Bacillus velez, its use and feed

Abstract

The present application relates to the field of biotechnology, and discloses a strain of Bacillus velezensis, which is classified and named Bacillus velezensis ZM2024, and the deposit number is CCTCC NO: M2024695; the deposit date is April 15, 2024, and it is deposited in the China Center for Type Culture Collection. The Bacillus velezensis in the present application has good stress resistance and safety, high production of cellulase, and can significantly improve the nutritional content and feeding value of high-fiber plant feed raw materials such as palm meal; at the same time, the Bacillus velezensis can inhibit diarrhea-causing pathogens of livestock and poultry, and is suitable for livestock and poultry feed processing and feeding. In addition, the present application also discloses the use of the above-mentioned Bacillus velezensis and a feed.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a strain of Bacillus Velezii and its application and feed. Background Art

[0002] my country's unconventional feed resources are very rich in variety and quantity, and the price is relatively economical. It is estimated that the annual output of unconventional feed resources can reach 4 billion tons. At the same time, the nutritional value of unconventional feed raw materials is also rich, containing abundant carbohydrates, crude protein, vitamins and minerals. Therefore, the development of unconventional feed may be an effective measure to alleviate the shortage of feed resources in my country.

[0003] Crude fiber is the main component of plant-based unconventional feed raw materials, and its content is generally 18.41% to 44.5%. Cellulose is the main substance in crude fiber. It is a macromolecular polysaccharide composed of β-1,4-glycosidic bonds. It is easy to form crystals and fibril structures and is the main anti-nutritional factor of plant-based feed raw materials. High-fiber plant-based feed is not easily digested by monogastric animals such as pigs and chickens, which increases the viscosity of digestive tract chyme, hinders the digestion and absorption of other nutrients, increases the burden on the digestive system and increases the risk of intestinal diseases. At the same time, high-fiber plant-based feed raw materials usually have a rough texture and poor palatability for pigs and chickens, which has an adverse effect on growth, development and health, further limiting the application of plant-based unconventional feed raw materials in pig and chicken feed. Therefore, reducing the fiber content can promote the application of plant-based unconventional feed raw materials and alleviate the shortage of feed resources in my country.

[0004] Microbial fermentation is an effective measure to degrade fiber in plant-based unconventional feed raw materials. Compared with traditional physical, chemical and enzymatic methods, it has the advantages of low cost, no toxic residues, mild reaction conditions and simple operation. It can degrade anti-nutritional factors and produce beneficial metabolites, thereby improving the nutritional value of feed. Microbial strains are the main factor determining the efficiency of fiber fermentation degradation. Therefore, screening and obtaining cellulose-degrading bacteria that can efficiently degrade crude fiber in unconventional feed raw materials is of great significance to alleviate the shortage of feed resources in my country and reduce costs and increase efficiency in animal husbandry.

[0005] Diarrhea-causing bacterial infections such as enterotoxigenic Escherichia coli, Staphylococcus aureus, Salmonella, Shigella dysenteriae and Shigella flexneri cause diarrhea and even death in animals, causing huge economic losses to the animal husbandry industry. In the past livestock and poultry farming process, antibiotics were used extensively to prevent and control bacterial infections in animals and improve growth performance in order to improve the production efficiency of livestock and poultry farming. However, long-term irregular and unscientific use has led to problems such as antibiotic residues, drug-resistant bacteria and environmental pollution. Therefore, antibiotics as feed additives have been strictly prohibited, and there is also a call to reduce the amount of antibiotics used for treatment. Therefore, it is urgent to develop new antibiotic substitutes. Probiotics have the effects of antibacterial and promoting animal growth, and are not toxic or side effects. They are gradually becoming the main force of antibiotic substitutes.

[0006] Chinese patent application 201910548471.9 discloses a cellulose-degrading Bacillus velezensis GX-1 from Minzhu. It is deposited in China Center for Type Culture Collection (CCTCC) on November 29, 2018, with a deposit number of CCTCC NO:M 2018841, and is classified as Bacillus velezensis GX-1. The strain of this scheme can produce cellulase, which can efficiently degrade cellulose and be used for biodegradation of cellulose. The cellulase produced by the GX-1 strain has good thermal stability and can maintain good enzyme activity in the range of 40-70°C; the strain GX-1 has the advantages of rapid growth, high enzyme production, low requirements for culture conditions and enzyme production conditions, and easy colonization in the pig intestine;

[0007] Further observation of the scheme shows that, although the strain described in the scheme is capable of producing cellulase, it does not show an effect on the activity of filter paper enzyme and β-glucosidase;

[0008] Secondly, although the scheme discloses in paragraph 51 of the specification that it can exhibit obvious inhibitory effects on microorganisms of the Actinobacteria and Proteobacteria phyla, it can be seen from paragraph 52 of the specification of the scheme that its inhibitory ability against microorganisms of the Firmicutes phylum is relatively weak, and Staphylococcus aureus is a microorganism of the Firmicutes phylum.

