Bacillus subtilis y32 and application thereof

Abstract

This invention relates to a Bacillus subtilis ( Bacillus subtle Y32, with accession number CGMCC No. 34819, is a strain that produces heat-resistant and highly stable xylanase, as well as various hydrolases such as β-mannanase and protease. It also produces extracellular polysaccharides, inhibits Botrytis cinerea, and is acid-resistant, resistant to porcine bile salts, and resistant to 10% NaCl. The optimal temperature for the xylanase produced by this strain is 55℃, and the optimal pH is 6.0. Under these conditions, the half-life is 12.5 h, and the relative enzyme activity remains above 80% after 3 h of incubation, demonstrating good thermal stability. This strain can also increase the yield of cucumbers and Pinellia ternata, and increase the egg production rate of 70-week-old Hy-Line Brown hens from 70.4% to 90.1%. This strain has promising applications in agriculture, food processing, and cosmetics production.

Description

Technical Field

[0001] This invention relates to Bacillus subtilis Y32 and its applications. Background Technology

[0002] Against the backdrop of rapid development in global agriculture and animal husbandry, the demand for efficient, environmentally friendly, and sustainable production methods is increasingly urgent. The plant cultivation sector faces numerous challenges, including frequent pest and disease outbreaks, soil degradation, and environmental pollution caused by the overuse of chemical fertilizers and pesticides. At the same time, the animal husbandry industry is also plagued by high feed costs, significant pressure in animal disease control, and food safety and antibiotic resistance issues resulting from antibiotic overuse.

[0003] Soil microorganisms, especially rhizosphere microorganisms, occupy a specific ecological niche in the soil and are widely recognized as a valuable second genome for plants in green agriculture. They directly or indirectly participate in regulating the material cycle and energy flow in the soil, exhibiting high environmental adaptability and metabolic multifunctionality. They promote plant growth through various pathways, such as activating soil nutrients, producing plant hormones, releasing volatile substances, and inhibiting pathogens. Microorganisms, especially gut microorganisms, play a positive role in helping the host digest complex carbohydrates, synthesize vitamins, maintain gut health, and enhance immunity. Improving the utilization efficiency of biomass resources, enhancing feed processing performance, and promoting green waste treatment are key aspects of achieving biomass resource recycling. Summary of the Invention

[0004] The purpose of this invention is to provide a Bacillus subtilis Y32 that can produce xylanase, β-mannanase and extracellular polysaccharides, solubilize phosphorus, antagonize Botrytis cinerea, and is acid- and choline-resistant, for use in plant cultivation, animal husbandry and other fields.

[0005] The present invention adopts the following technical solution:

[0006] A type of Bacillus subtilis ( Bacillus subtilis Y32, with accession number CGMCC No. 34819, was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center, located in Beijing, China.

[0007] Furthermore, the Bacillus subtilis Y32 is capable of producing xylanase and β-mannanase.

[0008] Furthermore, the xylanase produced by Bacillus subtilis Y32 has an optimal temperature of 55℃ and an optimal pH of 6.0. Under these conditions, its half-life is 12.5 h, and its relative enzyme activity remains above 80% after 3 h of incubation, demonstrating good thermal stability. Simultaneously, this xylanase exhibits a wide pH adaptability range, with Mg... 2+ Ca2+ Mn 2+ Co 2+ Zn 2+ K + It has an activating effect on all xylanases, especially Ca. 2+ This increased the relative enzyme activity of the xylanase by 30.3%.

[0009] Furthermore, the Bacillus subtilis Y32 is capable of lysing phosphorus.

[0010] Furthermore, the Bacillus subtilis Y32 is capable of producing extracellular polysaccharides.

[0011] Furthermore, the Bacillus subtilis Y32 is able to antagonize Botrytis cinerea.

[0012] Furthermore, the Bacillus subtilis Y32 is resistant to acid and choline.

[0013] Furthermore, the Bacillus subtilis Y32 can promote the growth of cucumber and Pinellia ternata and increase their yield.

[0014] A xylanase prepared from the above-mentioned Bacillus subtilis Y32.

[0015] An application of the above-mentioned Bacillus subtilis Y32 in inhibiting diseases caused by Botrytis cinerea.

[0016] An application of the above-mentioned Bacillus subtilis Y32 in promoting plant growth.

[0017] An application of the above-mentioned Bacillus subtilis Y32 in increasing the yield of cucumber or Pinellia ternata.

