Application of bacillus subtilis Z-14 in prevention of spoilage of silage corn straws

Abstract

The invention discloses application of bacillus subtilis Z-14 in prevention of spoilage of silage corn straws, and relates to the technical field of whole-plant corn straw silage. According to the invention, a bacillus subtilis strain Z-14 is screened, and the bacillus subtilis strain Z-14 has significant broad-spectrum inhibition of pathogenic fungi and activity of pathogenic bacteria, secretes protease, amylase, cellulase and other digestive enzymes, effectively inhibits the number of bacteria, fungi and escherichia coli in the silage corn straw, accelerates the reduction of the pH value of the silage corn straw, and improves the yield of the silage corn straw. The content of aflatoxin B1 in the silage corn is obviously reduced, the silage quality is improved, a foundation is laid for effectively inhibiting the spoilage of the silage corn straw, and the silage microbial additive has huge application potential.

Description

Technical Field

[0001] The invention relates to the technical field of whole-plant corn stalk silage, and more specifically to a Bacillus subtilis Z-14 and an application thereof in preventing silage corn stalk from spoiling and deteriorating. Background Art

[0002] Silage is one of the main feed resources for ruminants, and the quality of its fermentation will directly affect the growth and development of ruminants and the economic benefits of farmers. In the process of preparing silage, there is a lot of residual air inside, and the number of lactic acid bacteria attached to the leaves of grass grass is small in the natural state, and the aerobic respiration time is long, making it difficult to form an anaerobic environment in a short period of time, resulting in the growth of undesirable microorganisms such as yeast and mold, and serious loss of nutrients, which greatly affects the quality of feed and silage effect. At the same time, the contaminated and corrupted silage produces a large amount of mycotoxins. After being ingested by ruminants for a long time, it will not only inhibit the immune function of ruminants, reduce their production capacity or even cause death, seriously affecting the healthy development of animal husbandry, but also mycotoxins and their metabolites will be transmitted to humans through meat and dairy products, ultimately posing a threat to human health.

[0003] The appropriate amount of microbial agents added to the silage fermentation has a positive effect on accelerating the silage process and improving the nutritional quality of silage. Bacillus subtilis is an aerobic bacterium. As a microbial additive, it multiplies in large numbers in the early stage of silage fermentation, rapidly consumes oxygen in the raw materials, and makes the silage straw quickly enter an anaerobic state, inhibiting the proliferation of aerobic microorganisms such as fungi; at the same time, the rapidly formed anaerobic environment promotes the proliferation of lactic acid bacteria, produces a large amount of organic acids, and rapidly reduces the pH, further inhibiting the proliferation of pathogenic microorganisms; Bacillus subtilis produces a large amount of secondary metabolites during the proliferation process, which has significant antagonistic activity against a variety of pathogens, and the secreted digestive enzymes promote the decomposition of macromolecular nutrients and improve the quality of silage. In addition, its activity can also increase the temperature of the silage system and inhibit the growth and reproduction of other aerobic spoilage bacteria, and has great application prospects in animal husbandry production.

[0004] Therefore, screening a strain of Bacillus subtilis that can prevent silage corn stalks from spoiling and deteriorating is an urgent problem that needs to be solved by those skilled in the art. Summary of the invention

[0005] In view of this, the present invention is proposed.

[0006] One of the purposes of the present invention is to provide uses of Bacillus subtilis Z-14.

[0007] The second object of the present invention is to provide a preparation prepared by Bacillus subtilis Z-14 for antagonizing pathogenic bacteria and pathogenic fungi.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] The first aspect of the embodiment of the present invention provides the use of the Bacillus subtilis Z-14, wherein the Bacillus subtilis (Bacillus subtilis) is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration, with a deposit number of CGMCC No. 20821, a deposit date of September 25, 2020, and a classification name of Bacillus subtilis. The use includes:

[0010] 1) Application of Bacillus subtilis Z-14 in preventing silage corn stalks from spoiling, reducing the content of aflatoxin B1 in silage corn, antagonizing pathogenic bacteria and pathogenic fungi, and improving silage quality;

[0011] 2) Application of Bacillus subtilis Z-14 in large-scale fermentation to produce protease, amylase and cellulase.

