradation.

[0151] In summary, the Bacillus subtilis (Bacillus subtilis) provided by this invention... Bacillus subtilis YT1 is a multifunctional, efficient, and safe feed microorganism. Its most prominent advantage lies in achieving multiple goals—fiber degradation, fermentation promotion, and oxidative protection—with a single strain, a process traditionally requiring multiple additives. This strain can simultaneously secrete four key lignocellulose-degrading enzymes, effectively breaking down plant cell wall structures and providing substrates for lactic acid bacteria fermentation, thereby significantly improving the fermentation quality, nutritional value, and aerobic stability of silage. Experimental results show that Bacillus subtilis YT1 is suitable for various difficult-to-ensilage raw materials, including those from the Poaceae, Asteraceae, and Leguminosae families, and can optimize the silage microbial community structure. Furthermore, comprehensive safety evaluations confirm that it meets the standards for probiotics and food-grade microorganisms. This invention provides a novel, integrated solution to overcome industry challenges such as insufficient soluble sugars, low fiber utilization, and easy oxidation and deterioration after opening silage, and has significant application value for promoting cost reduction, efficiency improvement, and sustainable development in animal husbandry.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis YT1, characterized in that, The Bacillus subtilis YT1 is deposited at the China Center for Type Culture Collection, located at Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, with accession number CCTCCNO:M20242767 and deposit date of December 10, 2024.

2. A microbial preparation comprising live cells of Bacillus subtilis YT1 as described in claim 1.

3. The application of Bacillus subtilis YT1 as described in claim 1 or the microbial preparation as described in claim 2 in the preparation of silage.

4. The application as described in claim 3, characterized in that, The application includes at least one of the following aspects: a) Reduce the content of neutral detergent fiber, acid detergent fiber and acid detergent lignin in silage; b) Improve the antioxidant capacity of silage; c) Reduce the pH and ammonia nitrogen / total nitrogen ratio of silage; d) Increase the lactic acid content of silage; e) Optimize the microbial community structure of silage, enrich beneficial Lactobacillus microorganisms and fiber-degrading bacteria, and inhibit harmful bacteria.

5. The application as described in claim 4, characterized in that, The raw materials for the silage include at least one of the following: grasses, asteraceae grasses, or legume grasses.

6. A method for improving the quality of silage, characterized in that, Includes the following steps: (1) Cut the silage raw materials into shorter pieces; (2) Mix the silage raw material treated in step (1) with the Bacillus subtilis YT1 of claim 1 or the microbial preparation of claim 2 evenly; (3) Compact, seal and store the mixture obtained in step (2).

7. The method for improving the quality of silage as described in claim 6, characterized in that, In step (2), the amount of Bacillus subtilis YT1 or the microbial preparation added is 1×10⁻⁶. 4 cfu·g -1 -1×10 6 cfu·g -1 .

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