[0018] An application of the above-mentioned Bacillus subtilis Y32 in improving the egg production rate of laying hens.

[0019] A chicken feed additive comprising Bacillus licheniformis KD-1 and Bacillus subtilis Y32.

[0020] In the chicken feed additive, the ratio of Bacillus licheniformis KD-1 spores to Bacillus subtilis Y32 spores is 1:1.

[0021] In the chicken feed additive, the total number of spores of Bacillus licheniformis KD-1 and Bacillus subtilis Y32 is not less than 20 billion spores / gram.

[0022] The chicken feed additive also includes β-mannanase.

[0023] A chicken feed comprising Bacillus licheniformis KD-1 and Bacillus subtilis Y32.

[0024] The chicken feed contains 1 to 4 billion Bacillus licheniformis KD-1 spores and 1 to 4 billion Bacillus subtilis Y32 spores per kilogram of feed.

[0025] The chicken feed contains 500 U of β-mannanase per kilogram.

[0026] The beneficial effects of this invention are as follows:

[0027] (1) The Bacillus subtilis Y32 of this invention can produce a variety of hydrolases such as xylanase and β-mannanase. In particular, the optimal temperature for xylanase is 55°C, and the optimal pH is 6.0. Under these conditions, the half-life reaches 12.5 h, which is higher than the thermal stability of most xylanases. This enzyme also has a wide pH range of adaptability. 2+ The relative enzyme activity of xylanase can be increased by 30.3% in the presence of ions.

[0028] (2) The Bacillus subtilis Y32 of the present invention has the function of inhibiting gray mold, with an inhibition rate of 80.0%, and can be used to treat or inhibit plant diseases caused by gray mold.

[0029] (3) The Bacillus subtilis Y32 of the present invention has the function of producing extracellular polysaccharides and can be used in the food industry to produce thickeners or in the cosmetics industry to produce moisturizing products.

[0030] (4) The Bacillus subtilis Y32 of the present invention can also be applied to plant growth, especially to increase the yield of cash crops (such as cucumbers) and Chinese medicinal materials (such as Pinellia ternata).

[0031] (5) The Bacillus subtilis Y32 of the present invention can also be used in combination with Bacillus licheniformis KD-1. In experiments on laying hens at around 500 days old, the egg production rate was significantly increased from 70.4% to 90.1%. Attached Figure Description

[0032] Figure 1 The image shows the results of culturing strain Y32 on different culture media.

[0033] Figure 1 In the table, A is the screening medium for xylanase production; B is the screening medium for β-mannanase production; C is LB medium; D is nitrogen-fixing medium; E is calcium phytate medium; F is potassium-solubilizing medium; G is siderophore-producing medium; and H is LB medium containing 10% NaCl.

[0034] Figure 2 Phylogenetic tree of strain Y32.

[0035] Figure 3 The graph shows the results of the phosphorus solubility test for strain Y32.

[0036] Figure 4 The image shows the results of strain Y32 inhibiting the growth of Botrytis cinerea.

[0037] Figure 5 This is a diagram showing the growth of strain Y32 under different pH conditions.

[0038] Figure 6 This is a diagram showing the growth of strain Y32 under different bile salt conditions.

[0039] Figure 7 The figure shows the effect of temperature on xylanase Y32XynA.

[0040] Figure 8 The figure shows the effect of xylanase Y32XynA on the thermal stability.

[0041] Figure 9 The figure shows the effect of pH on xylanase Y32XynA.

[0042] Figure 10 The figure shows the effect of metal ions on xylanase Y32XynA.

[0043] Figure 11 The figure shows the effect of strain Y32 on cucumber growth;

[0044] Figure 11 In the figures, A represents the height of cucumber plants 30 days after transplanting under different treatments; B represents the number of cucumbers per plant under different treatments; C represents the yield per plant under different treatments; ** and *** indicate significant differences from the control, p<0.01 and p<0.001, respectively.

[0045] Figure 12 The graph shows the yield of Pinellia ternata after applying strain Y32.

[0046] Figure 13 Figure showing the effect of different treatments on the egg production rate of laying hens in the late laying period;

[0047] Figure 13 In this study, CK was the control group; A was the low-dose group; B was the medium-dose group; and C was the high-dose group. Detailed Implementation

[0048] The technical solution of the present invention will be described in detail below with reference to specific embodiments and accompanying drawings.

