Bacillus licheniformis JSF-9 and complex microbial inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus licheniformis JSF-9 and a complex microbial inoculant and application thereof.The strain JSF-9 is obtained by being separated from healthy cattle rectum contents by a research group, and it is detected that the strain has the good beta-1, 4-glucanase producing capacity, and the beta-1, 4-glucanase producing capacity is high. The antibacterial effect on staphylococcus aureus, salmonella typhimurium and shigella flexneri is achieved; the strain has a good bagasse fermentation capability, for example, after the strain, bacillus altitudinis MLF-1 and bacillus safensis MLL-5 are prepared into a mixed microbial inoculum, the bagasse fermentation capability is better, and verification shows that the complex microbial inoculum can effectively inhibit the reproduction of harmful bacteria, so that the preservation period of fermented feed is prolonged, and the yield of the fermented feed is improved. Animal feeding experiments find that the nutritional value and the disease resistance of the complex microbial inoculant fermented bagasse are remarkably improved, 10-15% of the complex microbial inoculant fermented bagasse is added into basic ration, and the cost-reducing and effect-increasing effects are achieved for feeding Jersey cattle.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to Bacillus licheniformis JSF-9, its compound bacterial agent and application. Background Art

[0002] Sugarcane is the main raw material for producing white granulated sugar in southern China and is also one of the main sugar crops in China. How to effectively utilize sugarcane bagasse as waste from white granulated sugar production is the focus of research in our region. Currently, the utilization of sugarcane bagasse is mostly used for preparing silage feed, organic fertilizer, and even for preparing beverages and other fields. There are also many microorganisms used in the field of preparing silage feed, and the most commonly used one is lactic acid bacteria. Lactic acid bacteria are not only a kind of fermenting bacteria but also a kind of probiotic that can effectively regulate the animal intestine. There are also reports in the prior art on the research of using Bacillus for fermenting sugarcane bagasse. However, some Bacillus will change the animal intestinal flora environment when applied to animals, resulting in animal diarrhea. Therefore, when we screen Bacillus, we need to pay more attention to its antibacterial performance against intestinal pathogens to achieve the purpose of effectively inhibiting the growth of intestinal pathogens. In addition, Bacillus also has a strong ability to decompose cellulose, which provides a new technical route for effectively utilizing sugarcane bagasse. Therefore, it is necessary for us to screen and verify the cellulose enzyme decomposition ability, select Bacillus suitable for fermenting sugarcane bagasse substrate, and improve the utilization rate of sugarcane bagasse by microorganisms.

[0003] Therefore, we need to conduct a large number of screenings and verifications on microorganisms, develop probiotics suitable for its fermentation for sugarcane bagasse substrate, produce sugarcane bagasse fermented feed, and verify the feasibility of this feed in feeding animals. Summary of the Invention

[0004] In view of the above, it is necessary to verify the antibacterial property and cellulose enzyme degradation ability, screen suitable Bacillus, develop microorganisms that can effectively ferment sugarcane bagasse substrate, provide theoretical support for the production of sugarcane bagasse fermented feed, and preliminarily verify the feasibility of sugarcane bagasse fermented feed in feeding animals.

[0005] To achieve the above object, the present invention has screened a new strain: Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9, and its taxonomic name is: Bacillus paralicheniformis JSF-9, the Chinese taxonomic name is: Bacillus licheniformis JSF-9, and the preservation number is GDMCC NO: 65786; this strain is preserved in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025.

[0006] The present invention also includes a compound bacterial agent containing the above-mentioned Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9.

[0007] Further, the compound bacterial agent further comprises Bacillus altitudinis ( Bacillus altitudini s) MLF-1 and / or Bacillus safensis ( Bacillus safensis ) MLL-5; the Bacillus altitudinis ( Bacillus altitudini s) MLF-1, its taxonomic name is: Bacillus altitudinis MLF-1, the Chinese taxonomic name is: Bacillus altitudinis MLF-1, and the preservation number is GDMCC NO: 65789; this strain is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025; the Bacillus safensis ( Bacillus safensis ) MLL-5, its taxonomic name is: Bacillus safensis MLL-5, the Chinese taxonomic name is: Bacillus safensis MLL-5, and the preservation number is GDMCC NO: 65790; this strain is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is February 10, 2025.

