Application of bacillus subtilis Z-14 in improving aerobic stability of silage

Abstract

The invention discloses application of bacillus subtilis Z-14 in improving aerobic stability of silage, and relates to the technical field of silage corn fermentation, and the bacillus subtilis Z-14 has the preservation number of CGMCC (China General Microbiological Culture Collection Center) No.20821. The bacillus subtilis strain Z-14 disclosed by the invention has antagonistic activity on various pathogenic fungi and pathogenic bacteria which are easy to pollute in an ensiling process, so that the silage is prevented from spoilage after a cellar is opened; meanwhile, digestive enzymes generated by the strain can reduce the content of acid detergent fibers and improve the digestion and absorption rate of the silage; furthermore, the strain can reduce the pH value of the silage and improve the stability of the quality of the silage, and the acting effect is lasting. The strain Z-14 is applied to silage processing, and a material basis and a theoretical basis are provided for efficient and safe production of silage.

Description

Technical Field

[0001] The invention relates to the technical field of silage corn fermentation, and more specifically to the application of Bacillus subtilis Z-14 in improving the aerobic stability of silage. Background Art

[0002] Silage is a simple, economical and roughage processing method that can better preserve the nutritional value of raw materials and prolong the storage time. The quality of silage fermentation directly affects the growth and development of ruminants and the economic benefits of farmers. At the same time, silage can also improve the quality of feed and maintain the fresh and juicy characteristics of green feed for a long time. After silage, the feed is soft and juicy, with a fragrant sour taste and good palatability. It can increase the appetite of ruminants and solve the problem of lack of fresh green feed in autumn and winter. Silage of agricultural and sideline products such as straw, wheat bran, and bran can also solve the problem of ecological environment pollution caused by on-site abandonment or non-fuel incineration.

[0003] However, after the silage is opened, it is fully exposed to the air, aerobic microorganisms multiply, pH rises, and aerobic corruption occurs easily, which seriously affects the quality and nutritional value of the feed. At the same time, aerobic corruption of silage will produce a large amount of mycotoxins, which will not only inhibit the immune function of ruminants, reduce their production capacity or even cause death after being ingested by ruminants for a long time, seriously affecting the healthy development of animal husbandry, but also mycotoxins and their metabolites will be transmitted to humans through meat and dairy products, posing a threat to human health.

[0004] Therefore, how to inhibit aerobic corruption and toxin production after silage pit opening is a problem that technical personnel in this field urgently need to solve. Summary of the invention

[0005] In view of this, the present invention provides the use of Bacillus subtilis Z-14 in improving the aerobic stability of silage.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] Application of Bacillus subtilis Z-14 in improving the aerobic stability of silage, wherein the preservation number of the Bacillus subtilis Z-14 is CGMCC No.20821.

[0008] Preferably, the addition amount of Bacillus subtilis Z-14 is 1.0×10 5 cfu / g~5.0×10 5 cfu / g.

[0009] Another object of the present invention is to provide an application of a bacterial agent containing Bacillus subtilis Z-14 in improving the aerobic stability of silage, wherein the deposit number of the Bacillus subtilis Z-14 is CGMCC No.20821.

[0010] Another object of the present invention is to provide a method for preparing silage, comprising the following steps: harvesting whole corn plants, crushing, adjusting the moisture content to 70-80%, placing the corn in a silage container, and mixing the corn in a silage container at a temperature of 1.0×10 5 cfu / g~5.0×10 5 cfu / g, add the above-mentioned Bacillus subtilis Z-14, seal it and ferment it anaerobically at 25-30°C for 50-70 days.

[0011] Preferably, the period for harvesting the whole corn plant is from the late milky stage to the early waxy stage of the corn.

[0012] Preferably, the crushing standard is 1 to 2 cm.

[0013] Another object of the present invention is to provide silage prepared by the above method.

[0014] Preferably, after the anaerobic fermentation is completed, the silage has a shelf life of 15 days after being opened.

[0015] Beneficial effects: Bacillus subtilis is non-toxic, non-residue, non-polluting, and environmentally friendly. Its application as a microbial additive for silage is in line with the development direction of modern animal husbandry. After the silage corn stalks are unbagged, aerobic conditions are conducive to the proliferation and metabolism of Bacillus subtilis, thereby producing broad-spectrum antibacterial active substances, inhibiting the growth and reproduction of pathogenic bacteria and fungi, and improving the aerobic stability of silage. Bacillus subtilis can also produce a variety of extracellular enzymes such as amylase and cellulase to decompose polysaccharides into monosaccharides, providing sufficient nutrients for the survival and proliferation of Bacillus subtilis. While improving the nutritional quality of corn stalks, it further enhances the function of the strain and reduces the corruption and deterioration of silage.