[0009] Chinese patent application 202110114861.2 discloses a strain of Bacillus Velezii K01 and its application. Strain K01 not only has the growth-promoting functions of solubilizing phosphate, producing indoleacetic acid, cellulase, protease, etc., but also can produce antibacterial active substances and volatile antibacterial substances, which can inhibit the growth of various pathogens. It has a good biocontrol function and can colonize well in the rhizosphere and interior of plants. It can be used for plant growth promotion and biological control of plant diseases, and has very good application prospects.

[0010] Further observation of the instructions of the scheme shows that the pathogens inhibited are gray mold, fusarium oxysporum, tomato early blight, small long beak mold, fusarium moniliforme, lily root rot, watermelon wilt, peach brown rot, wheat sheath blight, rice sheath blight, cotton verticillium wilt, celery spot blight, anthracnose, sunflower black stem pathogen, sunflower sclerotinia and rice seedling pathogen, etc.

[0011] However, the above pathogens are mostly plant fungi and do not include Escherichia coli, Staphylococcus aureus, Salmonella, Shigella dysenteriae and Shigella flexneri.

[0012] The problem to be solved in this scheme is: how to provide a Bacillus Velezii with good enzyme activity and good antibacterial ability. Summary of the invention

[0013] The purpose of the present application is to provide a Bacillus Velezii, which has a high production of cellulase. After optimization, the activities of carboxymethyl cellulase, filter paper enzyme, β-glucosidase and total cellulase are 203.78U / mL, 102.46U / mL, 197.90U / mL and 504.14U / mL, respectively. At the same time, the proliferation of diarrhea-causing pathogens (enterotoxigenic Escherichia coli K88, Staphylococcus aureus, Salmonella, Shigella dysenteriae and Shigella flexneri) is inhibited, and the diameters of the inhibition zones are 20.51±0.31mm, 18.07±0.31mm, 19.45±0.70mm, 27.08±0.54mm and 13.91±0.33mm, respectively. The Bacillus Velezii grows rapidly and has good safety and stress resistance.

[0014] To achieve the above-mentioned purpose, the present invention provides the following technical solution: a strain of Bacillus velezensis, classified and named Bacillus velezensis ZM2024, with a preservation number of CCTCC NO: M2024695, a preservation date of April 15, 2024, a preservation unit of China Center for Type Culture Collection, and a preservation address of No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0015] Preferably, the Bacillus Velez is extracted from healthy Ningxiang pig feces.

[0016] Preferably, the 16SrRNA gene sequence of Bacillus velez is as shown in SEQ ID NO:1.

[0017] In addition, the present application also discloses the use of the above-mentioned Bacillus Velezii as an active ingredient for inhibiting diarrhea-causing pathogens in feed.

[0018] Preferably, the diarrhea-causing pathogens are Escherichia coli, Staphylococcus aureus, Salmonella, Shigella dysenteriae and Shigella flexneri.

[0019] In addition, the present application also discloses the use of the above-mentioned Bacillus Velez subtilis as a fermentation bacterium in feed.

[0020] In addition, the present application also discloses a feed comprising 10 8 ~10 9 cfu / kg of Bacillus velez as described above.

[0021] Preferably, the feed is a feed containing plant components, and the plant components are plant components containing crude protein, acid-soluble protein and crude fiber.

[0022] Further preferably, the plant ingredient is selected from at least one of palm seeds, palm meal and palm cake.

