Bacillus velezensis with the ability of degrading vomitoxin and multiple enzyme activities and application thereof

Abstract

The present application belongs to the technical field of mycotoxin biodegradation and bacillus velezensis, and particularly relates to a bacillus velezensis strain with degrading vomitoxin and multiple enzyme activities and application thereof. The strain is isolated from pig intestinal contents, and is identified by morphological identification, physiological and biochemical identification, and 16S rRNA and gyrB gene molecular identification, and is confirmed as bacillus velezensis, and is named as bacillus velezensis YK-19 (Bacillus velezensis YK-19). The strain has a degrading rate of vomitoxin (deoxynivalenol, DON) as high as 93.79%, and has multiple enzyme activities such as protease, amylase, beta-mannanase, xylanase, cellulase and pectinase, and does not have hemolytic activity, and has good biological safety. The strain also shows good acid resistance, bile salt resistance, salt resistance and high temperature resistance, and has a wide application prospect in actual production.

Description

Technical Field

[0002] This invention belongs to the field of Bacillus belyssus and mycotoxin biodegradation technology, specifically involving a strain of Bacillus belyssus with degrading vomitoxin and multi-enzyme activity and its application. Background Technology

[0004] Deoxynivalenol (DON), also known as deoxynivalenol, is a type B trichothecene toxin produced by Fusarium spp. It widely contaminates grains such as wheat, corn, barley, and oats, as well as their processed products. DON is one of the most frequently occurring and widely contaminating mycotoxins globally, seriously endangering animal health and food safety.

[0005] DON has multifaceted toxic effects on animals. Acute toxicity mainly manifests as gastrointestinal symptoms such as anorexia, vomiting, and diarrhea; chronic toxicity can lead to stunted growth, immunosuppression, and a decline in overall production performance. Pigs are particularly sensitive to DON; a DON content exceeding 0.5 mg / kg in feed can cause significant decreases in feed intake and stunted growth. Furthermore, DON can cross the blood-brain barrier, affecting neuroendocrine function and inducing intestinal mucosal damage, disrupting the integrity of the intestinal barrier, and significantly increasing the risk of pathogen invasion, posing a serious threat to animal health and livestock production.

[0006] Currently, detoxification strategies for DON are mainly divided into three categories: physical methods, chemical methods, and biological methods. Physical methods include adsorption, heat treatment, irradiation, and ozone treatment, while chemical methods involve alkalization, acidification, and oxidant treatment. However, these physical and chemical methods generally suffer from limited detoxification efficiency, significant loss of feed nutrients, and the potential introduction of secondary pollutants, making them difficult to fully meet actual production needs. Biological methods utilize microorganisms or their produced enzymes to specifically biotransform and degrade DON. They have advantages such as high selectivity, mild conditions, minimal impact on feed palatability, and safety and environmental friendliness, and have become the most promising technical route in the field of DON control.

[0007] Bacillus is one of the most widely used probiotic species in the field of feed microbial additives. Among them, Bacillus velezensis has attracted widespread attention from researchers at home and abroad due to its comprehensive advantages, including broad-spectrum enzyme production, excellent intestinal colonization characteristics, good anti-pathogenic activity, and tolerance to processing conditions. However, most of the Bacillus strains reported to have DON degradation function currently possess only a single functional characteristic, making it difficult to simultaneously meet the multiple functional requirements of efficient DON degradation, production of multiple digestive enzymes, broad-spectrum inhibition of pathogenic bacteria, and excellent probiotic properties, thus limiting its widespread application in the feed industry.