[0012] Preferably, the pathogenic bacteria are Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis; the pathogenic fungi are Helminthosporium, Fusarium solani and Helminthosporium curvatum.

[0013] The second aspect of the embodiment of the present invention provides a preparation for preventing silage corn stalks from spoiling and antagonizing pathogenic bacteria and pathogenic fungi, comprising the Bacillus subtilis Z-14

[0014] An enzyme preparation, comprising a sterile supernatant of Bacillus subtilis Z-14, wherein the sterile supernatant contains amylase, cellulase and protease

[0015] Preferably, the preparation process of the sterile supernatant of Bacillus subtilis Z-14 is as follows:

[0016] 1) Inoculate Bacillus subtilis Z-14 onto NA plates, incubate at 37°C overnight, pick out single colonies with good growth conditions and inoculate into NB medium, incubate at 37°C overnight on a shaker to obtain fermentation broth;

[0017] 2) The fermentation broth was transferred to fresh NB medium at a 10% inoculation rate, and cultured in a shaker at 37°C for 48 h; centrifuged at 10,000 rpm for 5 min, and the supernatant was recovered and filtered through a hydrophilic microporous filter membrane with a pore size of 0.22 μm. The recovered filtrate was the sterile supernatant.

[0018] It can be seen from the above technical scheme that compared with the prior art, the present invention has discovered a new function of Bacillus subtilis strain Z-14, which has significant broad-spectrum inhibitory activity against pathogenic fungi and pathogenic bacteria, secretes digestive enzymes such as protease, amylase, cellulase, etc., effectively inhibits the number of bacteria, fungi and Escherichia coli in silage corn stalks, accelerates the decrease in pH of silage corn stalks, significantly reduces the content of aflatoxin B1 in silage corn, improves silage quality, and lays a foundation for effectively inhibiting the corruption and deterioration of silage corn stalks. It has great application potential as a microbial additive for silage feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The attached figure shows the antagonistic activity of the sterile supernatant of Bacillus subtilis strain Z-14 against pathogenic fungi; A to C are Helminthosporium, Fusarium solani and Helminthosporium flexudata, respectively; a is a fresh NB medium control, and b is the fermentation supernatant of strain Z-14.

[0021] Figure 2 The attached figure shows the inhibitory effect of the sterile supernatant of Bacillus subtilis strain Z-14 on pathogenic bacteria; A to C are Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis, respectively; a is a fresh NB culture medium control, and b is the fermentation supernatant of Z-14 strain.

[0022] Figure 3 The attached figure shows the activity analysis of Bacillus subtilis synthetic bioenzymes; A, B, and C are the protease, amylase, and cellulase activities of Bacillus subtilis strain Z-14, respectively.

[0023] Figure 4 The attached figure shows the characteristic detection of corn stover fermented by Bacillus subtilis strain Z-14. DETAILED DESCRIPTION

[0024] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. 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.

[0025] Materials used in the examples:

[0026] Culture medium

[0027] PDA culture medium: Weigh 200.0 g of peeled potatoes, chop them, heat and boil them, then filter the filtrate with 4 layers of gauze to recover the filtrate, add 20.0 g of glucose and 20 g of agar, add deionized water to make the volume to 1000 mL, set the pH to natural, divide into 250 mL Erlenmeyer flasks, 100 mL in each bottle, and steam sterilize at 115°C for 30 min.

[0028] NA medium: peptone 10g; beef extract 5g; NaCl 5g; agar 20g, add deionized water to make up to 1000mL, pH 7.0-7.5, steam sterilize at 121℃ for 15min after packaging.

[0029] NB medium: peptone 10g, beef extract 5g, NaCl 5g, add deionized water to 1000mL, pH 7.0-7.5, steam sterilize at 121℃ for 15min after packaging.

[0030] Milk agar medium: 500 mL skim milk, 500 mL deionized water, 20 g agar, natural pH, steam sterilize at 115°C for 30 min after packaging.