[0023] The beneficial effects of this application are:

[0024] The Bacillus Velez ZM2024 provided by the present invention is separated from healthy Ningxiang pigs, grows rapidly, has good stress resistance and safety, and produces a high amount of cellulase, and can significantly improve the nutritional components and feeding value of high-fiber plant feed raw materials such as palm meal; at the same time, the Bacillus Velez can inhibit diarrhea-causing pathogens of livestock and poultry, and is suitable for livestock and poultry feed processing and feeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the strain with transparent circle isolated from Ningxiang pig feces;

[0026] Figure 2 is a schematic diagram of the glucose standard curve;

[0027] Figure 3 is a schematic diagram of the phylogenetic tree of strain C36;

[0028] Figure 4 Schematic diagram of the colony morphology of Bacillus velez ZM2024 in NA medium;

[0029] Figure 5 is a schematic diagram of the Gram staining results of Bacillus velez ZM2024;

[0030] Figure 6 is a schematic diagram of the growth curve of Bacillus velez ZM2024;

[0031] Figure 7 It is a schematic diagram of the survival rate of Bacillus Velez ZM2024 at different pH;

[0032] Figure 8 It is a schematic diagram of the survival rate of Bacillus Velez ZM2024 after being treated at different temperatures;

[0033] Fig. 9 Schematic diagram of the survival rate of Bacillus Velez ZM2024 in artificial gastric juice and intestinal juice;

[0034] Fig.10 It is a schematic diagram of the survival rate of Bacillus velez ZM2024 in different concentrations of bile salts;

[0035] Fig.11 is a schematic diagram of the drug sensitivity test results of Bacillus velez ZM2024;

[0036] Fig.12 It is a schematic diagram of the antibacterial results of Bacillus velez ZM2024;

[0037] Fig.13 Schematic diagram of the effect of different temperatures on the cellulase production of Bacillus Velez ZM2024;

[0038] Fig.14 Schematic diagram of the effect of different inoculation amounts on the cellulase production of Bacillus Velez ZM2024;

[0039] Fig.15 Schematic diagram of the effect of different culture time on the cellulase production of Bacillus Velez ZM2024. DETAILED DESCRIPTION

[0040] In the description of the present invention, it should be noted that, if the specific conditions are not specified in the examples, the experiments were carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0041] The present invention will be described clearly and completely below in conjunction with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] Example 1

[0043] 1. Isolation, screening and identification of cellulose-degrading bacteria

[0044] 1.1 Culture media and reagents

[0045] Nutrient broth (NB): peptone 10 g / L, beef extract powder 3.0 g / L, NaCl 5.0 g / L, pH 7.2±0.2, autoclave at 121°C for 15 min.

[0046] Nutrient agar medium (NA): peptone 10 g / L, beef extract powder 3.0 g / L, NaCI 5.0 g / L, agar 15.0 g / L, pH 7.3±0.1, high pressure sterilization at 121°C for 15 min.

[0047] Cellulase enrichment medium: CMC-Na 10g / L, MgSO4 0.5g / L, NaNO3 3.0g / L, KCI 0.5g / L, K2HPO4 0.4g / L, FeSO4 0.01g / L, high pressure sterilization at 121℃ for 15min.

[0048] Isolation medium: CMC-Na 10 g / L, MgSO4 0.5 g / L, NaNO3 3.0 g / L, KCI 0.5 g / L, K2HPO4 0.4 g / L, FeSO4 0.01 g / L, agar 15 g / L, high pressure sterilization at 121°C for 15 min.

[0049] LB Broth (LB): 10.0 g / L tryptone, 5.0 g / L yeast extract powder, 10.0 g / L NaCl, pH 7.0±0.1, autoclave at 121°C for 15 min.

[0050] Mueller-Hinton Agar (MHA): beef extract powder 6.0 g / L, soluble starch 1.5 g / L, acid hydrolyzed casein 17.5 g / L, agar 17.0 g / L, pH 7.3±0.1, autoclave at 121°C for 15 min.

[0051] Congo red, sodium carboxymethylcellulose, salicin, acetic acid, and sodium acetate were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0052] DNS reagent, pepsin, trypsin, porcine bile salt, and bacterial genomic DNA extraction kit were purchased from Beijing Solebow Technology Co., Ltd.;

[0053] Sodium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and concentrated hydrochloric acid were provided by Sinopharm Chemical Reagent Co., Ltd.;

[0054] Antibiotic susceptibility paper was purchased from Changde Beekman Biotechnology Co., Ltd.;

[0055] Gram stain was purchased from Qingdao High-Tech Industrial Park Haibo Biotechnology Co., Ltd.;

[0056] Whatman No. 1 filter paper was purchased from GlaxoScience Life Sciences (Shanghai) Co., Ltd.

[0057] Artificial gastric juice: Add concentrated hydrochloric acid to 0.9% saline to adjust the pH to 1.8, add 1 g of pepsin to every 100 mL of hydrochloric acid solution, mix well, filter through a 0.22 μm sterile filter membrane to sterilize, and store at 4°C for use.