[0008] The prior art patent document CN202210335855.4 discloses a strain of Bacillus velezensis and its application in fermenting feed to degrade microbial toxins. Specifically, it involves a strain of Bacillus velezensis BL-14 screened by mutagenesis and its liquid mixed bacterial solution for efficiently degrading vomitoxin (DON), aflatoxin (AFB1), and zearalenone (ZEN) in feed raw materials. This strain BL-14 has a rich hydrolytic enzyme system and can produce amylase, protease, and cellulase, etc. When fermented with a mixed bacterial agent composed of Lactobacillus plantarum 1.2158 and Bacillus coagulans 1.6565, it can degrade vomitoxin by 83.25%, aflatoxin by 90.09%, and zearalenone by 80.76%. The lactic acid content in the fermented feed reaches 36.5 g / kg. Patent document CN202510313897.1 discloses a strain that reduces the vomitoxin function of wheat and corn and its application. It describes preparing a stock solution (AB) by mixing 10%–90% of *Bacillus belyssiensis* XNYF-1 fermentation broth (Liquid A) and 10%–90% of *Pseudomonas fluorescens* XNYF-2 fermentation broth (Liquid B). Then, well-rotted organic fertilizer or mineral nutrients such as nitrogen, phosphorus, potassium, trace elements, and humic acid are added, and the mixture is processed into functional organic fertilizer and organic-inorganic compound fertilizer. XNYF-1 and XNYF-2 are two functional microbial strains that have undergone screening and mutagenesis, producing chitinase, ituronidin, various phenazine derivatives, and biosurfactants, respectively. Their metabolites exhibit good biocontrol effects. While the technical solutions in these two patent documents disclose the function and enzyme production characteristics of *Bacillus belyssiensis* in degrading vomitoxin, the types and performance of its enzymes, as well as its effectiveness in degrading vomitoxin, still require further improvement.

[0009] Therefore, screening Bacillus berleis strains that possess efficient DON degradation capabilities, multiple feed enzyme production capabilities, and excellent probiotic properties is of significant theoretical importance and has broad application prospects for developing novel multifunctional feed microbial preparations and promoting efficient biological detoxification of mycotoxins in feed. Summary of the Invention

[0011] The purpose of this invention is to provide a strain of Bacillus velezensis with degrading vomitoxin and multi-enzyme activity. The screened and isolated Bacillus velezensis YK-19 has application potential in terms of multi-enzyme activity, including the production of protease, amylase, β-mannanase, xylanase, cellulase and pectinase. At the same time, this Bacillus velezensis YK-19 has biological characteristics, including good bile salt tolerance, acid resistance, salt tolerance, artificial gastrointestinal fluid tolerance and high temperature tolerance, so it can be further used in feed, food and environmental fields.

[0012] To achieve the above objectives, the present invention adopts the following technical solution: a strain of Bacillus velezensis YK-19 with vomitoxin degradation and multi-enzyme activity, which is classified as Bacillus velezensis YK-19 and was deposited at the China Center for Type Culture Collection (CCTCC) on April 20, 2026, with accession number CCTCCNO: M 2026664.

[0013] The Bacillus berreatus YK-19 was isolated from the intestinal contents of pigs.

[0014] The colony morphology of Bacillus belyssus YK-19 is as follows: it forms milky white, rough, wrinkled crater-shaped colonies on LB plates; under scanning electron microscopy, short rod-shaped bacteria with blunt ends are visible, mostly existing singly or in short chains; it is Gram-positive, and the bacteria are blue-purple rod-shaped; malachite green spore staining reveals green spores and red vegetative cells.

[0015] The physiological and biochemical characteristics of the Bacillus belyssus YK-19 were as follows: positive for VP reaction, positive for citrate utilization, positive for D-xylose fermentation, positive for gelatin liquefaction, positive for growth under 7wt% NaCl conditions, positive for growth under pH 5.7 conditions, positive for nitrate reduction, and positive for starch hydrolysis; negative for propionate, L-arabinose, and D-mannitol fermentation.

[0016] The molecular biological characteristics of Bacillus velezensis YK-19 were as follows: its whole genome DNA was extracted, and PCR amplification and sequencing were performed using universal primers for bacterial 16S rRNA and specific primers for the gyrB gene. After BLAST homology comparison with the NCBI database and phylogenetic analysis, the strain was finally identified as Bacillus velezensis and named Bacillus velezensis YK-19.