[0008] Further, the compound bacterial agent is prepared by mixing Bacillus altitudinis ( Bacillus altitudini s) MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 and Bacillus safensis ( Bacillus safensis ) MLL-5 in a volume ratio of (1-2):(2-6):(1-3).

[0009] Further, the compound bacterial agent is prepared by mixing Bacillus altitudinis ( Bacillus altitudini s) MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 and Bacillus safensis ( Bacillus safensis ) MLL-5 in a volume ratio of 1:6:3.

[0010] The present invention also includes the application of the Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 or the compound bacterial agent in the preparation of bagasse fermented feed.

[0011] The present invention also includes the application of the Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 in the production of β-1,4-glucanase.

[0012] The present invention also includes the application of the Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 in the preparation of an intestinal pathogen bacteriostatic agent, and the intestinal pathogen is Staphylococcus aureus ( Staphylococcus aureus ), Salmonella typhimurium ( Salmonella typhimurium.) and / or Shigella flexneri( Shigella flexneri ).

[0013] The present invention also includes a fermented feed containing the Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 or the composite microbial agent. The preparation method of the fermented feed is as follows: inoculate the Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 or the composite microbial agent into bagasse at an inoculation amount of 100 ml / kg and ferment for 30 days to obtain the fermented feed.

[0014] The present invention also includes the application of the fermented feed in feeding Jersey cows.

[0015] The present invention has the following beneficial effects: 1. The strain JSF-9 of the present invention is isolated from healthy rectal contents by the research group. After detection, this strain has good β-1,4-glucanase production ability and has antibacterial effects on Staphylococcus aureus, Salmonella typhimurium and / or Shigella flexneri; this strain has good fermentation ability for bagasse. For example, after preparing a composite microbial agent with this strain, Bacillus altitudinis MLF-1 and Bacillus safensis MLL-5, the fermentation ability for bagasse is better. And through intestinal flora verification, it is found that this composite microbial agent can effectively inhibit the reproduction of harmful bacteria, thereby extending the shelf life of the fermented feed. After feeding the fermented feed prepared with this composite microbial agent to Jersey cows, it is found that: the bagasse fermented by the composite microbial agent has significantly improved both nutritional value and disease resistance. Adding 10%-15% to the basal diet plays a role in reducing costs and increasing efficiency in feeding Jersey cows.

[0016] 2. The present application also conducts an orthogonal experiment on the composite microbial agent to obtain the optimal volume ratio of the composite microbial agent. Using this composite microbial agent for co-fermentation of bagasse further improves the utilization rate of the feed. The research on this strain, composite microbial agent and fermentation method provides good support and technical means for solving the waste of sucrose processing enterprises. Description of the Drawings

[0017] Figure 1 It is the colony morphology diagram of strain MLF-1.

[0018] Figure 2 It is the colony morphology diagram of strain JSF-9.

[0019] Figure 3 It is the colony morphology diagram of strain MLL-5.

[0020] Figure 4 It is the decolorization result of strain JSF-9 on Congo red-stained carboxymethyl cellulose sodium screening medium.

[0021] Biological Material Deposit Information

[0022] The strain information preserved in this application is: Bacillus altitudinis ( Bacillus altitudini s) MLF-1, and its taxonomic name is: Bacillus altitudinis MLF-1, the Chinese taxonomic name is: Bacillus altitudinis MLF-1, and the preservation number is GDMCC NO: 65789; this strain is preserved in the Guangdong Provincial Culture Collection of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025.

[0023] The strain information preserved in this application is: Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9, and its taxonomic name is: Bacillus paralicheniformis JSF-9, the Chinese taxonomic name is: Bacillus licheniformis JSF-9, and the preservation number is GDMCC NO: 65786; this strain is preserved in the Guangdong Provincial Culture Collection of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025.