[0058] Artificial intestinal fluid: Take 5.59g of K2HPO4 and 0.41g of KH2PO4 and dissolve them in 1000mL of deionized water, pH = 8.0. Add 1g of trypsin to 100mL of the solution, mix well, filter through a 0.22μm sterile filter membrane for sterilization, and store at 4℃ for use.

[0059] 1.2 Strain Isolation

[0060] Equal masses of fecal samples from three Ningxiang pigs fed with green roughage were mixed, and then 1 g of the mixed fecal sample was weighed and placed in a centrifuge tube containing 10 mL of cellulase enrichment medium. Glass beads were added and vortexed to mix thoroughly, and then cultured at 37°C for 24 h.

[0061] Take 100 μL of bacterial solution and inoculate it into 10 mL of cellulase enrichment medium, continue to culture at 37°C for 24 hours, repeat twice. Take 1 mL of bacterial solution and add it into 9 mL of normal saline. The dilution concentration is 10 -1 , and then diluted to 10 at a ratio of 1:10 -9 , take 100 μL of bacterial suspension from each dilution gradient and apply it to the separation medium, culture it at 37℃ for 24-72h until most colonies grow, pick colonies and streak them in NA medium until a single colony is purified. Then pick the purified strain and inoculate it in NB medium for amplification for 24h, and store it at 4℃ for later use.

[0062] The activated strain was inoculated in the separation medium by three-point inoculation method and inverted at 37℃ for 48h. After the strain culture was completed, 10mL of 0.5% Congo red solution was slowly poured from the edge of the culture dish, and gently shaken to cover the entire plate with the Congo red solution. After standing for 10min, the liquid in the culture dish was poured out, and the plate was rinsed once with 1mol / L NaCl solution, and then 10mL of 1mol / L NaCl solution was added to the culture dish. After standing for "decolorization" for 15min, the liquid in the culture dish was poured out, and the transparent circle diameter (D) and colony diameter (d) on the culture medium plate were observed and measured, and the ratio of the transparent circle diameter (D) to the colony diameter (d) was calculated. The strains with a diameter ratio of >2 were rescreened for cellulase determination.

[0063] Isolation results of cellulose-degrading strains Figure 1 As shown, 7 strains with obvious hydrolysis circles were isolated from Ningxiang pig feces, referring to Table 1, among which the diameter ratios of C36, C37 and C44 were greater than 2, and cellulase activity was determined.

[0064] Table 1

[0065] Strain number Decolorization circle diameter D(mm) Strain diameter d(mm) Diameter ratio D / d C8 5.25±0.4 9.9±0.22 1.89±0.18 C24 11.73±2.2 19.22±1.25 1.67±0.23 C34 5.62±0.6 10.8±0.38 1.93±0.15 C36 6.56±0.53 29.41±0.19 4.48±0.36 C37 8.26±0.53 23.63±0.97 2.87±0.23 C44 7.1±0.99 26.54±2.62 3.83±0.77 C51 5.09±0.14 9.94±0.09 1.95±0.04

[0066] 1.3. Cellulase activity assay

[0067] 1.3.1. Preparation of glucose standard curve

[0068] Preparation of glucose standard solution: Weigh 100 mg of analytical pure anhydrous glucose, dissolve it in distilled water, and make up to 100 mL. The concentration is 1 mg / mL. Add reagents according to Table 2.

[0069] Table 2

[0070]

[0071]

[0072] After mixing the solutions in each tube, heat them in a boiling water bath for 10 minutes, then take them out, immediately cool them to room temperature with cold water, and dilute them to 10 mL with distilled water. Measure the absorbance at 520 nm. Use EXCEL software to draw a standard curve with glucose (mg) as the horizontal axis and light absorption value as the vertical axis.

[0073] Glucose standard curve Figure 2 As shown, the glucose content (mg) is the horizontal axis, and OD 520nm The equation of the glucose standard curve is: y = 0.1424x-0.0167, R 2 =0.995, which meets the requirements of the standard curve.

[0074] 1.3.2. Cellulase activity assay

[0075] The strain obtained in the initial screening was activated and inoculated into the cellulase enrichment medium at an inoculation rate of 2% (v / v). After constant temperature culture at 37°C for 48 hours, the culture was centrifuged at 6010×g and 4°C for 10 minutes, and the supernatant was the crude enzyme solution.

