Bacillus altitudinis MLF-1 as well as complex microbial inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus altitudinis MLF-1 and a complex microbial inoculant and application thereof.The bacillus altitudinis strain MLF-1 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 is achieved on enterobacter aerogenes, staphylococcus aureus, salmonella typhimurium and escherichia coli; the bacillus altitudinis MLF-1 has good fermentation capacity on the siraitia grosvenorii residues, after the bacillus altitudinis MLF-1 is mixed with the bacillus licheniformis strain JSF-9 and the bacillus safensis strain MLL-5 to prepare the complex microbial inoculant, the nutritional value and the disease resistance of the siraitia grosvenorii residues fermented by the complex microbial inoculant are remarkably improved, 10%-15% of the complex microbial inoculant is added into a basic ration, and the content of the complex microbial inoculant in the siraitia grosvenorii residues fermented by the complex microbial inoculant is reduced. The Jersey cattle feed has the effects of reducing cost and improving efficiency for feeding Jersey cattle.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, and particularly to Bacillus altitudinis MLF-1, its compound bacterium agent and application. Background Art

[0002] Momordica grosvenori, commonly known as "fairy fruit", is a perennial vine plant of the Cucurbitaceae family. Its fruit can be used as medicine and contains medicinal components such as mogroside, various amino acids and vitamins. Guangxi is an important production area of Momordica grosvenori. After being pressed, Momordica grosvenori is processed into preparations, lozenges, beverages and other products, and the remaining waste fruit residue is less studied and utilized. Currently, Momordica grosvenori residue is usually burned as fuel, and most of the fruit residue is discarded, which not only pollutes the environment but also wastes precious resources. In the prior art, there are also some methods of preparing fermented feed by fermenting Momordica grosvenori residue. However, in our actual work, it is found that due to the presence of more components such as mogroside and flavonoids in the substrate, it has a certain inhibitory effect on some probiotics. Some microorganisms reported to have good fermentation effects cannot effectively ferment Momordica grosvenori residue when applied to Momordica grosvenori residue, and the utilization rate of the substrate is not high. While directly composting, due to the uncertain composition of microorganisms in nature, some intestinal pathogenic bacteria may breed, which will affect the utilization rate of Momordica grosvenori residue.

[0003] Therefore, it is necessary to conduct a large number of screenings and verifications on microorganisms, develop probiotics suitable for the fermentation of Momordica grosvenori residue substrate, produce Momordica grosvenori residue 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 conduct a large number of screenings and verifications on microorganisms, develop probiotics suitable for the fermentation of Momordica grosvenori residue substrate, produce Momordica grosvenori residue fermented feed, and verify the feasibility of this feed in feeding animals.

[0005] To achieve the above object, the present invention has screened out a new strain: Bacillus altitudinis ( Bacillus altitudinis ) MLF-1, whose 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 Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025.

[0006] The present invention also includes a compound bacterium agent containing the above-mentioned Bacillus altitudinis ( Bacillus altitudinis ) MLF-1.

[0007] Furthermore, the compound bacterium agent also includes Bacillus licheniformis ( Bacillus paralicheniformis) JSF-9 and / or Bacillus safensis ( Bacillus safensis ) MLL-5; the Bacillus licheniformis ( Bacillus paralicheniformi s) JSF-9, whose taxonomic name is: Bacillus paralicheniformis JSF-9, Chinese taxonomic name: Bacillus licheniformis JSF-9, deposit number: GDMCC NO: 65786; this strain is deposited in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou, deposit date: January 14, 2025; the Bacillus safensis ( Bacillus safensis ) MLL-5, whose taxonomic name is: Bacillus safensis MLL-5, Chinese taxonomic name: Bacillus safensis MLL-5, deposit number: GDMCC NO: 65790; this strain is deposited in the Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, 100th Yard, Xianlie Middle Road, Guangzhou, deposit date: February 10, 2025.

[0008] Further, the compound bactericide is prepared by mixing Bacillus altitudinis ( Bacillus altitudinis ) MLF-1, Bacillus licheniformis ( Bacillus paralicheniformi s) JSF-9 and Bacillus safensis ( Bacillus safensis ) MLL-5 in a volume ratio of (4-6):(1-3):(1-3).

[0009] Further, the compound bactericide is prepared by mixing Bacillus altitudinis ( Bacillus altitudinis ) MLF-1, Bacillus licheniformis ( Bacillus paralicheniformi s) JSF-9 and Bacillus safensis ( Bacillus safensis ) MLL-5 in a volume ratio of 4:3:1.