[0016] The Bacillus subtilis strain Z-14 of the present invention has antagonistic activity against a variety of pathogenic fungi and pathogenic bacteria that are easily contaminated during the silage process, preventing the silage from spoiling after the silage is opened; at the same time, the digestive enzyme produced by the strain can reduce the content of acidic detergent fiber and improve the digestion and absorption rate of the silage; further, the strain can also reduce the pH value of the silage, improve the stability of the silage quality, and the effect is long-lasting. The strain Z-14 is applied to the processing of silage, providing a material basis and theoretical basis for the efficient and safe production of silage. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] 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.

[0018] Figure 1 This is the antagonistic activity of Bacillus subtilis strain Z-14 against pathogenic fungi, from left to right: Fusarium oxysporum, Aspergillus flavus, and Fusarium graminearum.

[0019] Figure 2 The graph shows the inhibitory effect of Bacillus subtilis strain Z-14 on pathogenic bacteria, wherein A is Escherichia coli, B is Shigella dysenteriae, C is Staphylococcus aureus, and D is Salmonella enteritidis.

[0020] Figure 3 Analysis of digestive enzyme activity of Bacillus subtilis strain Z-14.

[0021] Figure 4 The effect of Bacillus subtilis strain Z-14 on the total bacterial count in corn straw silage.

[0022] Figure 5 This is the effect of Bacillus subtilis strain Z-14 on the total number of Escherichia coli, a representative of contaminating bacteria in corn straw silage.

[0023] Figure 6 The effect of Bacillus subtilis strain Z-14 on the number of fungi in corn straw silage.

[0024] Figure 7 The effect of Bacillus subtilis strain Z-14 on the number of lactic acid bacteria in corn straw silage.

[0025] Figure 8 The effect of Bacillus subtilis strain Z-14 on pH value in corn straw silage.

[0026] Fig. 9 The effect of Bacillus subtilis strain Z-14 on the acid detergent fiber content in corn straw silage. DETAILED DESCRIPTION

[0027] 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.

[0028] Biological materials: Bacillus subtilis strain Z-14, named as Bacillus subtilis according to its classification, was deposited in the General Microbiology Center of China Microorganism Culture Collection on September 25, 2020, with the deposit number CGMCC No. 20821; the deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0029] Culture medium:

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] Amylase identification medium: soluble starch 2g, beef extract 5g, peptone 10g, NaCl 5g, agar powder 20g, distilled water 1000mL, pH 7.0-7.5, steam sterilize at 115℃ for 30min after packaging.

[0036] EMB medium: peptone 10g, lactose 10g, K 2 HPO 4 ·3H2 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.

[0037] 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.

[0038] Example 1

[0039] Cultivation of Bacillus subtilis strain Z-14 and preparation of sterile supernatant

[0040] The strain Z-14 stored at -80℃ was inoculated onto NA plates by streaking method and incubated at 37℃ overnight. A single colony with good growth was picked and inoculated into NB medium, incubated at 37℃ overnight in a shaker, and the fermentation liquid was transferred to fresh NB medium at 10% inoculation volume, and incubated at 37℃ for 48h. The fermentation liquid was centrifuged at 10000rpm for 5min, and the bacteria and supernatant were recovered respectively. The bacteria were suspended with an appropriate amount of sterile water, the viable bacteria content was detected by plate counting method, and the bacteria were diluted with sterile water to the required viable bacteria content for use. The supernatant was filtered with a hydrophilic microporous filter membrane with a pore size of 0.22μm, and the filtrate was recovered to obtain a sterile supernatant.