[0076] Determination of carboxymethyl cellulase activity (CMCase): After the crude enzyme solution is appropriately diluted, 1 mL is taken, and 1 mL of a buffer solution (pH 4.8, 0.2 mol / L HAc-NaAC buffer) containing 1% CMC-Na is added. The reaction is carried out at 50°C for 30 min, and 2 mL of DNS reagent is quickly added, followed by a boiling water bath for 5 min. After cooling, the OD value of the solution is measured at 540 nm, and the reducing sugar content produced by the reaction is calculated using the glucose standard curve.

[0077] Determination of filter paper enzyme activity (FPA): Add 50 mg of filter paper to a test tube containing 1 mL of appropriately diluted crude enzyme solution and 1 mL of 0.2 mol / L acetate buffer at a pH of 4.8. React at 50°C for 60 min, then add 2 mL of DNS reagent to terminate the reaction. Boil in a boiling water bath for 5 min, cool with cold water, and compare color at 540 nm.

[0078] Determination of β-glucosidase activity: Add 1 mL of salicin solution with a salicin mass fraction of 0.5% and 1 mL of appropriately diluted enzyme solution into a centrifuge tube in sequence, mix well, react at 50°C for 30 min, add 2 mL of DNS reagent to terminate the reaction, boil in a boiling water bath for 5 min, cool with cold water and compare color at 540 nm.

[0079] Definition of enzyme activity: The amount of cellulase required to produce 1 μmol of reducing sugar by decomposing substrate per minute is defined as one cellulase activity unit, expressed as U / mL.

[0080] The calculation formula is shown in Formula 1:

[0081]

[0082] In the formula, 5.56 is the μmoL number of 1mg of glucose, that is, 1000 / 180=5.56

[0083] Cellulase activity As shown in Table 3, the activities of carboxymethyl cellulase, filter paper enzyme and β-glucosidase of strain C36 were 125.93±0.94U / mL, 125.63±0.86U / mL and 71.01±0.66U / mL, respectively, which were higher than those of C37 and C36. Therefore, C36 was identified as a high-producing cellulase strain for subsequent experiments.

[0084] Table 3

[0085]

[0086] 1.4. Identification of bacterial species

[0087] The genomic DNA of the strain was extracted using a bacterial genomic DNA extraction kit produced by Beijing Solebow Technology Co., Ltd. The extracted DNA sample was appropriately diluted and used as a PCR template, and the universal primers of bacterial 16S rDNA were amplified using Qingke 1×TSE101 Gold Mix.

[0088] The 27F primer sequence was: AGAGTTTGATCMTGGCTCAG;

[0089] The 1492R primer sequence is: GGTTACCTTGTTACGACTT.

[0090] The components of the amplification system are shown in Table 4:

[0091] Table 4

[0092] 1×TSE101 Gold Mix 45uL 27F(10P) 2uL 1492R(10P) 2uL DNA template 1uL

[0093] The above amplification system was amplified according to the following amplification program, as shown in Table 5:

[0094] Table 5

[0095]

[0096] After PCR amplification, the strain was identified by 2% agarose gel electrophoresis (2uL sample + 6uL bromophenol blue, 300V voltage for 12 minutes) and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The whole genome sequence of the strain is shown in SEQ ID No: 1; the sequence results were compared in the NCBI database, and the results showed that strain C36 was Bacillus velezensis ( Figure 3 ). The strain was named Bacillus velez ZM2024 and was deposited in the China Center for Type Culture Collection on April 15, 2024 with the deposit number CCTCC NO:M 2024695. The deposit address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, China.

[0097] Example 2

[0098] 2. Biological characteristics of Bacillus velez ZM2024

[0099] 2.1 Colony morphology observation

[0100] The strain was inoculated on NA medium by the plate streak method, and the colony morphology was observed after inverted culture at 37°C for 16 h, and the colonies were picked for Gram staining.

[0101] The colonies of Bacillus Velezii ZM2024 in NA culture are semi-transparent and round with irregular edges, wrinkled surface and protrusions ( Figure 4Further Gram staining showed positive results, the cells were rod-shaped, the spores were centrally or terminally borne, the sporangium capsules were not swollen, and the spores were columnar ( Figure 5 ).