[0010] The present invention also includes the application of the Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 or the compound bactericide in the preparation of fermented feed from Momordica grosvenori residue.

[0011] The present invention also includes the application of the Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 in the production of β-1,4-glucanase.

[0012] The present invention also includes the application of the Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 in the preparation of an intestinal pathogen bacteriostatic agent, and the intestinal pathogens are Enterobacter aerogenes ( Enterobacter aerogenes ), Staphylococcus aureus ( Staphylococcus aureus ), Salmonella typhimurium ( Salmonella typhimurium. ) and / or Escherichia coli ( Escherichia coli ).

[0013] The present invention also includes a fermented feed containing the Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 or the compound microbial agent as described above. The preparation method of the fermented feed is as follows: inoculate the Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 or the compound microbial agent into Momordica grosvenori residue 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 MLF-1 of the present invention is isolated from healthy rectal contents by the research group. After detection, this strain has good ability to produce β-1,4-glucanase and has antibacterial effects on Enterobacter aerogenes, Staphylococcus aureus, Salmonella typhimurium and / or Escherichia coli; Bacillus altitudinis MLF-1 has good fermentation ability for Momordica grosvenori residue. After feeding the Momordica grosvenori residue fermented feed to Jersey cows, it is found that the Momordica grosvenori residue fermented by the compound microbial agent has significantly improved both nutritional value and disease resistance. Adding 10%-15% to the basal diet can reduce costs and increase efficiency in feeding Jersey cows.

[0016] 2. The present application also mixes Bacillus altitudinis MLF-1 with Bacillus licheniformis strain JSF-9 and Bacillus safensis strain MLL-5 to prepare a compound microbial agent, and obtains the optimal volume ratio of the compound microbial agent through adjustment of the Momordica grosvenori residue fermentation experiment. The fermented feed prepared by fermenting Momordica grosvenori residue with this compound microbial agent can effectively improve the utilization rate of the feed. The research on this strain, compound microbial agent and fermentation method provides good support and technical means for solving the waste of Momordica grosvenori 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 MLF-1 on Congo red-stained carboxymethyl cellulose sodium screening medium.

[0021] Biological Material Deposit Information

[0022] The strain information deposited in the present application is as follows: Bacillus altitudinis ( Bacillus altitudinis ) MLF-1, and its taxonomic naming is:Bacillus altitudinis MLF-1, classified and named in Chinese as: Bacillus altitudinis MLF-1, with the preservation number of GDMCC NO: 65789; this strain is preserved in Guangdong Provincial Microbial Culture Collection Center, 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 paralicheniformi s) JSF-9, and its classification and naming are: Bacillus paralicheniformis JSF-9, classified and named in Chinese as: Bacillus licheniformis JSF-9, with the preservation number of GDMCC NO: 65786; this strain is preserved in Guangdong Provincial Microbial Culture Collection Center, 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 classification and naming are: Bacillus safensis MLL-5, classified and named in Chinese as: Bacillus safensis MLL-5, with the preservation number of GDMCC NO: 65790; this strain is preserved in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the preservation date is February 10, 2025. Detailed implementation mode

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

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

[0027] Example 1

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

[0029] Isolation and purification of strains: A total of 67 samples of healthy bovine rumen contents and rectal contents were collected, placed into sterile cryotubes containing glycerol, the bottle mouths were wrapped with sealing film, and then they were put into 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 serially diluted 10-fold 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 after increasing the dilution, a sterile pipette or tip was changed. 100 μl of diluted solutions at 3 - 4 appropriate gradients 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 storage 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 of the diameter of the transparent circle (D) to the diameter of the colony (d) (D / d), with 3 replicates for each strain. A total of 15 strains producing cellulase were screened out. Strains with larger ratios of the size of the transparent circle to the size of the colony were selected through the ratio screening, and the enzyme production conditions were optimized. The specific results are shown in Table 1.

[0030]

[0031] Three strains with larger D / d ratios 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 PCR amplification and sequencing were performed on the 16S region. 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 had a relatively close genetic relationship with Bacillus altitudinis and, combined with morphological identification, the strain was classified and named as Bacillus altitudinis .

[0033] (2)Identification of strain JSF-9: ①Morphological identification is as Figure 2 shown: The JSF-9 strain 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; ②After 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 was closely related to Bacillus paralicheniformis , and combined with morphological identification, this strain was classified and named as Bacillus paralicheniformis .

[0034] (3)Identification of strain MLL-5: ①Morphological identification is as Figure 3 shown: The MLL-5 strain 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; ②After 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 was closely related to Bacillus safensis , and combined with 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 of strain MLF-1 and the optimal enzyme production conditions.