[0041] Example 2

[0042] Antibacterial Activity Detection of Bacillus subtilis Strain Z-14

[0043] 1. Antagonism against pathogenic fungi

[0044] Fusarium oxysporum Fusarium oxysporum )、 Aspergillus flavus ( Aspergillus flavus ) and a sickle bacteria( Fusarium graminearum) is a pathogenic fungus that is easily infected during silage.Inoculate the pathogenic fungi Fusarium oxysporum, Aspergillus flavus and Fusarium graminearum into the PDA slant, and culture them in a 25°C incubator for 7 days. Inject an appropriate amount of sterile water into the cultured fungal slant, and use a bamboo stick to scrape the fungal spores into the sterile water 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. After the plates solidify, inoculate the Z-14 strain onto the plates, invert the plates and culture them at a constant temperature for 7 days, and observe the formation of inhibition zones (see Appendix). Figure 1 ) was used to detect the antagonistic activity of Z-14 strain against pathogenic fungi.

[0045] The results showed that Bacillus subtilis strain Z-14 had significant antagonistic activity against the three indicator strains tested, Fusarium oxysporum, Aspergillus flavus and Fusarium graminearum. Among them, the antagonistic activity against Fusarium oxysporum was the most significant, with an inhibition zone diameter of 2.8 cm, showing extremely strong antibacterial activity; the antagonistic activity against Fusarium graminearum was the weakest, with an inhibition zone diameter of 2.0 cm, but still had strong antibacterial activity; the antagonistic activity against Aspergillus flavus was between Fusarium oxysporum and Fusarium graminearum, with an inhibition zone diameter of 2.4 cm, and the antagonistic effect was significant.

[0046] 2. Antagonism of pathogenic bacteria

[0047] Escherichia coli, Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis are pathogenic bacteria that are easily transmitted during the silage process and cause harm to the animal digestive tract. The bacterial pathogenic indicator strains of Escherichia coli, Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis were streaked onto NA medium plates and incubated inverted at 37°C overnight. Single colonies with good growth conditions were picked and inoculated into NB medium and incubated at 37°C overnight with constant temperature shaking. Heat 100 mL of NA solid medium to melt and cool to about 40°C, add 5 mL of bacterial culture solution, shake and mix, then quickly invert the plate. After the plate solidifies, 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 of Bacillus subtilis strain Z-14 to each of the 3 holes, and add 50 μL of fresh NB medium to the 4th hole as a negative control. Place the plate upright in a 37°C constant temperature incubator for incubation. After all the added liquid is immersed in the culture medium, invert the plate for incubation for 24 hours, and observe the formation of the inhibition zone (see Appendix). Figure 2 ) was used to detect the antagonistic activity of Z-14 strain against pathogenic bacteria.

[0048] The results showed that Bacillus subtilis strain Z-14 had significant antagonistic activity against the four indicator strains tested, namely Escherichia coli, Shigella dysenteriae, Staphylococcus aureus and Salmonella enteritidis. Among them, 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 Escherichia coli and Shigella dysenteriae was the weakest, with an inhibition zone diameter of 1.8 cm, but still had strong antibacterial activity; the inhibition zone diameter against Salmonella enteritidis was 2.3 cm, with a significant antagonistic effect.

[0049] Example 3

[0050] Detection of digestive enzyme activity of Bacillus subtilis strain Z-14

[0051] Use a sterile cork puncher to evenly punch 4 holes with a diameter of 7 mm on the milk agar medium, CMC medium, and amylase identification medium plates, add 50 μL of Z-14 sterile supernatant to each hole, and make 3 parallel groups for each culture medium. Incubate in a 37°C constant temperature incubator for 24 hours to observe whether there is a hydrolysis circle. Analyze the protease activity by directly observing whether there is a hydrolysis circle around the milk agar medium hole. Add 10mL of 1.0mg / mL Congo red solution to the CMC medium plate that has been cultured for 24 hours and stain for 1 hour. Pour out the Congo red solution and soak it in 10mL of 1.0mol / L NaCl solution for 1 hour to decolorize. Observe whether there is a hydrolysis circle around the hole to analyze the cellulase activity. Add 50-fold diluted Lugol's iodine solution to the amylase identification medium plate that has been cultured for 24 hours, and observe whether there is a starch hydrolysis circle around the hole to analyze the amylase activity.

[0052] The activities of protease, amylase and cellulase of strain Z-14 were tested by punching method. The results are shown in the attached figure. Figure 3 As shown, strain Z-14 has significant protease, amylase and cellulase activities. In milk agar medium, CMC medium and amylase identification medium, the sterile supernatant of Z-14 showed obvious transparent circles with diameters of 1.8 cm, 3.1 cm and 2.5 cm, respectively, indicating that strain Z-14 has strong protease, amylase and cellulase 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 nutrients such as protein, starch, and cellulose.