[0049] Example 1 Screening of strains

[0050] Soil samples were obtained from a cornfield in Baoding City, Hebei Province. Xylan-degrading strains were screened using the method described by Lin Xiaohong et al. (Screening, identification and enzymatic properties of xylanase-producing strains [J]. Chinese Journal of Food Science, 2016, 16(01): 115-122.). The screening medium for xylanase-producing strains was as follows: xylan 0.5g, NH4NO3 0.5g, K2HPO4 0.2g, MgSO4·7H2O 0.05g, agar 2g, distilled water 100mL, pH 7.0.

[0051] Based on this, the selected strains were further screened using the method of Tian Geng et al. (Screening, identification and enzymatic properties of thermostable β-mannanase-producing strains [J]. Food Industry Technology, 2020, 41(19): 127-131.). The screening medium for β-mannanase-producing strains was: konjac gum 5 g / L, peptone 5 g / L, KH2PO4 1 g / L, MgSO4 0.1 g / L, natural pH.

[0052] The results showed that strain Y32 could grow on a medium in which xylan was the sole C source ( Figure 1 A), this bacterium has a visible transparent ring around it; and it is also a strain that can grow on konjac gum as the sole C source ( Figure 1 B), indicating the ability of strain Y31 to produce both xylanase and β-mannanase.

[0053] Example 2: Determination of physiological and biochemical properties of strain Y32

[0054] The morphology, Gram staining, and spores of strain Y32 were observed under a microscope. Indicators such as acidic growth, catalase reaction, proteolysis, starch hydrolysis, cellulose hydrolysis, tyrosine decomposition, D-glucose acid production, D-mannitol acid production, L-arabinose acid production, propionate utilization, citrate utilization, and nitrate reduction were measured. Gram staining was performed on bacteria cultured for 18–20 h, while VP and nitrate reduction experiments were conducted on bacterial cultures cultured for 3 days.

[0055] Nitrogen fixation capacity assay of strain Y32: The nitrogen fixation capacity of strain Y32 was determined using bacterial streak and fungal spot inoculation methods on nitrogen-fixing medium (mannitol 10 g, calcium sulfate 0.1 g, sodium chloride 0.2 g, calcium carbonate 5 g, potassium dihydrogen phosphate 0.2 g, magnesium sulfate heptahydrate 0.2 g, agar 15 g, distilled water 1000 mL). The bacteria were incubated at 28℃ for 3–7 days. Growth was observed; colony growth indicated nitrogen fixation capacity.

[0056] The ability of strain Y32 to solubilize phosphorus and produce phytase was determined by inoculating it into a calcium phytate organic phosphorus selection medium. If the strain could grow, it indicated that it possessed the ability to solubilize phosphorus and produce phytase. The calcium phytate organic phosphorus selection medium contained 10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, 1.0 g MnSO4·H2O, and 5.0 g calcium phytate (C6H6Ca6O3) per liter. 24 P6), 15g agar, pH 7.0.

[0057] The potassium-solubilizing ability of the strain was determined using the Alexander Alexandrite plate method. Strain Y32 was inoculated onto potassium-solubilizing medium (5 g / L glucose, 0.5 g / L magnesium sulfate heptahydrate, 0.1 g / L calcium carbonate, 2 g / L calcium phosphate, 2 g / L potassium feldspar powder, 0.005 g / L ferric chloride, 5 g / L bromothymol blue, and 17 g / L agar) and incubated at 28°C for 5 days. The presence and size of a yellow halo were observed and measured.

[0058] Siderogenic capacity determination: Strain Y32 was cultured at 28℃ for 5-7 days via spot inoculation. The siderogenic capacity was determined using siderogenic medium (Crazin S (CAS) 60.5 mg, cetyltrimethylammonium bromide (HDTMA) 72.9 mg, ferric chloride hexahydrate 2.645 mg, sodium dihydrogen phosphate dihydrate 295.25 mg, disodium hydrogen phosphate dodecahydrate 1213.5 mg, ammonium chloride 125 mg, potassium dihydrogen phosphate 37.5 mg, sodium chloride 62.5 mg, agar 9000 mg, distilled water 1000 mL). When the sample contained siderogenic material, a distinct yellow or orange-red transparent halo was observed in the solid sample test.