[0024] The strain information preserved in this application is: Bacillus safensis ( Bacillus safensis ) MLL-5, and its taxonomic name is: Bacillus safensis MLL-5, the Chinese taxonomic name is: Bacillus safensis MLL-5, and the preservation number is GDMCC NO: 65790; this strain is preserved in the Guangdong Provincial Culture Collection of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is February 10, 2025. Detailed implementation manners

[0025] All features disclosed in this specification, or steps in all methods or processes disclosed, except for mutually exclusive features and / or steps, can be combined in any manner.

[0026] Any feature disclosed in this specification (including any additional claims, abstract) is, unless otherwise stated, just an example of a series of equivalent or similar features.

[0027] Example 1

[0028] This example is for the isolation and identification of strains.

[0029] Isolation and purification of strains: A total of 67 samples of healthy bovine rumen contents and rectal contents were collected, placed in sterile cryotubes containing glycerol, the bottle mouths were wrapped with sealing film, and then placed in a liquid nitrogen tank and taken back to the laboratory for storage at -80 °C for later use. The rumen contents stored at -80 °C were thawed at low temperature in a sterile operating table and diluted 10-fold gradient in sterile PBS, and shaken well (mixed with a vortex mixer) to form a 1:10 sample homogenate. Another sterile pipette or micropipette tip was taken, and according to the above operation sequence, 10-fold increasing sample homogenates were made. Each time the dilution increased, a sterile pipette or tip was changed. 100 μl of 3-4 appropriate gradient diluents were respectively taken and spread on the surface of NA agar medium. After spreading, the plates were left standing to allow the inoculum to be completely absorbed by the medium, then the petri dishes were inverted and cultured at 39 °C. After the colonies grew out, colonies with different morphologies were picked and purified by the streaking method (2-3 times), and stored using magnetic bead strain preservation tubes. A total of 256 strains were purified. The purified strains were inoculated on the surface of sodium carboxymethyl cellulose medium plates and cultured at 39 °C for 18-24 h. After staining with 0.1% congo red solution for 15 min, the staining solution was discarded, and then an appropriate amount of 1 mol / L NaCl solution was added for decolorization for 30 min. Strains producing cellulase were determined according to the ratio (D / d) of the diameter of the transparent circle (D) to the diameter of the colony (d), with 3 replicates for each strain. A total of 15 strains producing cellulase were screened out. Strains with a large ratio of the size of the transparent circle to the size of the colony were screened, and the enzyme production conditions were optimized. The specific results are shown in Table 1.

[0030]

[0031] Three strains with a large D / d ratio were selected from Table 1 for strain identification.

[0032] (1) Identification of strain MLF-1: ① Morphological identification is as Figure 1 shown: The MLF-1 strain was inoculated on an LB plate medium and cultured for 24 h. The colonies were round, white, with a diameter of 1.5-3.5 mm, irregular edges, thick and moist, and easy to pick. ② After extracting the strain DNA and sequencing, molecular identification was carried out. The DNA sequence of the strain was amplified, and the 16S region was amplified by PCR and sequenced. The primers used were the forward sequence 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ and the reverse sequence 1492R: 5’-TACGGCTACCTTGTTACGACTT-3’. After sequencing the PCR product, the obtained 16S sequence was as shown in Sequence Listing SEQ ID NO: 1. After BLAST alignment and additional measurement of the housekeeping gene, it was determined that the strain was closely related to Bacillus altitudinis and, combined with morphological identification, the strain was classified and named as Bacillus altitudinis .

[0033] (2)Identification of strain JSF-9: ① Morphological identification is as shown in Figure 2 : The strain JSF-9 was inoculated on an LB plate medium and cultured for 24 h. The colony was round, milky white, with a smooth surface and regular edges; ② After extracting the strain DNA and sequencing, molecular identification was carried out. The DNA sequence of the strain was amplified, and the 16S region was amplified by PCR and sequenced. The primers used were the forward sequence 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ and the reverse sequence 1492R: 5’-TACGGCTACCTTGTTACGACTT-3’. After sequencing the PCR product, the obtained 16S sequence was as shown in Sequence Listing SEQ ID NO: 2. After BLAST alignment and additional measurement of the housekeeping gene, it was determined that this strain had a relatively close genetic relationship with Bacillus paralicheniformis . Combining morphological identification, this strain was classified and named as Bacillus paralicheniformis .