[0017] The degradation activity of *Bacillus belyssus* YK-19 against vomitoxin was assessed by inoculating strain YK-19 into LB liquid medium containing 1 μg / mL vomitoxin and incubating at 37℃ and 220 rpm for 48 h with shaking. The residual DON content was then detected using ELISA, and the degradation rate was calculated. The rescreening results showed that strain YK-19 achieved a degradation rate of 93.79% for vomitoxin, significantly superior to other screened strains.

[0018] The growth characteristics of *Bacillus belyssus* YK-19 described in this invention were determined through the following experiments: Activated *Bacillus belyssus* YK-19 was inoculated into liquid culture medium and cultured with shaking at 37°C. Odionation levels (OD) of the culture were measured every 2 hours. 600 The viable cell count was determined using plate counting, and a growth curve was plotted. The results showed that strain YK-19 experienced a lag phase, a logarithmic growth phase, and a stationary phase. Initially, the cell count increased slowly, followed by a rapid proliferation phase, and finally stabilized, achieving a high viable cell count.

[0019] The tolerance characteristics of Bacillus belyssus YK-19 described in this invention were determined through the following experiments:

[0020] (1) Bile salt tolerance test: Bacillus beryl YK-19 bacterial suspension was inoculated into culture medium containing different concentrations of bovine bile salts and cultured statically at a suitable temperature for 1h, 2h and 3h. The number of viable bacteria before and after treatment was determined by plate counting method and the survival rate was calculated.

[0021] (2) Acid resistance test: The bacterial suspension of Bacillus belye YK-19 was adjusted to the set pH value with hydrochloric acid solution of different pH values, and treated at a suitable temperature for 1h, 2h and 3h. The number of viable bacteria before and after treatment was determined by plate counting method, and the survival rate was calculated.

[0022] (3) Salt tolerance test: Bacillus belye YK-19 was inoculated into liquid culture medium containing different concentrations of NaCl and cultured with shaking at a suitable temperature for 24 h. The number of viable bacteria was determined by plate counting method. The survival rate was calculated with the culture medium without added NaCl as the control.

[0023] (4) Gastrointestinal fluid tolerance test: Prepare simulated gastric fluid and simulated intestinal fluid, add the bacterial suspension of Bacillus belyssus YK-19 to the simulated gastric fluid and simulated intestinal fluid respectively, and let it stand at 37°C for different times (e.g., 1~6h). Take samples and use plate counting method to determine the number of viable bacteria and calculate the survival rate.

[0024] (5) High temperature tolerance test: Bacillus beryl YK-19 spores were collected, resuspended and placed in water baths at different temperatures (75~95℃) for 15 min. After cooling immediately, the number of viable bacteria was determined by plate counting method. The untreated group was used as the control and the survival rate was calculated.

[0025] Experimental results show that strain YK-19 has good tolerance to bile salts, acid, salt, gastrointestinal fluids, and high temperatures.

[0026] The enzyme-producing characteristics of *Bacillus belyssiensis* YK-19 described in this invention were determined through the following experiments: Using the clear zone method, *Bacillus belyssiensis* YK-19 was inoculated onto selective culture medium plates containing substrates for protease, amylase, β-mannanase, xylanase, cellulase, or pectinase, respectively. After incubation at a suitable temperature for a certain period, the diameters of the clear hydrolysis zones and colonies on each plate were observed and measured. The ratio of the hydrolysis zone diameter to the colony diameter (D / d) was calculated to evaluate the strain's ability to produce different hydrolytic enzymes. The experimental results showed that this strain can produce a variety of hydrolytic enzymes, including protease, amylase, β-mannanase, xylanase, cellulase, and pectinase.