[0053] Example 4

[0054] Application of Bacillus subtilis strain Z-14 in whole-plant corn silage fermentation experiment

[0055] 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 and early waxy stages (when the corn milk line exceeded 2 / 3), crushed to 1-2 cm by a crusher, and the moisture content was adjusted to 70%-80%. After mixing, they were packed into silage bags (40 cm×50 cm, 24 silks). The experiment was divided into five treatments, namely, the blank control group CK0 without the addition of bacterial agents; the treatment group T0 with the addition of lactic acid bacteria, which 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; adding 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, and vacuum packaging machine was used to evacuate and anaerobically ferment at 25-30℃ for 50-70 days, then the bags were opened and placed. The number of major microorganisms, pH and acid detergent fiber content were measured on the 0th, 3rd, 6th, 9th, 12th and 15th days after the bags were opened.

[0056] 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 acid detergent fiber content is determined using an ANKOM automatic fiber analyzer. The number of microorganisms is determined by the plate count method. The filtrate is serially diluted by the 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.

[0057] Excel 2010 was used to organize the basic data, SPSS 18.0 statistical software was used for one-way analysis of variance, and the Duncan method was used for significance analysis of differences among groups, with a significance level of 0.05.

[0058] The results are attached Figure 4 As shown in the figure, on the 0th day after bag opening, the aerobic bacteria content in group T3 was the highest, significantly higher than that in other treatment groups, and that 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 bacterial content; there was no significant difference between groups T1, CK0, and T0, indicating that the addition of 0.2×10 5cfu / g Z-14 bacterial agent and lactic acid bacteria preparation had no significant effect on the bacterial content. Thereafter, as the bag opening time prolonged, the bacterial content of each treatment group increased significantly. On the 3rd, 6th, and 9th days after bag opening, the bacterial content of group T3 was the lowest, and group T2 was lower than group T1, but higher than group T3, indicating that the Z-14 bacterial agent inhibited the growth of bacteria and ensured the quality of feed. After 12 days of bag opening, there was no significant difference in the bacterial content of each group, indicating that the Z-14 bacterial agent had a significant effect on inhibiting bacterial proliferation in the short term (9 days), but after a long time of bag opening (12 days), the effect decreased significantly until it disappeared. There was no significant difference between the T0 group and the control group with the addition of lactic acid bacteria preparation, indicating that its inhibitory effect on bacterial proliferation was not significant.

[0059] As attached Figure 5 As shown in the figure, on the 0th day after bag opening, the number of E. coli in group T0 was lower than that in group CK0, indicating that the addition of lactic acid bacteria had a certain inhibitory effect on E. coli, but the difference between the two was not significant. The number of E. coli in group T1 was lower than that in group CK0 and group T0, indicating that the addition of Z-14 bacteria had a further inhibitory effect on E. coli, but the difference was still not significant. Groups T2 and T3 were significantly lower than group T1, and T3 was lower than T2. ​​With the increase in the amount of Z-14 bacteria added, the number of E. coli gradually decreased, indicating that Z-14 bacteria had a significant inhibitory effect on E. coli. As the bag opening time increased, the number of E. coli in each treatment group increased significantly, and lactic acid bacteria had no significant effect on the number of E. coli. In the first 12 days after bag opening, as the amount of Z-14 additive increased, the number of E. coli decreased successively, and the T3 group was significantly lower than the CK0 group, indicating that Z-14 had a significant inhibitory effect on E. coli. On the 15th day after bag opening, there was no significant difference in the number of E. coli among the treatment groups, indicating that the inhibitory effect of Z-14 on E. coli was weakened and eventually disappeared after long-term bag opening and exposure to aerobic environment.

[0060] As attached Figure 6 As shown, on the 0th day after bagging, the fungal content in group T3 was the lowest, indicating that the addition of Z-14 significantly inhibited the proliferation of fungi. The fungal content in group T2 was lower than that in group T1, but higher than that in group T3, indicating that with the increase in the dosage of Z-14, the inhibitory effect on fungi gradually increased, and the effect was significant. As the bagging time prolonged, the fungal content in each treatment group increased significantly. After adding Z-14, the number of fungi was significantly lower than that in group CK0, among which the number of fungi in group T3 was the lowest. The number of fungi in group T2 was higher than that in group T3 but lower than that in group T1. After 12 days of bagging, the inhibitory effect of Z-14 on fungi gradually weakened, but it was still lower than that in the control group, indicating that the addition of Z-14 was effective in inhibiting the proliferation of fungi in silage straw. There was no significant difference between group T0 and group CK0, indicating that the addition of lactic acid bacteria had no significant inhibitory effect on fungi after bagging of silage corn straw.