[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)The strain MLF-1 was inoculated into the seed liquid medium and cultured with shaking at 39 °C and 160 r / min until OD 600It was 0.6 to obtain the seed solution.

[0040] (4) The seed solution 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 fermentation broth was centrifuged at 4 °C and 6000 r / min for 10 min, and the supernatant was taken as the crude enzyme solution. Finally, it was obtained that Bacillus altitudinis The enzyme production of strain MLF-1 was as follows Figure 4 shown: The figure shows that strain MLF-1 can produce a clear zone on the Congo red-stained carboxymethyl cellulose sodium screening medium, indicating that this strain can produce β-1,4-glucanase. The specific enzyme production situation is shown in Table 2.

[0041]

[0042] As can be seen from Table 2, the optimal enzyme production conditions for strain MLF-1 were: an inoculation amount of 9%, shake flask fermentation for 4 d, and the best enzyme production effect of endo-β-1,4-glucanase was 687.14 U / ml; an inoculation amount of 7%, shake flask fermentation for 4 d, and the best enzyme production effect of exo-β-1,4-glucanase was 805.33 U / ml.

[0043] Example 3

[0044] This example was about the antibacterial effect of strain MLF-1 on common intestinal pathogenic bacteria.

[0045] The agar diffusion method with hole punching was used to determine the antibacterial activity of strain MLF-1 against Staphylococcus aureus ( Staphylococcus aureus ), Escherichia coli ( Escherichia coli ), Salmonella typhimurium ( Salmonella typhimurium. ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ), 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, a, b, c: indicate significant differences in the data of the same column (p < 0.05).

[0048] As can be seen from Table 3, strain MLF-1 against Enterobacter aerogenes ( Enterobacter aerogenes ), Staphylococcus aureus ( Staphylococcus aureus ), Salmonella typhimurium (Salmonella typhimurium. ) and / or Escherichia coli ( Escherichia coli ) have antibacterial effects, and have no antibacterial effects on Yersinia enterocolitica ( Yersinia enterocolitica ) and Shigella flexneri ( Shigella flexneri ); The antibacterial zone Aerobacter aerogenes ( Enterobacter aerogenes ) > Staphylococcus aureus ( Staphylococcus aureus ) > Salmonella typhimurium ( Salmonella typhimurium. ) > Escherichia coli ( Escherichia coli ) indicates that the strain MLF-1 has the best antibacterial effect on Aerobacter aerogenes ( Enterobacter aerogenes ), followed by Staphylococcus aureus ( Staphylococcus aureus ), then Salmonella typhimurium ( Salmonella typhimurium. ) and / or Escherichia coli ( Escherichia coli ).

[0049] Example 4

[0050] This example is about the fermentation effects of different bacterial agents screened on the Momordica grosvenori residue fermented feed.

[0051] Experimental design: The experiment set 4 treatment groups: CK group: Without adding any bacterial agent, directly ferment the Momordica grosvenori residue naturally; MLF-1 group: Add the strain MLF-1 and inoculate it into the Momordica grosvenori residue at an inoculation amount of 100 ml / kg for fermentation for 30 d; JSF-9 group: Add the strain JSF-9 and inoculate it into the Momordica grosvenori residue at an inoculation amount of 100 ml / kg for fermentation for 30 d; MLL-5 group: Add the strain MLL-5 and inoculate it into the Momordica grosvenori residue at an inoculation amount of 100 ml / kg for fermentation for 30 d; The viable bacteria count of each group of bacterial agents was 10 7 -10 8 cfu / ml; After fermentation was completed, discard the samples in the upper 10 cm, and the remaining samples were sampled by the quartering method, and the nutritional components and fermentation quality after fermentation were detected. The indexes for the determination of nutritional components included dry matter, crude protein, crude fiber, neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents, and the results were shown in Table 4; The indexes for the determination of fermentation quality included lactic acid, acetic acid, propionic acid, butyric acid and ammonia nitrogen, and the results were shown in Table 5.

[0052]

[0053] Note: Different lowercase letters in the table indicate significant differences in the same column of data (p < 0.05), and the same letters indicate no significant differences (p > 0.05). The same applies to the following tables.