[0059] Salt tolerance test of the strain: Y32 monoclonal strains were inoculated into LB liquid medium and cultured at 30℃ and 200 r / min on a shaker until the OD of the bacterial culture was reached. 600 To achieve a concentration of 0.5, take 1 mL of culture medium and perform serial dilutions (10⁻⁵). -1 ~10 -5 ), 10 μL of each dilution was spread onto the surface of solid LB medium containing different concentrations of NaCl (0, 2%, 4%, 6%, 8%, 10%), and incubated upside down in a 30℃ incubator for 1 day to observe the growth of the strain under different salt stresses.

[0060] The results of culturing strain Y32 on LB medium are as follows: Figure 1As shown in C, the colonies are round with serrated edges, white, moist, and can be stretched into threads when picked up with a toothpick, indicating the ability to produce extracellular polysaccharides. Strain Y32 is a Gram-positive, rod-shaped bacterium capable of producing spores. It has the ability to hydrolyze cellulose, starch, and pectin, and can produce urease, protease, xylanase, β-mannanase, and phytase. It also has nitrogen-fixing capabilities. Figure 1 D), phosphorus solubility ( Figure 1 E) Potassium solubility ( Figure 1 F), Iron production capacity ( Figure 1 G), can grow in LB medium containing 10% NaCl ( Figure 1 H), whose comprehensive properties are shown in Table 1.

[0061] Table 1. Physiological and biochemical characteristics of strain Y32

[0062] .

[0063] Example 3 Identification and preservation of strain Y32

[0064] Strain Y32 was sequenced, and its 16S rDNA sequence is shown in SEQ ID No. 1. The phylogenetic tree constructed based on the 16S rDNA sequence is shown below. Figure 2 Based on comprehensive morphological, physiological and biochemical characteristics and 16S rDNA sequence analysis, strain Y32 was identified as Bacillus subtilis (Bacillus subtilis). Bacillus subtilis ).

[0065] Strain Y32 was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center, Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 34819.

[0066] Example 4 Phosphate-solubilizing ability of strain Y32

[0067] Pick a single colony of Y32 with an inoculation needle and place it into sterile LB liquid medium. Incubate at 37 °C with shaking at 180 rpm for 12 h. Dilute the bacterial culture to OD200. 600 The inoculum concentration was 0.8; 1% was inoculated into 50 mL of calcium phytate liquid culture medium (each liter of medium contained 10.0 g glucose, 0.5 g ammonium sulfate, 0.3 g NaCl, 0.3 g KCl, 0.3 g MgSO4·7H2O, 0.03 g FeSO4·7H2O, 1.0 g MnSO4·H2O, and 5.0 g calcium phytate (C6H6Ca6O)). 24Fermentation was carried out in 250 mL Erlenmeyer flasks (P6, pH 7.0) at 180 rpm and 37 ℃ for 7 days. 1 mL samples were taken every 24 h, centrifuged at 8000 rpm for 10 min, and the soluble phosphorus content and pH of the supernatant were determined using the molybdenum antimony colorimetric method at different time points.

[0068] The results are as follows Figure 3 As shown, on the second day of fermentation, Y32 affects C6H6Ca6O 24 P6 exhibits the highest solubility, at 114.74 mg / L. Figure 3 Afterward, the solubility decreased and remained stable. This strain's phosphorus-solubilizing ability was higher than that reported for most microorganisms in dissolving calcium phytate. Simultaneously, it was observed that the pH of the culture supernatant gradually decreased with prolonged culture time, reaching 5.8 on day 2, and then stabilized. It is speculated that during the growth of Y32, phytase was produced to degrade calcium phytate, and organic acids may also have been generated, causing the pH of the fermentation broth to decrease, which facilitated the dissolution of solid calcium phytate and increased the soluble phosphorus content.

[0069] Example 5: Extracellular polysaccharide production capacity of strain Y32

[0070] The Y32 strain was cultured at 37 °C for 16 h on an extracellular polysaccharide (EPS) medium (20 g sucrose, 0.2 g K2HPO4, 0.5 g KH2PO4, 10 g NaCl, 0.5 g MgSO4∙7H2O, 3 g yeast extract, 15 g agar powder, 1000 mL ddH2O). The strain surface was moist and transparent, and the colonies were stringy when picked up, indicating that the strain could produce extracellular polysaccharides.