[0027] The biosafety of Bacillus belyssus YK-19 is as follows: the hemolytic test results showed no transparent hemolytic zone around the colony, indicating non-hemolysis (γ-hemolysis); the thermostable DNase test results showed no hydrolysis zone around the colony, indicating that strain YK-19 does not possess thermostable DNase activity and has good biosafety.

[0028] The present invention relates to the application of Bacillus vesiculosus YK-19 in the preparation of a feed compound enzyme preparation, wherein the feed compound enzyme preparation comprises at least one of protease, amylase, β-mannanase, xylanase, cellulase, and pectinase, and the feed compound enzyme preparation has a D / d value of 2.17±0.11 for protease, 1.40±0.02 for amylase, 2.76±0.15 for β-mannanase, 1.87±0.13 for xylanase, 2.27±0.30 for cellulase, and 1.95±0.12 for pectinase.

[0029] The application of Bacillus berberis YK-19 described in this invention in the preparation of probiotic products.

[0030] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0031] 1. This invention screened a strain of Bacillus belyssus YK-19 from pig intestinal contents, which has a highly efficient ability to degrade DON (vomiting toxin). Its DON degradation rate is as high as 93.79%, which is significantly better than other similar strains reported previously.

[0032] 2. The Bacillus berberis YK-19 of the present invention has multiple enzyme activities, including protease, amylase, β-mannanase, xylanase, cellulase and pectinase. While efficiently degrading DON, it can decompose large molecular nutrients in feed into easily digestible small molecular substances, effectively improving the nutritional value of feed.

[0033] 3. The Bacillus berberis YK-19 of the present invention is non-hemolytic, has no heat-stable DNA enzyme activity, has good biocompatibility, and can be used directly as a feed additive.

[0034] 4. The Bacillus berberis YK-19 spore-producing strain described in this invention has good high-temperature tolerance. After treatment at 75°C for 5 minutes, the survival rate can still reach 90.26%, which is convenient for industrial production and high-temperature treatment in feed pelleting process, and is suitable for practical production applications.

[0035] 5. The Bacillus berberis YK-19 described in this invention has certain acid resistance and bile salt resistance characteristics, and can tolerate the animal digestive tract environment to a certain extent, thus possessing the potential to be used as a probiotic feed additive. Attached Figure Description

[0037] Figure 1 The following are the morphological characteristics of strain YK-19: A: YK-19 colony morphology; B: SEM scanning electron microscopy morphology; C: Gram staining morphology; D: Malachite green spore staining morphology.

[0038] Figure 2 For molecular identification and phylogenetic analysis of strain YK-19, A: PCR electrophoresis diagram of 16S rRNA gene; B: PCR electrophoresis diagram of gyrB gene; C: Phylogenetic tree based on 16S rRNA gene; D: Phylogenetic tree based on gyrB gene (★marking strain YK-19).

[0039] Figure 3 The growth curve of strain YK-19 is shown.

[0040] Figure 4 For hemolysis agar plates: A: Hemolysis agar plate for strain YK-19; B: Hemolysis agar plate for Staphylococcus aureus ATCC 6538.

[0041] Figure 5 These are heat-stable DNase plates: A: Toluidine blue-DNase plate for strain YK-19; B: Toluidine blue-DNase plate for Staphylococcus aureus ATCC 6538.

[0042] Figure 6Plates for the production of hydrolases by strain YK-19: A: plate for producing protease; B: plate for producing amylase; C: plate for producing β-mannanase; D: plate for producing xylanase; E: plate for producing cellulase; F: plate for producing pectinase. Detailed Implementation

[0044] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0045] Example 1

[0046] Isolation and identification of Bacillus belyssus YK-19:

[0047] (1) Isolation of the target strain

[0048] Chicken and pig intestinal contents were collected and thoroughly mixed with sterile phosphate buffer to prepare sample suspensions. Under aseptic conditions, 100 μL of the suspension was plated onto the surface of LB agar medium containing 1 μg / mL vomitoxin and incubated at 37°C for 24–48 h under aerobic conditions. Single colonies were selected for streak purification based on colony morphology, color, and size, and the purification was repeated three times. A total of 68 preliminary screening strains were isolated from intestinal contents samples from different animals. Strains from chicken intestinal contents were numbered sequentially as LY-1, LY-2, LY-3, etc., and strains from pig intestinal contents were numbered sequentially as YK-1, YK-2, YK-3, etc. After numbering, all preliminary screening strains were stored at -20°C for subsequent experiments.