[0034] (3)Identification of strain MLL-5: ① Morphological identification is as shown in Figure 3 : The strain MLL-5 was inoculated on an LB plate medium and cultured for 24 h. The colony was round, yellowish white, with a smooth surface and regular edges; ② After extracting the strain DNA and sequencing, molecular identification was carried out. The DNA sequence of the strain was amplified, and the 16S region was amplified by PCR and sequenced. The primers used were the forward sequence 27F: 5’-AGAGTTTGATCCTGGCTCAG-3’ and the reverse sequence 1492R: 5’-TACGGCTACCTTGTTACGACTT-3’. After sequencing the PCR product, the obtained 16S sequence was as shown in Sequence Listing SEQ ID NO: 3. After BLAST alignment and additional measurement of the housekeeping gene, it was determined that this strain had a relatively close genetic relationship with Bacillus safensis . Combining morphological identification, this strain was classified and named as Bacillus safensis .

[0035] Example 2

[0036] This example is for the determination of the enzyme production situation and the optimal enzyme production conditions of strain JSF-9.

[0037] (1)Seed liquid medium: Peptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, distilled water 1 L, natural pH, sterilized at 121 °C for 20 min.

[0038] (2)Liquid fermentation medium: Bagasse 50 g, peptone 10.0 g, sodium chloride 10.0 g, distilled water 1 L, natural pH, sterilized at 121 °C for 20 min.

[0039] (3)Inoculate strain JSF-9 into the seed liquid medium and culture it with shaking at 39 °C and 160 r / min until OD 600It was 0.6 to obtain the seed liquid.

[0040] (4) The seed liquid was inoculated into 250 ml Erlenmeyer flasks containing 100 ml of liquid fermentation medium at inoculation amounts of 3%, 5%, 7%, and 9% (V / V) for shake flask fermentation. Bagasse was used as the inducer to induce the strain to secrete cellulase. The shaking speed of the shaker was 160 r / min, and fermentation was carried out at 39 °C for 2 d, 3 d, 4 d, and 5 d. The fermented broth was centrifuged at 4 °C and 6000 r / min for 10 min, and the supernatant was taken as the crude enzyme solution. The enzyme production of strain JSF-9 was finally obtained as Figure 4 shown in the figure: The figure shows that strain JSF-9 can produce a clear zone on the Congo red-stained carboxymethyl cellulose sodium screening medium, indicating that the strain can produce β-1,4-glucanase. The specific enzyme production is shown in Table 2.

[0041]

[0042] As can be seen from Table 2, for strain JSF-9, at an inoculation amount of 9% and shake flask fermentation for 3 d, the best enzyme production effect of endo-β-1,4-glucanase was 2546.29 U / ml; at an inoculation amount of 9% and shake flask fermentation for 4 d, the best enzyme production effect of exo-β-1,4-glucanase was 615.93 U / ml.

[0043] Example 3

[0044] This example was about the antibacterial effect of strain JSF-9 against common intestinal pathogenic bacteria.

[0045] The agar diffusion method with punching was used to determine the antibacterial activity of strain JSF-9 against Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Salmonella typhimurium ( Salmonella typhimurium. ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ), and Enterobacter aerogenes ( Enterobacter aerogenes ). Each sample was repeated 3 times; the results obtained are shown in Table 3.

[0046]

[0047] Note: In the table, "-" indicates no inhibitory effect. Different superscript letters in the same column of data indicate significant differences (P < 0.05); the same superscript letters or no letter markings indicate no significant differences (P > 0.05).