[0031] CMC medium: CMC-Na 20 g, (NH 4 ) 2 SO 4 20 g, KH 2 PO 4 1.0 g, NaCl 0.5 g, MgSO 4 7H 2 O0.5g, Congo red 0.2g, distilled water 1000mL, agar 20g, natural pH, steam sterilize at 121℃ for 15min after packaging.

[0032] Amylase identification medium: soluble starch 2g, beef extract 5g, peptone 10g, NaCl5g, agar powder 20g, distilled water 1000mL, pH 7.0-7.5, steam sterilize at 115℃ for 30min after packaging.

[0033] EMB medium: peptone 10g, lactose 10g, K 2 HPO 4 ·3H 2 O 2g, eosin 0.4g, methylene blue 0.065g, distilled water 1000mL, agar 20g, pH 7.0-7.5, steam sterilize at 115℃ for 30min after packaging.

[0034] MRS medium: peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g, glucose 20g, Tween 80 1mL, sodium acetate 5g, K 2 HPO 4 ·3H 2 O 2g, MgSO 4 7H 2 O 0.58g, MnSO 4 ·H 2 O 0.25g, agar 20g, distilled water 1000mL, pH 6.2-6.6, steam sterilize at 115℃ for 30min after packaging.

[0035] Data analysis

[0036] The basic data were sorted using Excel 2010, and SPSS18.0 statistical software was used for one-way analysis of variance. The Duncan method was used for analysis of the significance of differences between the groups, with a significance level of 0.05.

[0037] Bacillus subtilis strain Z-14 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 25, 2020, with the deposit number CGMCC No.20821. The deposit address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named Bacillus subtilis:

[0038] Example 1 Cultivation of Bacillus subtilis strain Z-14 and preparation of sterile supernatant

[0039] Bacillus subtilis strain Z-14 was isolated and preserved by our laboratory. The strain Z-14 preserved at -80°C was inoculated onto NA plates by streaking method and inverted at 37°C overnight. Single colonies with good growth were picked and inoculated into NB medium, incubated at 37°C overnight, and the fermentation liquid was transferred to fresh NB medium at 10% inoculation volume, and incubated at 37°C for 48 hours. The fermentation liquid was centrifuged at 10000rpm for 5 minutes, and the cells and supernatant were recovered respectively. The cells were suspended with an appropriate amount of sterile water, the viable bacterial content was detected by plate count method, and the cells were diluted with sterile water to the required viable bacterial content for use. The supernatant was filtered with a hydrophilic microporous filter membrane with a pore size of 0.22μm, and the recovered filtrate was the sterile supernatant.

[0040] Example 2 Detection of the Antagonistic Activity of Bacillus subtilis Strain Z-14 Sterile Supernatant against Pathogenic Fungi

[0041] The pathogenic Bipolaris maydis, Fusarium proliferat um and Bipolaris papendorfii were inoculated onto PDA slants and cultured in a 25°C incubator for 7 days. An appropriate amount of sterile water was injected into the cultured fungal slants, and the fungal spores were scraped into the sterile water with a bamboo stick to prepare a fungal spore suspension. Heat 100 mL of PDA culture medium to melt and cool to about 40°C, add 5 mL of fungal spore suspension to the PDA culture medium, mix quickly and pour into plates, pour 6 plates for every 100 mL of culture medium, and after the plates solidify, use a sterile hole puncher to evenly punch 4 holes with a diameter of 7 mm on each plate, add 50 μL of sterile supernatant to each of the 3 holes, and add 50 μL of fresh NB culture medium to the 4th hole as a negative control, place the plates upright in a 25°C constant temperature incubator for culture, and after all the added liquid is immersed in the culture medium, invert the plates and culture at a constant temperature for 5 days, observe the formation of inhibition zones, and thereby detect the antagonistic activity of the sterile supernatant of strain Z-14 against pathogenic fungi.