[0049] (2) Secondary screening of degrading strains

[0050] After activation, the 45 strains obtained from the initial screening were inoculated into LB liquid medium containing 1 μg / mL vomitoxin. Aerobic strains were cultured at 37℃ and 220 rpm for 48 h with shaking. Using sterile PBS solution containing the same concentration of vomitoxin as a blank control, the residual amount of vomitoxin in the culture medium was determined using a vomitoxin ELISA kit, and the degradation rate of each strain was calculated. The rescreening results showed that one strain achieved a vomitoxin degradation rate of 93.79%, and this strain was selected as the target strain for subsequent experiments.

[0051] The results of the comparative analysis of the rescreening are shown in Table 1. A strain with the highest degradation efficiency of vomitoxin was obtained from the intestinal contents of pigs. This strain was numbered YK-19.

[0052]

[0053] (3) Morphological identification of strains

[0054] Strain YK-19 forms milky-white, rough-surfaced, wrinkled, crater-like colonies on LB plates, such as... Figure 1 As shown in Figure A; under a scanning electron microscope, short rod-shaped bacterial cells with blunt ends are visible, such as... Figure 1 As shown in Figure B; Gram staining is positive, and the bacteria are blue-purple rod-shaped, as shown in Figure B. Figure 1 As shown in C; malachite green spore staining reveals green spores and red vegetative cells, as shown in Figure C. Figure 1 As shown in D.

[0055] (4) Physiological and biochemical identification of the strain

[0056] The physiological and biochemical characteristics of strain YK-19 were identified according to Bergey's Manual of Bacteriological Identification, and the results are shown in Table 2 below.

[0057]

[0058] Note: + indicates positive; - indicates negative.

[0059] Physiological and biochemical identification results showed that strain YK-19 was positive for VP reaction, citrate utilization, D-xylose fermentation, gelatin liquefaction, growth with 7wt% NaCl, growth at pH 5.7, nitrate reduction, and starch hydrolysis, while it was negative for propionate, L-arabinose, and D-mannitol fermentation. This biochemical phenotype is similar to that of Bacillus.

[0060] (5) Identification of 16S rRNA and gyrB gene of strain YK-19

[0061] The whole genome DNA of strain YK-19 was extracted and amplified by PCR using universal primers for bacterial 16S rRNA and specific primers for the gyrB gene, respectively. The amplified products were detected by agarose gel electrophoresis and then sequenced. Figure 2 As shown in Figures A and B. The assembled sequences were subjected to BLAST homology alignment in the NCBI database. Sequences with high homology were selected, and genetic distances were calculated using software to construct a phylogenetic tree, thus completing the molecular identification of the strain. Figure 2 As shown in C and D. The strain YK-19 was ultimately identified as *Bacillus velezensis*, and named *Bacillus velezensis* YK-19. This strain was deposited at the China Center for Type Culture Collection (CCTCC) on April 20, 2026, with accession number CCTCC NO: M 2026664.

[0062] Example 2

[0063] Determination of biological characteristics of Bacillus belyssus YK-19:

[0064] (1) Growth curve determination

[0065] The target strain YK-19 was picked and cultured in LB liquid medium at 37℃ and 220 r / min with shaking to obtain a seed culture. The culture was expanded by 1% inoculum for 36 h, and OD was measured every 2 h. 600 The viable cell count (CFU / mL) was performed, and a growth curve was plotted. Results are as follows: Figure 3 As shown, strain YK-19 grows through a sluggish phase, a logarithmic growth phase, and a stationary phase (decline phase). Growth is slow from 0 to 4 hours, rapid proliferation occurs from 4 to 24 hours, and the number of cells tends to stabilize after 24 hours.