[0054] As can be seen from Table 4, after the fermentation of Momordica grosvenori residue by strains MLF-1, JSF-9, and MLL-5, the content of dry matter decreased significantly (p<0.05), among which, the content of dry matter fermented by strain MLF-1 was the lowest; after the fermentation of Momordica grosvenori residue by strain MLF-1, the content of crude protein increased significantly (p<0.05), and there was no significant difference in crude protein between strains JSF-9 and MLL-5 (p>0.05); after the fermentation of Momordica grosvenori residue by strain MLF-1, the contents of crude fiber, neutral detergent fiber (NDF), and acid detergent fiber (ADF) decreased significantly (p<0.05), and there was no significant difference in crude fiber, neutral detergent fiber (NDF), and acid detergent fiber (ADF) between strains JSF-9 and MLL-5 (p>0.05); it shows that for the substrate of Momordica grosvenori residue, the cellulose degradation effect and protein improvement effect of strain MLF-1 are the most significant, indicating that the fermentation effect of strain MLF-1 on the substrate of Momordica grosvenori residue is the best.

[0055]

[0056] As can be seen from Table 5, after the fermentation of Momordica grosvenori residue by strains MLF-1, JSF-9, and MLL-5, the amount of lactic acid increased significantly (p<0.05). In terms of the improvement effect, strain MLF-1 > strain MLL-5 > strain JSF-9; the acetic acid content in the fermentation group of strain MLF-1 increased significantly (p<0.05), and there was no significant difference in the acetic acid content between the fermentation groups of other strains JSF-9 and MLL-5 and the control group (p>0.05); the improvement effects of the three strains on the contents of propionic acid and butyric acid in the Momordica grosvenori residue feed were not obvious (p>0.05); the three strains had a significant effect on reducing the content of ammonia nitrogen in the Momordica grosvenori residue feed (p<0.05). In terms of the reduction effect, strain MLF-1 = strain MLL-5 < strain JSF-9; it shows that strains MLF-1 and MLL-5 have the most obvious effect on reducing ammonia nitrogen in Momordica grosvenori residue, and the effect of strain JSF-9 on reducing ammonia nitrogen is the second.

[0057] Combined with the nutritional components and quality of the fermented feed, the fermentation effect of using strain MLF-1 on Momordica grosvenori residue is the best. Strains JSF-9 and MLL-5 have a certain fermentation effect on Momordica grosvenori residue, but the fermentation effect is not as good as that of strain MLF-1, indicating that in the substrate of Momordica grosvenori residue, the most suitable fermentation strain is MLF-1.

[0058] Example 5

[0059] This example is about the fermentation effect of a compound microbial agent on Momordica grosvenori residue fermented feed.

[0060] According to the experimental results of Example 4, we found that: Strain MLF-1 can increase the crude protein content, reduce the crude fiber, neutral detergent fiber, and acid detergent fiber levels of Momordica grosvenori residue, increase the lactic acid and acetic acid contents, and reduce the ammonia nitrogen content; Strain JSF-9 can increase lactic acid and reduce the ammonia nitrogen content; Strain MLL-5 can increase lactic acid and reduce the ammonia nitrogen content. In order to effectively improve the quality of Momordica grosvenori residue fermented feed and increase strain diversity, the research group considered preparing a compound microbial agent from strains MLF-1, JSF-9, and MLL-5 and then fermenting it with Momordica grosvenori residue, and using an orthogonal experiment to optimize the strain ratio of the compound microbial agent. The specific method is as follows: Mix strains MLF-1, JSF-9, and MLL-5 to prepare a compound microbial agent; then inoculate the compound microbial agent into Momordica grosvenori residue at an inoculation amount of 100 ml / kg and ferment for 30 d. 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; The optimal experimental group was selected with crude protein, lactic acid, and ammonia nitrogen content as the optimization indexes, as shown in Tables 6 and 7 specifically.

[0061]

[0062]

[0063] As can be seen from Table 7, from the perspective of crude protein, the improvement effects of Tests 1-4 and Test 9 are not as good as that of single strain MLF-1, and the improvement effect of Test 6 is the most obvious; from the perspective of lactic acid content, the improvement effects of Tests 1-4 and Test 9 are not as good as that of single strain MLF-1, and the improvement effect of Test 6 is the most obvious; from the perspective of ammonia nitrogen content, except that the reduction in Tests 3 and 9 is not obvious, the reduction effects of other experimental groups are close to that of strain MLF-1; Therefore, comprehensively, choosing the strain volume ratio of Tests 5-8 to prepare the compound microbial agent has a good fermentation effect on Momordica grosvenori residue, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 is (4-6):(1-3):(1-3); The optimal experimental group is Test 6, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 is 4:3:1.