[0042] Results Figure 1 , indicating that the sterile supernatant has significant antagonistic activity against the three indicator strains tested, namely, Helminthosporium, Fusarium solani and Helminthosporium flexuvolum. The antagonistic activity against Helminthosporium flexuvolum was the most significant, with an inhibition zone diameter of 3.6 cm, showing strong antibacterial activity; the antagonistic activity against Helminthosporium flexuvolum was the weakest, with an inhibition zone diameter of 1.7 cm, still showing strong antibacterial activity; the antagonistic activity against Fusarium solani was between Helminthosporium flexuvolum and Helminthosporium flexuvolum, with an inhibition zone diameter of 1.8 cm, showing significant antagonistic effect. The sterile supernatant of Z-14 has significant broad-spectrum antagonistic activity against pathogenic fungi.

[0043] Example 3 Detection of the antagonistic activity of the sterile supernatant of Bacillus subtilis strain Z-14 against pathogenic bacteria

[0044] Pathogenic bacterial indicator strains Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis were streaked onto NA medium plates and incubated at 37°C overnight with inversion. Single colonies with good growth were picked and inoculated into NB medium and incubated at 37°C overnight with shaking. Heat 100mL NA solid culture medium to melt and cool to about 40℃, add 5mL bacterial culture solution, shake and mix, then quickly pour the plate. After the plate solidifies, use a sterile hole puncher to evenly punch 4 holes with a diameter of 7mm on each plate, add 50μL sterile supernatant to each of the 3 holes, and add 50μL fresh NB culture medium to the 4th hole as a negative control. Place the plate upright in a 37℃ constant temperature incubator for culture. After all the added liquid is immersed in the culture medium, invert the plate for 24h and observe the formation of the inhibition zone to detect the antagonistic activity of the sterile supernatant of strain Z-14 against pathogenic bacteria. Results are shown in Figure 2 The sterile supernatant had significant antagonistic activity against the three indicator strains of Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis. The antagonistic activity against Staphylococcus aureus was the most significant, with an inhibition zone diameter of 2.8 cm, showing extremely strong antibacterial activity; the antagonistic activity against Shigella dysenteriae was the weakest, with an inhibition zone diameter of 1.8 cm, still showing strong antibacterial activity; the antagonistic activity against Salmonella enteritidis was between Staphylococcus aureus and Shigella dysenteriae, with an inhibition zone diameter of 2.3 cm, showing significant antagonistic effect. Z-14 sterile supernatant had significant broad-spectrum antagonistic activity against pathogenic bacteria.

[0045] Example 4 Detection of digestive enzyme activity produced by Bacillus subtilis strain Z-14

[0046] Z-14 was activated by streaking on NA plates, and single colonies were picked up with sterilized bamboo sticks and inoculated into milk agar medium, CMC medium, and amylase identification medium plates by cross-streaking method. Four "crosses" were drawn on each plate, and three parallel groups were made for each medium. Cultured at 37°C for 48 hours, and the formation of hydrolysis circles was observed. Protease activity was analyzed by directly observing whether there were hydrolysis circles around the bacterial moss in the milk agar medium. 10mL of 1.0mg / mL Congo red solution was added to the CMC medium plate that had been cultured for 24 hours and stained for 1 hour. The Congo red solution was poured out and soaked in 10mL of 1.0mol / L NaCl solution for 1 hour for decolorization. Whether there were hydrolysis circles around the bacterial moss was observed to analyze cellulase activity. 50-fold diluted Lugol's iodine solution was added to the amylase identification medium plate that had been cultured for 24 hours, and whether there were starch hydrolysis circles around the bacterial moss was observed to analyze amylase activity. Results are shown in Figure 3, Z-14 has significant protease, amylase and cellulase activities. In milk agar medium, CMC medium and amylase identification medium, Z-14 showed obvious transparent bands, indicating that strain Z-14 has strong protease, amylase and cellulase synthesis and secretion capabilities, and the secreted biological enzymes have a strong inhibitory effect on pathogens. At the same time, it has a strong digestion ability for macromolecular nutrients, promoting the digestion and decomposition of proteins, starch, cellulose and other substances.