[0102] 2.2 Growth curve

[0103] After the strain is activated, it is inoculated into 1 mL LB medium at a 3% (v / v) inoculum and cultured in a shaker at 37°C and 150 rpm. The strains are taken out at 0h, 2h, 4h, 6h, 8h, 10h, and 12h of culture, and the absorbance is measured at 600nm. The culture time is used as the horizontal axis, and the OD 600 Draw the growth curve with y as the vertical axis.

[0104] The growth curve of Bacillus velez ZM2024 is as follows Figure 6 The growth period of Bacillus Velezii C36 was 0-2h in the fermentation culture, and it entered the logarithmic growth period after 2h and the stable period after 10h.

[0105] 2.3 Acid resistance test

[0106] The activated strains were inoculated at 3% (v / v) in natural pH and NB medium with pH 2, 3, 4, and 5, respectively, and cultured at 37°C and 150 r / min for 24 hours, and the absorbance was measured at 600 nm. The OD600 of the strains grown in natural pH NB medium was used as the control group, and the rest were used as the experimental groups. The survival rate (%) = OD value of the experimental group / OD value of the control group × 100.

[0107] The results of the tolerance of Bacillus velez ZM2024 to different acidic environments are as follows Figure 7 As shown. At pH = 5, the survival rate reaches 99%, which has almost no effect on the growth and reproduction of the strain. At pH = 4, the survival rate reaches 70%, at pH = 3, the survival rate reaches 44%, and at pH = 2, the survival rate can still reach 39%. When the pH decreases, the survival rate of the strain decreases significantly.

[0108] 2.4 High temperature tolerance test

[0109] The activated strain was inoculated into 1 mL NB medium at a 3% (v / v) inoculum, and placed in a water bath at 37°C, 60°C, and 80°C for 10 min, followed by incubation at 37°C, 150 r / min for 24 h, and the absorbance was measured at 600 nm. The OD of the strain treated at 37°C was 600 is the control group, and the rest are experimental groups. Survival rate (%) = OD value of experimental group / OD value of control group × 100.

[0110] Depend on Figure 8It can be seen that the survival rate of Bacillus Velez ZM2024 at 60°C is 97%, and the survival rate at 80°C is still 45%.

[0111] 2.5 Gastric and intestinal fluid tolerance test

[0112] The activated strain was inoculated into 1 mL of intestinal fluid, gastric fluid and saline at a 3% (v / v) inoculation volume, cultured at 37°C and 150 r / min for 4 h, and the absorbance was measured at 600 nm. 600 For the control group, survival rate (%) = OD value of experimental group / OD value of control group × 100.

[0113] Tolerance of Bacillus Velezii ZM2024 to intestinal and gastric juices Fig. 9 The survival rate in intestinal fluid was 57% and in gastric fluid was 40%.

[0114] 2.6 Bile salt tolerance test

[0115] The activated strains were inoculated into NB medium with a final bile salt concentration of 0%, 0.1%, 0.3%, and 0.5% at a 3% (v / v) inoculation volume, and cultured at 37°C and 150 r / min for 4 hours. The number of viable bacteria was then determined by the plate count method, and the survival rate was calculated. The OD600 of the strain treated with 0% bile salt was used as the control group, and the rest were used as the experimental group. The survival rate (%) = OD value of the experimental group / OD value of the control group × 100.

[0116] The results of the bile salt tolerance of Bacillus velez ZM2024 are as follows Fig.10 The survival rates of Bacillus velez ZM2024 in 0.1%, 0.3% and 0.5% bile salt concentrations were 77%, 55% and 41%, respectively.

[0117] 2.7 Drug sensitivity testing

[0118] Pick the activated Bacillus Velez ZM2024 and add it to 1 mL of saline to make 1×10 8 cfu / mL bacterial suspension was evenly spread on MHA medium using a sterile cotton swab and placed at room temperature for a while until the surface was slightly dry. Use tweezers to evenly place 10 antibiotic paper discs on the surface of the medium, with two replicates. Incubate at 37℃ for 18-24h and observe the inhibition zone.

[0119] The results of the drug sensitivity test of Bacillus velez ZM2024 are as follows Fig.11Bacillus velez ZM2024 was sensitive to trimethoprim-sulfamethoxazole SXT, gentamicin GEN, penicillin PEN, tetracycline TET, ciprofloxacin CIP, erythromycin E, clobenicillin AMP, chloramphenicol C, and ceftriaxone CTR, and was moderately sensitive to lincomycin MY.