[0071] Single colonies of Y32 were selected and cultured in LB medium at 37 °C and 180 rpm for 24 h. The culture was then transferred at 5% to an extracellular polysaccharide-producing liquid medium and cultured for another 48 h under the same conditions to obtain EPS fermentation broth. 10 mL of the culture broth was centrifuged at 10,000 rpm for 10 min, the supernatant was removed, and the cells were dried and weighed. Simultaneously, 30 mL of 95% ethanol was added to the supernatant for alcohol precipitation, and the mixture was placed in a 4 °C refrigerator overnight. After centrifugation at 10,000 rpm for 10 min, the supernatant was removed, and the precipitate was dried at 60 °C to constant weight, weighed, and ground into a powder for determining the polysaccharide content. The yield of EPS from strain Y32 was determined using the phenol-sulfuric acid method. The results showed that strain Y32 produced 1585.1 mg / L of extracellular polysaccharide after 48 h, which is higher than the extracellular polysaccharide production capacity reported by most literature. EPS (extracellular EPS) retains moisture, protects microorganisms and the environment from drought. In the food industry, EPS can be used as a natural thickener and gelling agent. In the cosmetics industry, it is often used as a moisturizer. In agriculture, it has the ability to improve soil properties and structure. These results demonstrate the potential of strain Y32 for applications in multiple fields.

[0072] Example 6: Strain Y32's ability to inhibit Botrytis cinerea

[0073] Single clones of strain Y32 were picked and cultured in LB liquid medium at 37 ℃ and 180 rpm for 12 h with shaking in an Erlenmeyer flask. Then, 10% of the culture was transferred to fermentation medium (2% yeast extract, 2.5% peptone, 0.3% dipotassium hydrogen phosphate, 3% glucose) and cultured under the same conditions for another 3 days. The fermentation broth was centrifuged at 12000 rpm for 10 min, and the supernatant was used as the test culture to evaluate the ability of the strain to inhibit Botrytis cinerea.

[0074] The mycelial block inoculation method was adopted, using a sterilized 6 mm punch to inoculate activated culture of Botrytis cinerea (…). Botrytis cinerea Cut a piece of bacterial culture from the edge of the colony and place it in the center of a PDA medium. Place three sterilized circular filter paper discs with a diameter of 6 mm at equal intervals 1.5 cm away from the center of the bacterial culture disc. Add 8 μL of sterile water and the test bacterial solution to the filter paper discs respectively. Invert the discs and incubate them in a constant temperature incubator at 28 ℃ for 3 days. Test the size of the inhibition zone.

[0075] Inhibition rate (%) = (Control colony diameter - Treated colony diameter) / (Control colony diameter) × 100. The inhibition rate of strain Y32 against *Botrytis cinerea* was calculated based on this. The results showed that (as...) Figure 4 Strain Y32 can inhibit the growth of Botrytis cinerea, with an inhibition rate of 80.0%.

[0076] Example 7 Acid resistance of strain Y32

[0077] Strain Y32 was spread onto LB medium at pH 2.0, pH 3.0, and pH 4.0, respectively, and incubated upside down at 37°C. The pH tolerance of the strain was then tested, and the results are as follows: Figure 5 As shown in the figure. The results indicate that strain Y32 can grow well in LB medium at pH 2.0, indicating that strain Y32 is resistant to strong acid and can withstand the acidic environment of the animal's stomach to reach the intestine. Furthermore, the hydrolytic enzymes produced by Y32, such as xylanase, β-mannanase, amylase, and protease, can improve the utilization rate of feed. In addition, the xylooligosaccharides and mannooligosaccharides formed after the degradation of plant hemicellulose can increase intestinal probiotics, improve the intestinal environment, and enhance animal immunity.

[0078] Example 8: Bile salt tolerance of strain Y32

[0079] Strain Y32 was cultured in LB medium containing 0.1%, 0.5%, and 1.0% porcine bile salts, and incubated upside down at 37°C. The strain's bile salt tolerance was then tested, and the results are as follows: Figure 6 As shown in the figure. The culture results indicate that strain Y32 grows well in 1.0% porcine bile salt medium, demonstrating that strain Y32 exhibits bile salt tolerance. Bile salts emulsify fats in the small intestine, aiding in fat digestion and absorption. Strain Y32 can tolerate the bactericidal and inhibitory effects of bile, thus enabling it to colonize the intestinal mucosa and exert its probiotic function.

[0080] Example 9 Enzymatic Properties Analysis of Xylosylase Produced by Strain Y32

[0081] (1) Preparation of crude enzyme solution

[0082] Single clones of strain Y32 were selected and cultured in LB medium at 37 ℃ and 180 rpm for 12 h. Then, at a 10% ratio, they were transferred to xylanase induction medium (5 g / L beech xylan, 5 g / L peptone, 1 g / L KH₂PO₄, 0.1 g / L MgSO₄, natural pH) and cultured under the same conditions for 48 h. The fermentation broth was centrifuged at 12000 rpm for 10 min, and the collected supernatant was used as the crude enzyme solution to determine the enzymatic properties of xylanase.