[0047] Determination of Cellulase, Amylase and Protease Activities of Bacillus subtilis Strain Z-14

[0048] Drawing of standard curve

[0049] Take 6 clean test tubes, numbered 1 to 6, and add 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mL of 1 mg / mL glucose standard solution to the test tubes in turn, and make up to 2.0 mL with distilled water. After shaking, add 1.5 mL of 3,5-dinitrosalicylic acid (DNS) to each test tube, boil in a water bath for 5 minutes, and make up to 20 mL with distilled water. After fully mixing, use distilled water as a control and measure the absorbance value at 540 nm. Each group of experiments was repeated 3 times in parallel, and the standard curve was drawn using Origin 8.0 software after taking the average value.

[0050] Cellulase activity assay

[0051] Take 1.5mL of pH 5.0 0.05mol / L citric acid buffer (containing 0.51% sodium carboxymethyl cellulose) and place in a 50℃ constant temperature water bath for 8min, then add 0.5mL of sterile supernatant, mix well and place in a 50℃ water bath for 30min, then add 1.5mL of DNS reagent, shake the test tube and place in a boiling water bath for 5min, after the test tube cools, dilute to 20mL with distilled water, measure the absorbance at 540nm, and check the corresponding glucose content in the glucose standard curve to calculate the enzyme activity unit. Make 3 groups of parallels for each sample and calculate the average value. Enzyme activity calculation formula: X = (B×v×1000) / (0.5×t) (this method is the DNS method, which is essentially to determine the content of reducing sugar in the solution), B: glucose content, v: total volume, t: enzymolysis time.

[0052] Amylase activity assay

[0053] Take 0.9mL of 0.5% soluble starch solution and preheat at 65℃ for 10min, add 0.1mL of sterile supernatant, react at 65℃ for 10min, add 1.5mL of DNS solution, boil in water bath for 5min, cool rapidly in cold water, and measure the OD value at 540nm with a spectrophotometer. According to the glucose standard curve, calculate the amount of reducing sugar released, and thus calculate the amylase activity. The enzyme activity unit (U) is defined as: the amount of enzyme required to release 1μmol of reducing sugar per minute in the reaction system is 1 enzyme activity unit.

[0054] Protease activity assay

[0055] Drawing of standard curve

[0056] Take 7 clean test tubes, numbered 0 to 6, and add 0, 1, 2, 3, 4, 5, and 6 mL of 100 μg / mL standard tyrosine solution to the test tubes in turn, and make up to 10 mL with distilled water. Take 1 mL of tyrosine solution of different concentrations (do 3 replicates for each concentration), add 5 mL of 0.4 mol / L Na2CO3 solution, 1 mL of Folin phenol reagent (Shanghai Yuanye Biological Co., Ltd.), place in a 40°C water bath for color development for 20 minutes, and measure the OD value with a spectrophotometer at a wavelength of 680 nm. Draw a standard curve with the OD value as the ordinate and the tyrosine concentration as the abscissa. This curve should be a straight line passing through the zero point, and then calculate the K value [the tyrosine concentration (μg / mL) when the OD in the standard curve is 1].

[0057] Add 1mL of sterile supernatant to the test tube and preheat it in a 30℃ constant temperature water bath for 2min, add 1mL of 2% casein solution preheated in a 30℃ constant temperature water bath for 5min, shake well, react in a 30℃ constant temperature water bath for 10min, add 2.0mL of trichloroacetic acid, shake well, take out and let it stand for 10min, filter, take 1.0mL of filtrate and measure the absorbance value according to the standard curve method, do 3 replicates, and take another sterile inactivated supernatant as a blank control. Record the absorbance value and calculate the enzyme activity unit. Definition of neutral protease activity: Under pH 7.5 and 30℃ temperature conditions, 1μg of tyrosine produced by hydrolysis of casein per minute is defined as a protease activity unit (U). Protease activity X=(4 / l0)×K×OD. In the formula: 4 is 1mL taken out of 4mL reaction solution, 10 is the reaction time of 10min, and K is the tyrosine concentration (μg / mL) equivalent to OD of 1 in the standard curve.

[0058] The results showed that the protease activity of the sterile supernatant of Z-14 was 137.6 U / mL, the amylase activity was 68.3 U / mL, and the cellulase activity was 45.9 U / mL.