[0061] As attached Figure 7As shown in the figure, on the 0th day after bag opening, the number of lactic acid bacteria in the T0 group was significantly higher than that in the CK0 group, and also higher than that in the treatment group with the addition of Z-14 bacteria, indicating that the addition of lactic acid bacteria significantly increased the number of lactic acid bacteria in silage corn straw. However, there was no significant difference between the four treatments with the addition of bacteria. With the increase in the amount of Z-14 bacteria added, the number of lactic acid bacteria also increased, indicating that the addition of Z-14 bacteria promoted the proliferation of lactic acid bacteria, but there was no significant difference between the treatments. With the extension of the bag opening time, the number of lactic acid bacteria in each treatment group gradually decreased, and at the same time, there was no significant difference between the treatment groups, indicating that the aerobic environment after bag opening is not conducive to the growth of lactic acid bacteria, and the effect of the addition of bacteria on the number of lactic acid bacteria quickly disappeared.

[0062] As attached Figure 8 As shown in the figure, on the 0th day after bag opening, the lowest pH value of group T3 was 3.42, and the highest pH value of group T1 was 3.58, with no significant difference among the treatment groups. With the extension of bag opening time, the pH of each treatment group gradually increased, among which the pH of group T0 was significantly lower than that of group CK0 at all times of the test, indicating that the addition of lactic acid bacteria significantly reduced the pH value of silage corn and improved the stability of silage quality. At each test period, with the increase of Z-14 addition, the pH value gradually decreased, and the pH value of group T3 was significantly lower than that of groups T1, T2 and CK0. 15 days after bag opening, the pH of group T3 was still significantly lower than that of groups CK0 and T0, indicating that Z-14 significantly reduced the pH of silage straw, improved the stability of silage quality, and the effect was long-lasting.

[0063] As attached Fig. 9 As shown in the figure, on the 0th day after bag opening, the acid detergent fiber content of group T0 was lower than that of group CK0, but the difference was not significant. With the increase of the amount of Z-14 added, the acid detergent fiber content gradually decreased, and the T3 group was the lowest, with no significant difference from group T2, and significant difference from other groups, indicating that the addition of Z-14 promoted the reduction of acid detergent fiber content, promoted the decomposition of nutrients, and was beneficial to the absorption of feeding animals. With the increase of the number of days after bag opening, the acid detergent fiber content of each treatment group gradually decreased, but the decrease at this time does not mean the improvement of nutritional quality. It may be that after aerobic exposure, the growth of miscellaneous bacteria promoted the decomposition and utilization of nutrients. With the extension of bag opening time, the effect of Z-14 bacterial agent gradually weakened. On the 6th day after bag opening, there was no significant difference among the groups.

[0064] 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.

[0065] 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. Application of Bacillus subtilis Z-14 in improving the aerobic stability of silage, characterized in that: The deposit number of the Bacillus subtilis Z-14 is CGMCC No.20821.

2. The use according to claim 1, characterized in that: The addition amount of Bacillus subtilis Z-14 is 1.0×10 5 cfu / g~5.0×10 5 cfu / g.

3. The use of a bacterial agent containing Bacillus subtilis Z-14 in improving the aerobic stability of silage, characterized in that: The deposit number of the Bacillus subtilis Z-14 is CGMCC No.20821.

4. A method for preparing silage, characterized in that: The following steps are involved: Harvest the whole corn plant, crush it, adjust the moisture content to 70% to 80%, put it into silage containers, and store it in 1.0×10 5 cfu / g~5.0×10 5 cfu / g, add the Bacillus subtilis Z-14 of claim 1, and ferment anaerobicly at 25-30° C. for 50-70 days after sealing.

5. The method for preparing silage according to claim 4, characterized in that: The period for harvesting the whole corn plant is from the late milky stage to the early waxy stage of the corn.

6. The method for preparing silage according to claim 4, characterized in that: The standard of the crushing is 1 to 2 cm.

7. A silage, characterized in that: Prepared by the method described in any one of claims 4 to 6.

8. The silage according to claim 7, characterized in that After the anaerobic fermentation is completed, the shelf life after opening is 15 days.

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