[0066] (2) Bile salt tolerance test

[0067] The target strain YK-19 was resuspended in LB medium with different concentrations of bovine bile salts and cultured at 37°C for 3 h. The viable cell count results showed that the survival rate of strain YK-19 decreased with increasing bile salt concentration and time. At 3 h, the survival rates in 0.1wt%, 0.2wt%, and 0.3wt% bile salts were 62.93±4.01%, 46.23±3.68%, and 38.90±3.93%, respectively, showing good bile salt tolerance.

[0068] Table 3. Bile salt tolerance test of strain YK-19

[0069]

[0070] (3) Acid resistance test

[0071] The activated target strain YK-19 was cultured to the logarithmic growth phase. 1 mL of the bacterial culture was inoculated into sterile physiological saline solutions at pH 3, 4, and 5, and incubated at 37℃ for 3 hours. Samples were taken at 0h, 1h, 2h, and 3h, and after serial dilution, viable cell counts were performed (three replicates per group). The survival rate of the strain was calculated. The results showed that the survival rate of strain YK-19 gradually decreased with decreasing pH and prolonged incubation time. At 3h, the survival rates at pH=5, pH=4, and pH=3 were 64.80±2.56%, 50.46±3.14%, and 45.45±6.26%, respectively, indicating that strain YK-19 possesses a certain degree of acid tolerance.

[0072] Table 4 Acid resistance test of strain YK-19

[0073]

[0074] (4) Salt tolerance (osmotic pressure) test

[0075] The activated target strain YK-19 was cultured to the logarithmic growth phase and inoculated into LB liquid medium containing 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, and 6wt% NaCl at a 1% inoculum. The cultures were incubated at 37℃ and 220 rpm for 24 h with shaking. Samples were taken for viable cell counting (three replicates per group, counting method as described above), and the survival rate of strain YK-19 was calculated. The results showed that the survival rate of strain YK-19 gradually decreased with increasing NaCl concentration. The survival rates under 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, and 6wt% NaCl conditions were 84.48±4.83%, 77.72±3.03%, 65.76±2.88%, 57.25±4.08%, 52.36±4.12%, and 48.01±6.11%, respectively, indicating that strain YK-19 possesses a certain degree of salt (osmotic pressure) tolerance.

[0076] Table 5 Salt tolerance (osmotic pressure) test of strain YK-19

[0077]

[0078] (5) Gastric and intestinal fluid tolerance test

[0079] Artificial gastric fluid (containing 0.3wt% pepsin and 0.2wt% NaCl, adjusted to pH 2.0, and filtered for sterilization) and artificial intestinal fluid (containing 0.1wt% trypsin and 1.1wt% NaHCO3, adjusted to pH 8.0, and filtered for sterilization) were prepared. The activated target bacterial strain was centrifuged at 4℃ and 4000r / min for 10min, the supernatant was discarded, and the suspension was resuspended in artificial gastric fluid and artificial intestinal fluid, respectively, and incubated at 37℃. Samples were taken from the gastric fluid group at 0h, 1h, 2h, and 3h, and from the intestinal fluid group at 0h, 2h, 4h, and 6h. Viable bacteria were counted and the survival rate was calculated. The results showed that the survival rates of strain YK-19 after treatment in simulated gastric fluid for 1 h, 2 h, and 3 h were 59.30±5.58%, 53.61±4.22%, and 41.58±4.37%, respectively; and the survival rates after treatment in simulated intestinal fluid for 2 h, 4 h, and 6 h were 64.35±4.24%, 58.91±3.59%, and 51.30±3.35%, respectively, indicating that strain YK-19 has a certain tolerance to artificial gastrointestinal fluid.