[0059] Example 5 Experimental Design of Fermentation of Whole-plant Corn by Bacillus subtilis Strain Z-14

[0060] The whole corn plants were from the corn planting area of ​​the experimental farm of Hebei Agricultural University. The whole corn plants were harvested when the corn entered the late milky stage and early waxy stage (when the corn milk line exceeded 2 / 3), crushed by a crusher to 1-2 cm, adjusted to a suitable moisture content of 75%), mixed and packed into silage bags (40cm×50cm, 24 silks). The experiment was divided into 5 treatments, namely the blank control group CK0 without the addition of bacterial agents; the treatment group T0 with the addition of lactic acid bacteria, the bacterial agent was commercially available, and was a mixture of Lactobacillus plantarum and Lactobacillus buchneri in a ratio of 1:1, with an addition amount of 1.0×10 5 cfu / g; added 0.2×10 5 cfu / g Bacillus subtilis Z-14 inoculant treatment group T1; 1.0×10 5 cfu / g Z-14 treatment group T2; 5.0×10 5 cfu / g Z-14 treatment group T3. 30 bags of silage were prepared, 6 bags for each treatment, each bag was 2.0 kg, vacuumed with a vacuum packaging machine and anaerobically fermented at 28°C. The number of major microorganisms, pH and fermentation characteristics were measured on the 0th, 1st, 3rd, 6th and 15th day of fermentation.

[0061] Fermentation characteristic index detection method

[0062] Take 10g of silage, add 90mL of distilled water and grind it with a tissue grinder for 1min. After filtering through 4 layers of gauze, filter with qualitative filter paper to obtain the extract. The pH value is determined using a convenient pH meter. The content of aflatoxin B1 is detected by enzyme-linked immunosorbent assay. The number of microorganisms is determined by plate counting method. The filtrate is serially diluted by 10-fold dilution method to determine the number of lactic acid bacteria, aerobic bacteria, Escherichia coli, and fungi. Among them, lactic acid bacteria are counted in MRS medium at 37℃ for 48h; aerobic bacteria are counted in NA medium at 37℃ for 24h; Escherichia coli is counted in EMB medium at 37℃ for 48h; fungi are counted in PDA medium at 37℃ for 5d.

[0063] Results Figure 4 The effect of Z-14 on the silage effect of corn straw was detected using silage bags. The changes in the number of aerobic bacteria, fungi, lactic acid bacteria and Escherichia coli in silage corn straw were detected at 0, 1, 3, 6 and 15 days under anaerobic fermentation conditions at 28°C. The pH value and aflatoxin B1 content of silage corn straw were also detected.

[0064] On day 0, the bacterial content in group T3 was the highest, significantly higher than that in other treatment groups. The bacterial content in group T2 was significantly higher than that in group T1, but lower than that in group T3, indicating that the addition of Z-14 bacterial agent led to an increase in the content of aerobic bacteria. There was no significant difference between groups T1, CK0, and T0, indicating that the addition of 0.2×10 5cfu / g Z-14 agent and lactic acid bacteria preparation had no significant effect on the content of aerobic bacteria. Thereafter, as the fermentation time prolonged, the content of aerobic bacteria in each treatment group gradually decreased. On the 1st, 3rd, 6th, and 15th days of fermentation, the aerobic bacteria content in the T3 group was the highest, and the T2 group was higher than the T1 group, but lower than the T3 group. At each detection period, the CK0 group and the T0 group were significantly lower than the treatment group with the addition of Z-14 agent. With the increase in the amount of the agent added, the aerobic bacteria content of the different treatment groups with the addition of Z-14 agent gradually increased, indicating that the Z-14 agent increased the number of aerobic bacteria and could effectively survive and play a role in the straw. There was no significant difference between the CK0 group and the T0 group with the addition of lactic acid bacteria preparation at each detection period, indicating that the addition of lactic acid bacteria preparation had no significant effect on the number of aerobic bacteria.