[0083] (2) Development of the xylose standard curve

[0084] Xylose standard curve preparation: Mix 300 μL of xylose at different concentrations with 600 μL of DNS, boil for 5 minutes, cool, and then measure the OD using a SpectraMaxmi 3x multi-functional microplate reader. 540 The absorbance of the reaction solution at 540 nm was measured, and a standard curve was plotted. The mass of D-xylose (mg) was plotted on the x-axis, and the OD value was plotted on the y-axis. 540Plot a standard curve with y as the ordinate. Each experiment included three blank control trials and three replicate trials, with the average value of the replicate trials taken.

[0085] (3) Xylanase activity assay

[0086] Xylanase activity was determined using the 3,5-dinitrosalicylic acid method. Beechwood xylan was dissolved in pH 6.0 phosphate buffer to a substrate concentration of 0.5% (mass fraction, hereinafter the same). 30 μL of appropriately diluted enzyme solution was mixed with 270 μL of substrate and incubated at 55 °C for 10 minutes. Then, 600 μL of DNS was added, mixed thoroughly, boiled for 5 minutes, and immediately cooled in ice water. The OD was then measured. 540 Absorbance. The substrate was mixed with the diluted crude enzyme solution, immediately mixed with DNS, boiled, and cooled to serve as a blank control. Each crude enzyme solution was tested in triplicate.

[0087] At a given temperature and pH, the amount of enzyme required to form 1 μmol of xylose per minute is defined as one unit of enzyme activity (U).

[0088] (4) Effect of temperature on enzyme activity

[0089] The enzyme activity of xylanase Y32XynA at pH 6.0 was determined at different temperatures (45℃, 50℃, 55℃, 60℃, 65℃, and 70℃). The highest enzyme activity was set as 100%, and the relative enzyme activity at different temperatures was calculated.

[0090] The results showed that the optimal temperature for xylanase Y32XynA was 55 ℃. The enzyme activity of Y32XynA increased with increasing temperature from 45 to 55 ℃, reaching its highest relative activity at 55 ℃. Beyond 55 ℃, the relative enzyme activity of Y32XynA gradually decreased with increasing temperature. Figure 7 ).

[0091] Xylanase was incubated in pH 6.0 buffer at 55 °C, and the residual enzyme activity was measured at different times. The unincubated enzyme activity was set as 100%. The results are as follows: Figure 8 As shown, the enzyme's half-life is 12.5 h. With prolonged incubation, the enzyme activity gradually and slowly decreases. After 3 h of incubation, the relative enzyme activity still remains above 80%, indicating that the enzyme has good thermostability. Figure 8 ).

[0092] (5) Effect of pH on enzyme activity

[0093] Beechwood xylose was dissolved in solutions of different pH values ​​(pH 4.0, pH 5.0, pH 6.0, pH 6.5, pH 7.0, pH 8.0, pH 9.0) to prepare 0.5% (w / w) substrate solutions. The Y32XynA enzyme activity in these pH buffer systems was measured at 55 °C, with the enzyme activity measured at pH 6.0 recorded as 100%. Results are as follows: Figure 9 As shown, when the pH is below 6.0, the enzyme activity of Y32XynA increases with increasing pH, reaching its maximum at pH 6.0. As the pH continues to rise, the enzyme activity gradually decreases, and at pH 9.0, the relative enzyme activity of Y32XynA drops to 20%. This indicates that the enzyme has a wide pH adaptability range.

[0094] (6) Effects of metal ions on enzyme activity

[0095] To determine the effect of metal ions on Y32XynA, diluted crude enzyme solution was taken and mixed with an equal volume of 10 mmol Mg. 2+ Ca 2+ Mn 2+ Co 2+ Cu 2+ Zn 2+ K + Mixed, with water as a control, the enzyme activity of Y32XynA was determined at pH 6.0 and 55 ℃. The enzyme activity measured without the addition of metal ions was set as 100%. Results are shown below. Figure 10 Except Cu 2+ Besides inhibiting Y32XynA, the other metal ions studied all had an activating effect on Y32XynA, especially Ca. 2+ This increased the relative enzyme activity of Y32XynA by 30.3%.