[0080] Table 6. Gastric and intestinal fluid tolerance tests of strain YK-19

[0081]

[0082] (6) High temperature tolerance test

[0083] Spore suspensions of the target strain YK-19 were serially diluted 10-fold and the initial viable cell count was determined using the pour method. The remaining spore suspensions were then heat-treated in water baths at 75℃, 80℃, 85℃, 90℃, and 95℃ for 5 minutes each, followed by further serial dilutions and determination of the remaining viable cell counts. An equal volume of spore suspension mixed with an equal volume of physiological saline (37℃) served as a control. Each experiment was performed in triplicate, and the survival rate of the strain was calculated. The results showed that the survival rate of strain YK-19 gradually decreased with increasing heat treatment temperature. The survival rates at 75℃, 80℃, 85℃, 90℃, and 95℃ were 90.26±2.93%, 73.02±3.15%, 64.95±2.55%, 49.93±2.52%, and 43.67±4.20%, respectively, indicating that the YK-19 spores possess a certain degree of high-temperature tolerance.

[0084] Table 7. High-temperature tolerance test of strain YK-19

[0085]

[0086] Example 3

[0087] Analysis of hemolytic and enzyme-producing characteristics of Bacillus belyssus YK-19:

[0088] (1) Hemolytic test of Bacillus belyssus YK-19

[0089] The hemolytic activity of the bacterial strains was determined using the hemolytic agar method. Activated Bacillus belye YK-19 and Staphylococcus aureus ATCC 6538 (positive control) were separately spotted onto blood agar plates and incubated at 37℃ for 24 h. The formation of hemolytic rings around the colonies was observed. The results are as follows: Figure 4 The results showed that Bacillus vesiculosus YK-19 colonies did not have a clear hemolytic zone, indicating non-hemolysis; while the positive control Staphylococcus aureus ATCC 6538 colonies formed a clear hemolytic zone, indicating that this strain does not have hemolytic activity and has good biosafety.

[0090] (2) Thermostable DNAase

[0091] The toluidine blue-DNase plate method was used for detection: 100 μL of activated Bacillus belye YK-19 and Staphylococcus aureus ATCC6538 (positive control) were each inoculated onto plates and incubated at 37℃ for 24 h. The formation of hydrolysis zones around the colonies was observed. The results are as follows: Figure 5 The results showed that the positive control Staphylococcus aureus ATCC6538 colonies formed a distinct pink hydrolysis zone (producing thermostable DNase), while the YK-19 colonies did not have a hydrolysis zone, indicating that this strain does not possess thermostable DNase activity, suggesting that it has good biosafety.

[0092] (4) Protease

[0093] Activated Bacillus belye strain YK-19 was inoculated onto casein agar selection medium and incubated at 37°C for 24 hours. The presence of casein hydrolysis zones around the colonies was observed. The presence of casein hydrolysis zones around the YK-19 colonies indicates its ability to produce proteases. The results are shown in Table 8.

[0094] Table 8. Screening results of protease-producing strains

[0095]

[0096] (5) Amylase

[0097] The activated Bacillus berreatus YK-19 strain was inoculated onto soluble starch selection medium and incubated at 37℃ for 24 hours. After incubation, the plate was evenly covered with Lugol's iodine solution and allowed to stand for a while. The presence of a clear zone around the colony was then observed to determine the enzyme production of the strain. The diameter of the clear zone (D), the diameter of the colony (d), and the ratio of the two (D / d) were measured. The experimental results are shown in Table 9.

[0098] Table 9. Screening results of amylase-producing strains

[0099]

[0100] (6) β-Mannanase

[0101] The activated Bacillus belye strain YK-19 was inoculated on β-mannanase selection medium (with β-mannan as the sole carbon source) and incubated at 37℃ for 24 h. The enzyme production of the strain was judged by observing whether a clear zone was formed around the colony. The diameter of the clear zone (D), the diameter of the colony (d), and the ratio of the two (D / d) were measured. The experimental results are shown in Table 10.