[0048] As can be seen from Table 3, strain JSF-9 against Staphylococcus aureus ( Staphylococcus aureus ), Salmonella typhimurium ( Salmonella typhimurium.) and / or Shigella flexneri ( Shigella flexneri ) has antibacterial effect, and has no antibacterial effect on Escherichia coli ( Escherichia coli ), Yersinia enterocolitica ( Yersinia enterocolitica ) and Enterobacter aerogenes ( Enterobacter aerogenes ); The antibacterial circle of Staphylococcus aureus ( Staphylococcus aureus ) > Salmonella typhimurium ( Salmonella typhimurium. ) > Shigella flexneri ( Shigella flexneri ) > Escherichia coli ( Escherichia coli ) indicates that the strain JSF-9 has the best antibacterial effect on Staphylococcus aureus ( Staphylococcus aureus ), followed by Salmonella typhimurium ( Salmonella typhimurium. ), and then Shigella flexneri ( Shigella flexneri ).

[0049] Example 4

[0050] This example is about the fermentation effect of different bacterial agents screened on bagasse fermented feed.

[0051] 1. Experimental design: This experiment adopted a completely randomized experimental design, with 5 groups set, and 3 replicates in each group: the control group without any bacterial agent (CK group), the group added with Bacillus safensis MLL-5 (T1, Bacillus safensis 4.6×10 7 cfu / ml), the group added with Bacillus licheniformis JSF-9 (T2, 1×10 7 cfu / ml Bacillus licheniformis), and the group added with Bacillus altitudinis MLF-1 (T3, Bacillus altitudinis 6.2×10 7 cfu / ml). The water content of bagasse was adjusted to about 60% with distilled water, and the corresponding bacterial agent was added at 100 ml / kg according to the experimental design. After mixing evenly, it was filled into polyethylene bags (230 mm×400 mm) with a one-way exhaust valve, 500 g per bag, 3 replicates for each treatment, sealed with a vacuum sealer and fermented at room temperature in the dark for 30 d.

[0052] 2. Determination indexes and methods.

[0053] (1)Analysis of conventional nutrient components: The sample was dried at 105°C for 2 h and then at 65°C until constant weight to calculate the dry matter (DM) content (Zhang Liying. Feed Analysis and Feed Quality Detection Technology [M]. 2nd Edition. Beijing: China Agricultural University Press, 2003.); it was pulverized and passed through a 40-mesh sieve for the determination of crude protein (CP), water-soluble carbohydrates (WSC), neutral detergent fiber (NDF), and acid detergent fiber (ADF). The methods for determining CP, NDF, and ADF were referred to (van Soest P J, Sniffen C J, Mertens D R. A net protein system for cattle: The rumen submodel for nitrogen [C] / / Owens F N. Protein Requirements for Cattle: Proceedings of an International Symposium. 1981. Stillwater: Oklahoma State University: 265.); WSC was determined by the anthrone-colorimetric method.

[0054] (2)Determination of the fermentation quality of fermented sugarcane bagasse: 10 g of fresh sample was taken in a conical flask, 90 mL of distilled water was added, shaken well, sealed with a sealing film, placed in a 4°C refrigerator for 24 h. After the extract was filtered through gauze, it was centrifuged at 8000 r / min for 10 min to obtain the supernatant for the detection of fermentation parameters. The pH was measured using a pH meter, and ammonia nitrogen (NH3-N), lactic acid (LA), acetic acid (AA), propionic acid (PA), and butyric acid (BA) were detected using a high-performance liquid chromatograph. Total nitrogen = crude protein / 6.25.

[0055] The results obtained by testing using the above scheme are shown in Tables 4 - 6.

[0056]

[0057] Note: Different superscript letters in the same row of data indicate significant differences (P < 0.05); the same superscript letters or no letter annotation indicate no significant differences (P > 0.05). The same below.