[0065] As the fermentation time prolonged, the number of fungi in each treatment group gradually decreased. On the 0th day of fermentation, there was no significant difference in the number of fungi in each treatment group. On the 1st day of fermentation, there was no significant difference in the number of fungi in each treatment group, but in the treatment group with the addition of Z-14 agent, the number of fungi gradually decreased with the increase in the amount of additives, indicating that Z-14 agent began to inhibit the proliferation of fungi. On the 3rd day of fermentation, the number of fungi in the T3 group was significantly lower than that in the other treatment groups, and there was no significant difference in the treatment groups except T3. However, the number of fungi in the T2 group was lower than that in the other treatments except T3, but the difference was not significant. There was no significant difference between the T0 group and the CK0 group. On the 15th day of fermentation, there was no significant difference in the number of fungi between the T0 group and the CK0 group, indicating that the addition of lactic acid bacteria had no significant effect on the number of fungi. The number of fungi in each treatment group with added Z-14 fungus agent was significantly lower than that in T0 and CK0 groups. The number of fungi in T2 group was higher than that in T3 group, but there was no significant difference between the two. It was lower than that in T1 group and the difference between the two was significant, indicating that Z-14 fungus agent significantly inhibited the proliferation of fungi, and the effect became more significant with the increase of the amount of additive.

[0066] On the 0th day of fermentation, except for the T0 group, the number of lactic acid bacteria in other treatment groups was low and there was no significant difference. The lactic acid bacteria content in the T0 group was high and significantly higher than that in other treatments, indicating that the addition of lactic acid bacteria preparations significantly increased the number of lactic acid bacteria in the T0 group. On the 1st day of fermentation, the number of lactic acid bacteria in the T0 group was still the highest, and the number of lactic acid bacteria in other treatment groups increased, indicating that anaerobic fermentation increased the number of lactic acid bacteria. With the increase in the amount of Z-14 bacterial agent additive, the number of lactic acid bacteria gradually increased, indicating that the addition of Z-14 promoted the proliferation of lactic acid bacteria. The number of lactic acid bacteria in the Z-14 bacterial agent treatment group was higher than that in the CK0 group, but there was no significant difference between the groups. On the 3rd day of fermentation, there was little difference between the T0 group and the T3 group, but it was higher than the T2 group and the T1 group, the T2 group was higher than the T1 group, and there was no significant difference between the T0 group and the treatment groups with the addition of Z-14 bacterial agent. The T0 group and the T3 group were significantly higher than the CK0 group, and the T1 group and the T2 group were higher than the CK0 group, but there was no significant difference between the three. On the 15th day of fermentation, the number of lactic acid bacteria in the T0 and T3 groups was the highest, and there was no significant difference between them, but both were significantly higher than the CK0 and T1 groups, while there was no significant difference between the CK0 and T1 groups. The T2 group was higher than the CK0 and T1 groups, and lower than the T0 and T3 groups, but there was no significant difference between it and the other four treatments.

[0067] On the 0th day of fermentation, there was no significant difference in the number of E. coli in each treatment group. As the fermentation time increased, the number of E. coli in each treatment group gradually decreased, indicating that anaerobic fermentation reduced the number of E. coli in silage. On the 1st and 3rd days of fermentation, there was no significant difference in the number of E. coli in each treatment group, but the number of E. coli in each treatment group with Z-14 bacterial agent was lower than that in the CK0 group. At the same time, as the amount of additives increased, the number of E. coli gradually decreased, indicating that Z-14 bacterial agent inhibited the number of E. coli and was positively correlated with the amount of additives. On the 6th and 15th days of fermentation, the number of E. coli in each treatment group with Z-14 bacterial agent was lower than that in the CK0 group. At the same time, as the amount of additives increased, the number of E. coli gradually decreased. The number of E. coli in the T3 group was significantly lower than that in the CK0 group. There was no significant difference in the number of E. coli between the other treatments except the T3 group.