[0120] 2.8 Antibacterial test

[0121] The activated diarrhea-causing indicator bacteria (enterotoxigenic Escherichia coli K88, Salmonella, Staphylococcus aureus, Shigella dysenteriae, Shigella flexneri) were streaked on NA plates and cultured at 37°C for 12 to 16 hours. The colonies were picked and inoculated into LB medium and cultured at 37°C for 6 to 12 hours. The activated Bacillus Velez ZM2024 bacterial solution was inoculated into NB medium at a 3% (v / v) inoculation rate, cultured at 37°C and 150r / min for 12 hours, and set aside. The antibacterial test was carried out by double-layer agar diffusion method. First, pour about 10mL of MHA medium into the culture dish, wait for it to solidify, and evenly place the Oxford cup on the MHA medium. Then take an appropriate amount of the cultured indicator bacterial solution and add it to the MHA medium at about 50°C to make the bacterial solution concentration about 1×10 8 cfu / mL, and then pour 20mL of MHA medium containing indicator bacteria into the culture dish. After the MHA medium solidifies, pull out all Oxford cups, add 100uL of cultured Bacillus Velezii ZM2024 to each well, set up a negative control of physiological saline and a positive control of 5ug / mL gentamicin sulfate. Culture at 37℃ for 16-24h, observe whether there is an inhibition zone, if there is, measure the diameter of the inhibition zone, measure 3 directions in each well, and calculate the average value as the size of the inhibition zone.

[0122] The sizes of inhibition zones of Bacillus Velez ZM2024 against enterotoxigenic Escherichia coli K88, Staphylococcus aureus, Salmonella, Shigella dysenteriae and Shigella flexneri are shown in Table 6. The diameters of the inhibition zones are 20.51±0.31mm, 18.07±0.31mm, 19.45±0.70mm, 27.08±0.54mm and 13.91±0.33mm, respectively. The inhibition effects are significantly better than those of the 5ug / mL gentamicin sulfate positive control treatment. The plate inhibition zones are shown in Table 6. Fig.12 shown.

[0123] Table 6

[0124]

[0125] 2.9 Statistical methods

[0126] All experiments were repeated three times and SPSS26.0 software was used for multiple comparisons using independent sample t-test and Duncan test in one-way analysis of variance. GraphPad Prism 9.5 was used for graphics. Data were expressed as mean ± standard error. P < 0.05 indicated statistical significance. The absence of the same letter on the shoulder of the data or at the top of the bar graph indicated a significant difference.

[0127] Example 3

[0128] 3. Enzyme production characteristics of Bacillus velez ZM2024

[0129] 3.1 Preparation of seed solution

[0130] After the strain is activated, it is inoculated into NB medium at an inoculum amount of 2% (v / v), and cultured at a constant temperature of 37° C. and 150 rpm for 10 hours to obtain seed solution.

[0131] 3.2 Effect of temperature on enzyme production

[0132] The seed liquid was inoculated into 10 mL of cellulase enrichment medium at an inoculum rate of 2% (v / v), and cultured in constant temperature incubators at 25°C, 30°C, 35°C, and 40°C for 48 hours, respectively. The enzyme activity was determined according to the method described in Example 1.

[0133] Effect of temperature on enzyme production of Bacillus velez ZM2024 Fig.13 As shown in the figure, at 25℃, the activities of carboxymethyl cellulase, filter paper enzyme, β-glucosidase and total enzyme of the strain were significantly higher than those at other temperatures, which were 165.02U / mL, 89.20U / mL, 170.75U / mL and 424.97U / mL respectively.

[0134] 3.3 Effect of inoculation amount on enzyme production

[0135] The seed liquid was inoculated into 10 mL of cellulase enrichment medium at an inoculum amount of 2%, 5%, 8%, and 11% (v / v), and cultured in a 37° C. constant temperature incubator for 48 h. The enzyme activity was determined according to the method described in Example 1.

[0136] Effect of inoculation amount on enzyme production of Bacillus velez ZM2024 Fig.14 When the inoculation amount was 8%, the carboxymethyl cellulase, filter paper enzyme, β-glucosidase and total enzyme activities of the strain were the highest, which were 170.48U / mL, 94.31U / mL, 172.83U / mL and 437.62U / mL respectively; followed by 5%, which were 169.33U / mL, 92.44U / mL, 172.84U / mL and 434.61U / mL respectively, and there was a certain difference between the two.