[0058] As can be seen from Table 5, in terms of DM content, there was no significant difference between CK and the experimental groups of T1, T2, and T3, indicating that Bacillus can ferment with less dry matter loss and can well preserve the nutritional components of bagasse; in terms of CP content, the CP content of the T2 group was significantly higher than that of other experimental groups (P<0.05), and there was no significant difference between the experimental groups of T1 and T3 and CK (P>0.05), indicating that the fermentation by strain JSF-9 can significantly increase the protein content of bagasse; in terms of WSC, after fermentation treatment, the WSC content of each group decreased compared with the raw material (1.18% DM); the ADF and NDF contents of the experimental groups of T1, T2, and T3 were significantly lower than those of the CK group (P<0.05), indicating that Bacillus has good cellulose degradation ability. In terms of the degradation rate, the content of the T2 group was the lowest and was significantly different from that of other experimental groups (P<0.05); the above results show that: strain JSF-9 has good fermentation ability for bagasse, and after fermentation, it can effectively increase the protein content of fermented bagasse and effectively degrade the contents of neutral detergent fiber and acid detergent fiber in bagasse.

[0059]

[0060] As can be seen from Table 6, in terms of pH value, CK and the experimental groups of T1, T2, and T3 were all lower than 4.2, but there was no significant difference among groups (P>0.05). pH and organic acids are important indicators reflecting the quality of feed fermentation. The pH of high-quality fermented feed is generally below 4.2. In this experiment, the pH values of the three additive treatment groups were all lower than 4.2, indicating that the pH values of the above-mentioned feeds can reach good quality after fermentation; in terms of LA content, the LA content of the CK group was significantly lower than that of the experimental groups of T1, T2, and T3 (P<0.05). Among the experimental groups of T1, T2, and T3, the LA content of the T2 experimental group was the highest, followed by the T3 group and then the T1 group; in terms of AA content, the AA content of the CK group was significantly higher than that of the experimental groups of T1, T2, and T3 (P<0.05). Among the experimental groups of T1, T2, and T3, the LA content of the T2 experimental group was the lowest, followed by the T3 group and the T1 group; in terms of PA content, T1 was significantly higher than the CK group, followed by the T3 group and the T2 group (P<0.05); in terms of BA content, CK was significantly higher than the groups of T1, T2, and T3 (P<0.05), and there was no significant difference among the three groups of T1, T2, and T3 (P>0.05); the fermentation results of organic acids show that: although the pH values of the CK group and the groups of T1, T2, and T3 are not very different, the compositions of organic acids are quite different. The lactic acid content of the experimental groups with added Bacillus was significantly higher than that of CK. This may be because Bacillus can quickly consume oxygen and form an anaerobic environment in a short time, which helps the reproduction of lactic acid bacteria and thus increases the lactic acid production; in terms of NH3-N content, there was no significant difference between CK and the experimental groups of T1 and T3 (P>0.05), while the NH3-N content of the T2 experimental group was significantly lower than that of other experimental groups.

[0061] In summary, when Bacillus licheniformis JSF-9 is used as a single microbial agent for fermentation, the fermentation effect of sugarcane bagasse is the best. After fermentation, it can effectively improve the protein content of fermented sugarcane bagasse, effectively degrade the neutral detergent fiber and acid detergent fiber content of sugarcane bagasse, increase the lactic acid content, and reduce the NH3-N content.

[0062] Example 5

[0063] This example is about the fermentation effect of a compound microbial agent on sugarcane bagasse fermented feed.