[0096] Example 10: Application of Bacillus subtilis Y32 in cucumber cultivation

[0097] (1) Preparation of spores

[0098] Single clones of strain Y32 were picked and cultured on LB medium at 37 ℃ and 180 rpm for 16 h. Then, they were transferred at a 2% ratio to sporulation medium ((NH4)2SO4 4 g / L, yeast extract 4 g / L, K2HPO4 1 g / L, MgSO4·7H2O 0.82 g / L, MnCl2 0.08 g / L, glucose 2 g / L, 0.16 g / L CaCl2·2H2O, natural pH). Fermentation was carried out in a 5 L fermenter from Shanghai Baoxing Bio-Equipment Engineering Co., Ltd. at 37 ℃ and 200 rpm with an aeration rate of 0.05 m³ / h. 3Fermentation was carried out under conditions of / h for 4 days to obtain Y32 spore fermentation broth. The fermented broth was placed in an 80 ℃ water bath for 20 min to inactivate the Y32 vegetative cells. After appropriate dilution, it was spread onto LB solid medium and incubated upside down at 37 ℃. A broth of the same dilution without high-temperature treatment was used as a control. After colonies grew, the number of spores was counted and the sporulation rate was calculated. The results showed that the sporulation rate of Y32 reached 95%, and the spore yield reached 260 million spores / mL.

[0099] (2) Preparation of Y32 spore liquid inoculum

[0100] The fermentation broth of cultured Y32 spores was centrifuged at 10,000 rpm for 10 min, and the supernatant was discarded to obtain Bacillus subtilis Y32 spores. The obtained spores were diluted with tap water to prepare a liquid inoculum of Y32 spores at a concentration of 10 billion spores / mL for use in plant cultivation.

[0101] (3) Application of Bacillus subtilis Y32 in cucumber cultivation

[0102] The prepared Y32 spore liquid inoculant was applied to cucumber planting holes at a concentration of 16 billion spores per square meter before transplanting. A control group without inoculant application was used. Twelve plants were randomly selected from each treatment. Plant height was measured 30 days after transplanting. Once the cucumbers reached approximately 25 cm in height, the number of cucumbers per plant and the yield were recorded for each treatment.

[0103] The results are as follows Figure 11 As shown, 30 days after cucumber transplanting, the average height of plants treated with Y32 spore-forming liquid inoculant reached 101.3 cm, an increase of 34.8% compared to the untreated group (CK). The application of Y32 spore-forming liquid inoculant increased the number of cucumbers by 156.0% and the yield by 192.9%. In addition, the application of the inoculant promoted cucumber growth, which advanced the harvest period by one week.

[0104] Example 11 Application of Bacillus subtilis Y32 in Pinellia ternata cultivation

[0105] The preparation of Bacillus subtilis Y32 spores and the preparation of liquid Bacillus subtilis spore inoculum are the same as in Example 10.

[0106] Two applications of Y32 spore-forming liquid inoculant were administered. The first application was before sowing of *Pinellia ternata* seeds, spraying the soil surface with a concentration of 16 billion Y32 spores per square meter, followed by soil tilling. The second application was four weeks after emergence, again spraying the leaves of *Pinellia ternata* with a concentration of 16 billion spores per square meter, followed by tap water to ensure the inoculant reached the soil surface. Other water and fertilizer management was the same as the control group (CK). Results showed that applying 16 billion Y32 spores per square meter to the soil resulted in a *Pinellia ternata* yield of 419.4 kg dry weight per mu (approximately 667 square meters), a 26.6% increase compared to the control. Figure 12 It is 0.475 m from two treatments.2 The dried Pinellia ternata samples are shown in the diagram, where CK represents the untreated sample and Y32 represents the sample treated with Y32 inoculant.