[0102] Table 10 Screening results of β-mannanase-producing strains

[0103]

[0104] (7) Xylanase

[0105] The activated Bacillus belye strain YK-19 was inoculated on xylanase selection medium (with xylan as the sole carbon source) and incubated at 37℃ for 24 hours. The enzyme production of the strain was judged by observing whether a clear zone was formed around the colony. The diameter of the clear zone (D), the diameter of the colony (d), and the ratio of the two (D / d) were measured. The experimental results are shown in Table 11.

[0106] Table 11 Screening results of xylanase-producing strains

[0107]

[0108] (8) Cellulase

[0109] Activated Bacillus belye YK-19 strain was inoculated onto CMC-Na cellulase selection medium and incubated at 37℃ for 48 h. After Congo red staining and decolorization with NaCl solution, a clear transparent hydrolysis zone appeared around the colony. The diameter of the hydrolysis zone (D) and the colony diameter (d) were measured, and their ratio (D / d) was calculated. The results are shown in Table 12.

[0110] Table 12 Screening results of cellulase-producing strains

[0111]

[0112] (9) Pectinase

[0113] Activated Bacillus belye strain YK-19 was inoculated onto pectinase screening medium (with pectin as the sole carbon source) and incubated at 37℃ for 24–48 h. After incubation, Congo red solution was added to the plates, and the plates were allowed to stand for 30 min before observing whether a transparent hydrolysis zone (pectin hydrolysis zone) appeared around the colonies. The diameter of the hydrolysis zone (D) and the colony diameter (d) were measured, and their ratio (D / d) was calculated. The results are shown in Table 13.

[0114] Table 13 Screening results of pectinase-producing strains

[0115]

[0116] The foregoing has shown and described the basic principles, main features and advantages of the present invention. Various changes and modifications can be made to the present invention without departing from the spirit and scope thereof, and all such changes and modifications fall within the scope of the present invention as claimed.

Claims

1. A strain of Bacillus belyceta var. YK-19 possessing vomitoxin degradation and multi-enzyme activity, characterized in that: The Bacillus velezensis YK-19 strain is classified as Bacillus velezensis YK-19 and was deposited at the China Center for Type Culture Collection (CCTCC) on April 20, 2026, with accession number CCTCC NO: M 2026664.

2. The Bacillus vesiculosus YK-19 with vomitoxin degradation and multi-enzyme activity according to claim 1, characterized in that: The Bacillus berreatus YK-19 was isolated from the intestinal contents of pigs.

3. The Bacillus vesiculosus YK-19 with vomitoxin degradation and multi-enzyme activity according to claim 1, characterized in that: The Bacillus berberis YK-19 exhibits a degradation rate of up to 93.79% for vomitoxin and possesses beneficial properties such as tolerance to 0.3wt% bile salts, tolerance to acidic environments with pH=3.0, tolerance to heat treatment at 75~95℃, and tolerance to simulated gastric and intestinal fluids.

4. The Bacillus vesiculosus YK-19 with vomitoxin degradation and multi-enzyme activity according to claim 1, characterized in that, The Bacillus berberis YK-19 strain is non-hemolytic and lacks DNase activity.

5. The use of Bacillus berreatus YK-19 according to any one of claims 1 to 4 in the preparation of feed compound enzyme preparations.

6. The application according to claim 5, characterized in that: The feed compound enzyme preparation contains at least one of protease, amylase, β-mannanase, xylanase, cellulase and pectinase.

7. The application according to claim 6, characterized in that: The feed-grade compound enzyme preparation has the following D / d values: protease D / d value 2.17±0.11, amylase D / d value 1.40±0.02, β-mannanase D / d value 2.76±0.15, xylanase D / d value 1.87±0.13, cellulase D / d value 2.27±0.30, and pectinase D / d value 1.95±0.

12.

8. The use of Bacillus berberis YK-19 according to any one of claims 1 to 4 in the preparation of probiotic products.

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