[0064] According to the experimental results of Example 4, we found that among the three Bacillus strains, Bacillus licheniformis JSF-9 has the best fermentation effect on sugarcane bagasse substrate. Bacillus safensis MLL-5 and Bacillus altitudinis MLF-1 have a certain fermentation effect on sugarcane bagasse, but it is not as significant as that of strain JSF-9. However, in the antibacterial experiment of pathogenic bacteria, we found that the antibacterial ability of Bacillus safensis MLL-5 and Bacillus altitudinis MLF-1 against intestinal pathogenic bacteria is better than that of strain JSF-9. Based on this, in order to improve the antibacterial function of fermented feed and further improve the quality of sugarcane bagasse fermented feed, we considered inoculating the three groups of Bacillus strains as a compound microbial agent into sugarcane bagasse for fermentation, and optimizing the strain ratio of the compound microbial agent by orthogonal experiment. The specific method is as follows: Mix strains MLF-1, JSF-9, and MLL-5 to prepare a compound microbial agent, and then prepare sugarcane bagasse fermented feed according to the method of Example 4 with an inoculation amount of 100 ml / kg of the compound microbial agent. Among them, the effective viable count of Bacillus licheniformis JSF-9 is 1.2×10 8 cfu / ml, the effective viable count of Bacillus altitudinis MLF-1 is 3.5×10 7 cfu / ml, and the effective viable count of Bacillus safensis MLL-5 is 7.5×10 7 cfu / ml; Select the optimal experimental group with crude protein (CP), lactic acid (LA), and ammonia nitrogen content as optimization indicators, as shown in Tables 7 and 8 specifically.

[0065]

[0066]

[0067] As can be seen from Table 8, in terms of crude protein, the improvement effects of Tests 1-3 and Test 5 are better than those of Group T2 (2.23%) in Table 5, and the improvement effect of Test 3 is the most obvious; in terms of lactic acid content, the improvement effects of Tests 1-4 are better than those of Group T2 (0.56 mg / g) in Table 6, and the improvement effect of Test 2 is the most obvious; in terms of ammonia nitrogen content, the reduction effects of Tests 1-4 are better than those of Group T2 (4.63%) in Table 5, and the reduction effects of other test groups are not as good as those of Group T2. The reduction effect of Test 3 is the most obvious. Therefore, comprehensively, selecting the strain volume ratios of Tests 1-4 to prepare the compound microbial agent has a good fermentation effect on bagasse, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 is (1-2):(2-6):(1-3); in combination, although the LA content of Test 3 is not as good as that of Test 2, the difference between the two is not obvious. Therefore, considering other indicators, the optimal test group we selected is Test 3, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 is 1:6:3.

[0068] From the perspective of the range, the strain MLF-1 has the greatest impact on the crude protein of the fermented bagasse feed, followed by the strain MLL-5, and the strain JSF-9 has the least impact; the strain MLF-1 has the greatest impact on the lactic acid of the fermented bagasse feed, followed by the strain MLL-5, and the strain JSF-9 has the least impact; the strain MLF-1 has the greatest impact on the ammonia nitrogen of the fermented bagasse feed, followed by the strain MLL-5, and the strain JSF-9 has the least impact.

[0069] Example 6

[0070] This example is a feeding experiment of bagasse fermented feed on Jersey cows.

[0071] Forty 7-month-old Jersey cows with good body condition and similar weights were selected and randomly divided into 4 groups, with 10 cows in each group. The control group (CK) was fed with whole-plant corn silage as the basic feed; Experimental Group 1: 10% fermented bagasse (fermented without microbial agent) was added to the basic feed; Experimental Group 2: 10% fermented bagasse (the compound microbial agent was added according to the addition ratio of Test 3 in Example 5) was added to the basic feed; Experimental Group 3: 15% fermented bagasse (the compound microbial agent was added according to the addition ratio of Test 3 in Example 5). The pre-feeding period was 7 days and the test period was 28 days. The whole test lasted for 35 days. The initial weight and final weight of the Jersey cows were measured and then the average daily gain was calculated; the daily feed intake was recorded and the average daily feed intake was calculated. The feed-to-gain ratio was calculated based on the average daily feed intake / average daily gain. The number of diarrhea occurrences was observed and counted (one episode of diarrhea was recorded as 1, and two episodes of diarrhea were recorded as 2). The specific results are shown in Table 9.

[0072]

[0073] Note: Different lowercase letters superscripted on the same column of data indicate significant differences (P < 0.05), while the same letter or no letter indicates no significant difference (P > 0.05).