[0107] Example 12 Application of Bacillus licheniformis KD-1 and Bacillus subtilis Y32 in laying hen breeding

[0108] A single colony of Bacillus licheniformis KD-1 (CGMCC No. 18964) was inoculated into 25 mL of LB medium and cultured at 180 rpm and 37 ℃ for 14 h. The culture was then transferred at a 6% inoculum to sporulation medium (corn starch 30 g / L, yeast extract 25 g / L, K₂HPO₄ 2 g / L, KH₂PO₄ 1 g / L, ZnSO₄ 0.1 g / L, MnSO₄ 2 mg / L, CuSO₄ 2 mg / L, Na₂MoO₄ 1 mg / L). Fermentation was carried out in a 5 L fermenter from Shanghai Baoxing Bio-Equipment Engineering Co., Ltd. at 37 ℃ and 200 rpm with an aeration rate of 0.05 m³ / h. 3 Fermentation was carried out under the condition of / h for 4 days to obtain Bacillus licheniformis KD-1 fermentation broth. The fermentation broth was placed in an 80 ℃ water bath for 20 min to inactivate the Bacillus licheniformis KD-1 vegetative cells. After appropriate dilution, it was spread on LB solid medium and incubated upside down at 37 ℃. After colony growth, the number of spores was counted. The results showed that the spore yield of Bacillus licheniformis KD-1 was 160 million spores / mL. The Bacillus licheniformis KD-1 fermentation broth was centrifuged at 10000 rpm for 10 min to collect the spores, dried at room temperature, and prepared into a solid inoculum of Bacillus licheniformis KD-1 with 20 billion spores / g using maltodextrin as an excipient.

[0109] The preparation of Bacillus subtilis Y32 spores was the same as in Example 10. The obtained spores were dried at room temperature, and maltodextrin was used as an excipient to prepare a solid Bacillus subtilis Y32 inoculum of 20 billion spores / g. Bacillus licheniformis KD-1 and Bacillus subtilis Y32 solid inoculum were mixed at a mass ratio of 1:1 to prepare a compound inoculum.

[0110] Three hundred and eighty Hy-Line Brown laying hens at 70 weeks of age and with consistent feeding conditions and similar weights were selected as the research subjects. They were randomly divided into four groups of 96 hens each (six replicates per group, with 16 hens per replicate, for an eight-week experimental period). β-Mannanase was a commercially available product. The basal feed for the laying hens consisted of corn:soybean meal:lime powder:premix at a mass ratio of 63:24:10:3. The experiment included four treatments: the basal feed group, Group A (low-dose group) with 0.1 g of compound microbial agent and 500 U of β-mannanase per kilogram of feed, Group B (medium-dose group) with 0.2 g of compound microbial agent and 500 U of β-mannanase per kilogram of feed, and Group C (high-dose group) with 0.4 g of compound microbial agent and 500 U of β-mannanase per kilogram of feed. Daily group and replicate counts of normal egg production were recorded; broken eggs and shell-less eggs were not counted within the normal egg production range.

[0111] The results showed that adding Bacillus licheniformis KD-1, Bacillus subtilis Y32, and β-mannanase to the feed of laying hens improved the egg production rate of Hy-Line Brown laying hens in the late laying period. Figure 13 Among them, the egg production rate of the medium-dose group was significantly higher than that of the control group, increasing from 70.4% to 90.1%, an increase of 19.7%; although the low-dose and high-dose groups also increased by 3.3% and 8.3% respectively compared with the control, they did not reach a significant level. The amount of β-mannanase added was consistent among the three treatment groups, so it can be considered that the differences among the three treatments were caused by the different amounts of Bacillus licheniformis and Bacillus subtilis added. The difference in egg production rate between group A and group B was significant, indicating that the different amounts of Bacillus licheniformis and Bacillus subtilis added led to a significant difference in egg production rate. Figure 13 The results indicate that the simultaneous addition of Bacillus licheniformis and Bacillus subtilis to the diet can effectively improve the egg production rate of laying hens in the late laying period. The optimal addition amount is 2 billion Bacillus licheniformis KD-1 spores and 2 billion Bacillus subtilis Y32 spores per kilogram of feed.

Claims

1. A type of Bacillus subtilis ( Bacillus subtilis Y32, characterized in that, The accession number is CGMCCNo.34819.

2. The Bacillus subtilis Y32 according to claim 1, characterized in that, It can produce xylanase, β-mannanase and extracellular polysaccharides.

3. The Bacillus subtilis Y32 according to claim 1, characterized in that, It is resistant to acid and choline.

4. The Bacillus subtilis Y32 according to claim 1, characterized in that, It can antagonize gray mold.

5. The Bacillus subtilis Y32 according to claim 1, characterized in that, It can promote the growth of cucumbers and pinellia and increase yield.

6. The application of Bacillus subtilis Y32 as described in claim 1 in inhibiting diseases caused by Botrytis cinerea.

7. The application of Bacillus subtilis Y32 as described in claim 1 in promoting the growth of cucumber and Pinellia ternata.

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