[0137] 3.4 Effect of culture time on enzyme production

[0138] The seed liquid was inoculated into 10 mL of cellulase enrichment medium at an inoculum rate of 2% (v / v), and the culture was placed in a 37°C constant temperature incubator for 12 h, 24 h, 48 h, 72 h, 96 h, and 120 h, respectively. The enzyme activity was determined according to the method described in Example 1.

[0139] Effect of culture time on enzyme production of Bacillus velez ZM2024 Fig.15 When the culture time was 12h, the carboxymethyl cellulase, filter paper enzyme, β-glucosidase and total enzyme activities of the strain were the highest, which were 203.78U / mL, 102.46U / mL, 197.90U / mL and 504.14U / mL, respectively, and were significantly higher than those at other culture times.

[0140] Example 4

[0141] 4. Improving the quality of palm meal by fermentation with Bacillus velez ZM2024

[0142] 4.1 Preparation of seed solution

[0143] After the strain is activated, it is inoculated into NB medium at an inoculum amount of 2% (v / v), and cultured at a constant temperature of 37° C. and 150 rpm for 10 hours to obtain seed solution.

[0144] 4.2 Palm meal fermentation

[0145] The palm kernels were crushed and sieved, and 500.0 g was accurately weighed, and seed liquid was added at a ratio of 5%, with a material-water ratio of 5:5. The mixture was put into a fermentation bag with a valve, mixed evenly, and fermented at 30°C for 3 days.

[0146] 4.3 Nutritional analysis of palm meal before and after fermentation

[0147] 100.0 g of unfermented and fermented palm meal were dried at 105 °C, passed through a 60-mesh sieve, and the sieve was collected. The crude protein, acid-soluble protein, and crude fiber of palm meal before and after fermentation were determined according to GB / T 6432-2018, NY / T 3801-2020, and GB / T 6434-2022. The data were analyzed by independent sample t-test in SPSS26.0, and P<0.05 indicated significant differences.

[0148] As shown in Table 7, after fermentation, the crude protein content increased by 19.60%, the acid-soluble protein content increased by 77.42%, and the crude fiber content decreased by 34.20%.

[0149] Table 7

[0150] project Palm meal Fermented palm meal Change Crude protein, % 17.57±0.99b 21.01±0.01a +19.60 Acid soluble protein, % 0.62±0.02b 1.10±0.02a +77.42 Crude fiber, % 22.60±0.23a 14.87±0.21b -34.20

[0151] Note: “+” indicates increase, “-” indicates decrease; Data in the same row without the same letter in the shoulder indicates P < 0.05.

[0152] Through the above experiments, we can find that:

[0153] 1. The Bacillus Velezii ZM2024 of the present invention is a strain naturally isolated from Ningxiang pigs, has excellent cellulase production ability and ability to inhibit diarrhea-causing pathogens of livestock and poultry, grows rapidly, is tolerant to high temperature, gastric juice, intestinal juice and bile salts, has no drug resistance, and is suitable for livestock and poultry feed processing and feeding.

[0154] 2. The palm meal fermented by Bacillus Velez ZM2024 of the present invention can significantly improve the nutritional value.

[0155] It will be apparent to those skilled in the art that the invention is not limited to the details of the exemplary embodiments described above and that the invention can be implemented in other specific forms without departing from the spirit or essential features of the invention. Therefore, the embodiments should be considered exemplary and non-limiting in all respects, and the scope of the invention is defined by the appended claims rather than the foregoing description, and it is intended that all variations falling within the meaning and range of equivalent elements of the claims be included in the invention. Any reference numeral in a claim should not be considered as limiting the claim to which it relates.

Claims

1. A strain of Bacillus Velez, characterized in that: Classification Name Bacillus velezensis ZM2024, the deposit number is CCTCC NO: M2024695; the deposit date is April 15, 2024, and it is deposited in the China Center for Type Culture Collection.

2. The Bacillus Velezii according to claim 1, characterized in that The 16SrRNA gene sequence of the Bacillus Velezii is shown in SEQ ID NO:

1.

3. Use of any one of the Bacillus Velezii described in claims 1-2 as a fermentation bacterium in feed.

4. A feed, characterized in that: Contains 10 8 ~10 9 cfu / kg of the Bacillus Velezii as described in any one of claims 1-2.

5. The feed according to claim 4, characterized in that The feed is a feed containing plant components, and the plant components are plant components containing crude protein, acid-soluble protein and crude fiber.

6. The feed according to claim 5, characterized in that The plant component is selected from at least one of palm seeds, palm meal and palm cake.

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