[0074] As can be seen from Table 9, in terms of average daily gain, there was no significant difference among Experimental Group 2, Experimental Group 3 and the control group (P > 0.05), and they were significantly higher than Experimental Group 1 (P < 0.05); in terms of feed conversion ratio, there was no significant difference among Experimental Group 2, Experimental Group 3 and the control group (P > 0.05), and they were significantly lower than Experimental Group 1 (P < 0.05); in terms of the number of diarrhea occurrences, the diarrhea incidence rate of Experimental Group 1 was higher than that of the control group and higher than those of Experimental Group 2 and Experimental Group 3. This shows that due to the antibacterial effect of the compound bacterium agent on a variety of intestinal bacteria, the prepared fermented bagasse can also have good anti-diarrhea ability, while the bagasse feed not fermented with the compound bacterium agent may have bred other pathogenic bacteria, resulting in a higher number of diarrhea cases than the control group. The above conclusions indicate that the bagasse fermented with the compound bacterium agent can effectively improve the nutritional value of the bagasse, replace the basal feed, and it is also possible that the compound bacterium agent fermented bagasse has improved the disease resistance ability of Jersey cows, enhanced their immunity, and increased their feed utilization rate; from the feeding results, after the compound bacterium agent fermentation, the bagasse has been significantly improved in both nutritional value and disease resistance ability. Adding 10% - 15% to the basal diet can play a role in reducing costs and increasing efficiency in the feeding of Jersey cows.

[0075] In summary, the Bacillus licheniformis JSF-9 self-screened by the applicant of the present invention has good β-1,4-glucanase-producing ability and antibacterial effects on Staphylococcus aureus, Salmonella typhimurium and / or Shigella flexneri; Bacillus licheniformis JSF-9 has good fermentation ability for bagasse. After feeding the prepared bagasse fermented feed to Jersey cows, it was found that: the compound bacterium agent fermented bagasse has been significantly improved in both nutritional value and disease resistance ability. Adding 15% to the basal diet can play a role in reducing costs and increasing efficiency in the feeding of Jersey cows.

[0076] The above-described embodiments only represent several implementation manners of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.

Claims

1. Bacillus licheniformis ( Bacillus paralicheniformis ), JSF-9, characterized in that The Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 has a preservation number of GDMCC NO: 65786.

2. A composite microbial agent containing the Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9.

3. The compound microbial agent according to claim 2, wherein The composite bacterial agent further includes Bacillus altitudinis ( Bacillus altitudini s) MLF-1 and / or Bacillus safensis ( Bacillus safensis ) strain MLL-5; the preservation number of Bacillus altitudinis ( Bacillus altitudini s) MLF-1 is GDMCC NO: 65789; the preservation number of Bacillus safensis ( Bacillus safensis ) strain MLL-5 is GDMCC NO: 65790.

4. The compound microbial agent according to claim 3, wherein, The composite bacterial agent is prepared by mixing Bacillus altitudinis ( Bacillus altitudini s) MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9, and Bacillus safensis ( Bacillus safensis ) strain MLL-5 in a volume ratio of (1-2):(2-6):(1-3).

5. The composite microbial agent according to claim 4, wherein The composite bacterial agent is prepared by mixing Bacillus altitudinis ( Bacillus altitudini s) MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9, and Bacillus safensis ( Bacillus safensis ) strain MLL-5 in a volume ratio of 1:6:

3.

6. The application of Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 or the compound microbial agent according to claim 2 in the preparation of bagasse fermented feed.

7. Use of Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 in the production of β-1,4-glucanase.

8. The application of Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 in the preparation of an intestinal pathogen bacteriostatic agent, characterized in that The intestinal pathogen is Staphylococcus aureus ( Staphylococcus aureus ), Salmonella typhimurium ( Salmonella typhimurium. ), and / or Shigella flexneri ( Shigella flexneri ).

9. Fermented feed containing Bacillus licheniformis Bacillus paralicheniformis JSF-9 described in claim 1 or the compound microbial agent described in claim 2, characterized in that The preparation method of the fermented feed is as follows: Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9 or the compound microbial agent is inoculated into bagasse at an inoculation amount of 100 ml / kg and fermented for 30 days to obtain the product.

21. Use of the fermented feed according to claim 9 in feeding Jersey cattle.

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