[0068] On the 0th day of fermentation, the pH values ​​of all treatment groups were neutral, with no significant differences. As the fermentation time prolonged, the pH values ​​of all treatment groups gradually decreased. On the 1st day of fermentation, the pH value of the T0 group was the lowest, which was significantly different from the CK0 group, indicating that the addition of lactic acid bacteria significantly accelerated the decrease in pH, and there was no significant difference from the treatment group with the addition of Z-14 bacteria. The addition of Z-14 bacteria also accelerated the decrease in pH, but there was no significant difference from the CK0 group. As the amount of Z-14 bacteria additive increased, the rate of pH decrease gradually increased. On the 3rd and 6th days of fermentation, the pH values ​​of all treatment groups continued to decrease, and the differences and significance between different treatments were similar to those on the 1st day of fermentation. On the 15th day of fermentation, the pH continued to decrease, but there was no significant difference between the treatment groups, indicating that the pH reached equilibrium and dropped to the lowest value.

[0069] On the 0th and 1st day of fermentation, there was no significant difference in the content of aflatoxin B1 in each treatment group. With the extension of fermentation time, the content of aflatoxin B1 in CK0 group gradually increased. On the 3rd day of fermentation, there was no significant difference in the content of aflatoxin B1 in T0 group and CK0 group, indicating that the addition of lactic acid bacteria had no significant effect on the content of aflatoxin B1. The content of aflatoxin B1 in each treatment group with the addition of Z-14 bacteria was significantly lower than that in CK0 group. At the same time, with the increase of the amount of Z-14 bacteria additive, the content of aflatoxin B1 decreased successively, but the difference among the three treatments was not significant. On the 6th day of fermentation, there was no significant difference between CK0 group, T0 group and T1 group, indicating that the addition of a small amount of Z-14 bacteria additive had no significant effect on the content of aflatoxin B1, but with the increase of the amount of Z-14 bacteria additive, the content of aflatoxin B1 decreased significantly. The content of aflatoxin B1 in T2 group was significantly lower than that in CK0 group, T0 group and T1 group, but significantly higher than that in T3 group. The content of aflatoxin B1 in T3 group was the lowest. After 15 days of fermentation, there was no significant difference between the CK0 group and the T0 group, and the two treatments were significantly higher than the three treatments with the addition of Z-14 inoculant. The T1 group was significantly lower than the CK0 group and the T0 group. The T2 group and the T3 group were significantly lower than the T1 group, and the T3 group was lower than the T2 group, but there was no significant difference between the two.

[0070] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0071] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of Bacillus subtilis Z-14, wherein the Bacillus subtilis is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No. 20821 and a deposit date of September 25, 2020, and is classified and named Bacillus subtilis, characterized in that: The uses include: 1) Application of Bacillus subtilis Z-14 in preventing silage corn stalks from spoiling, reducing the content of aflatoxin B1 in silage corn, antagonizing pathogenic bacteria and pathogenic fungi, and improving silage quality; 2) Application of Bacillus subtilis Z-14 in large-scale fermentation to produce protease, amylase and cellulase.

2. The use according to claim 2, characterized in that: The pathogenic bacteria are Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis; the pathogenic fungi are Helminthosporium, Fusarium solani and Helminthosporium curvatum.

3. A preparation for preventing silage corn stalks from spoiling and antagonizing pathogenic bacteria and pathogenic fungi, characterized in that: The invention comprises the Bacillus subtilis Z-14 according to claim 1.

4. An enzyme preparation, characterized in that The enzyme preparation comprises the sterile supernatant of Bacillus subtilis Z-14 according to claim 1, wherein the sterile supernatant contains amylase, cellulase and protease.

5. The enzyme preparation according to claim 4, characterized in that The preparation process of sterile supernatant of Bacillus subtilis Z-14 is as follows: 1) Inoculate Bacillus subtilis Z-14 onto NA plates, incubate at 37°C overnight, pick out single colonies with good growth conditions and inoculate into NB medium, incubate at 37°C overnight on a shaker to obtain fermentation broth; 2) The fermentation broth was transferred to fresh NB medium at a 10% inoculation rate, and cultured in a shaker at 37°C for 48 h; centrifuged at 10,000 rpm for 5 min, and the supernatant was recovered and filtered through a hydrophilic microporous filter membrane with a pore size of 0.22 μm. The recovered filtrate was the sterile supernatant.

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