[0064] In terms of the range, the strain MLL-5 had the greatest impact on the crude protein of the fermented Momordica grosvenori residue feed, followed by the strain MLF-1, and the strain JSF-9 had the least impact; the strain MLF-1 had the greatest impact on the lactic acid of the fermented Momordica grosvenori residue feed, followed by the strain MLL-5, and the strain JSF-9 had the least impact; the strain JSF-9 had the greatest impact on the ammonia nitrogen of the fermented Momordica grosvenori residue feed, followed by the strain MLL-5, and the strain MLF-1 had the least impact.

[0065] Example 6

[0066] This example is a feeding experiment of Momordica grosvenori residue fermented feed on Jersey cows.

[0067] 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% of Momordica grosvenori residue fermented feed (fermented without fungicide) was added to the basic feed; Experimental group 2: 10% of Momordica grosvenori residue fermented feed (the compound fungicide in the fermented feed was added according to the addition ratio of Test 6 in Example 5) was added to the basic feed; Experimental group 3: 15% of Momordica grosvenori residue fermented feed (the compound fungicide in the fermented feed was added according to the addition ratio of Test 6 in Example 5) was added to the basic feed; The pre-feeding period was 7 days and the test period was 28 days, with a total of 35 days for the whole test; 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, and the feed-to-gain ratio was calculated based on the average daily feed intake / average daily gain. The number of diarrhea episodes was observed and counted (one episode of diarrhea was recorded as 1, and two episodes of diarrhea were recorded as 2); The results are shown in Table 8.

[0068]

[0069] As can be seen from Table 8, in terms of average daily gain, there was no significant difference between the control group and Experimental Group 2 and Experimental Group 3 (p>0.05), but the average daily gain of these three groups was significantly higher than that of Experimental Group 1 (p<0.05); in terms of feed conversion ratio, there was no significant difference between the control group and Experimental Group 2 and Experimental Group 3 (p>0.05), but the feed conversion ratio of these three groups was significantly lower than that of Experimental Group 1 (p<0.05); this indicates that the Momordica grosvenori residue fermented with the compound microbial agent (Experimental Group 2 and Experimental Group 3) can effectively replace the basal diet, while the conversion effect of the Momordica grosvenori residue without the addition of the microbial agent is not good, and the feeding effect significantly decreases after replacing part of the basal diet; in terms of the number of diarrhea: the number of diarrhea in Experimental Group 2 and Experimental Group 3 was 0 times, which indicates that after several microbial agents with the function of inhibiting intestinal pathogenic bacteria were prepared into a compound microbial agent, the number of diarrhea in Jersey cows was effectively reduced. The number of diarrhea in the control group reached 6 times, and the number of diarrhea in Experimental Group 1 reached 4 times, slightly lower than that of the control group. This indicates that Momordica grosvenori in the Momordica grosvenori residue has certain antibacterial properties and is not easy to breed pathogenic bacteria. However, the single raw material of the Momordica grosvenori residue cannot completely inhibit all pathogenic bacteria, so it will still cause diarrhea in Jersey cows. Based on the above conclusions, adding 10%-15% of the Momordica grosvenori residue fermented with the compound microbial agent to the basal diet will not have an adverse impact on the growth performance and health status of cows, and the number of diarrhea significantly decreases, indicating that the nutritional value of the fermented Momordica grosvenori residue of this application has been improved after being fermented by strains, and the disease resistance function and immunity of Jersey cows have been enhanced.

[0070] In summary, the Bacillus altitudinis MLF-1 self-screened by the applicant of the present invention has good β-1,4-glucanase production ability and has antibacterial effects on Enterobacter aerogenes, Staphylococcus aureus, Salmonella typhimurium and / or Escherichia coli; Bacillus altitudinis MLF-1 has good fermentation ability for Momordica grosvenori residue. After feeding the Momordica grosvenori residue fermented feed to Jersey cows, it was found that: this feed can effectively replace the basal diet to reduce the feed cost, improve the immunity of Jersey cows and reduce the number of diarrhea.

[0071] The above-described embodiments merely represent several implementation manners of the present invention, and the description thereof is relatively specific and detailed, but it should not be construed as a limitation on 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 altitudinis ( Bacillus altitudinis ), with the preservation number of GDMCC NO: 65789.

2. A composite microbial agent containing the Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 as claimed in claim 1.

3. The composite microbial agent according to claim 2, characterized in that, The composite microbial agent further comprises Bacillus licheniformis ( Bacillus paralicheniformi s) JSF-9 and / or Bacillus safensis ( Bacillus safensis ) MLL-5; the preservation number of Bacillus licheniformis ( Bacillus paralicheniformi s) JSF-9 is GDMCC NO: 65786; the preservation number of Bacillus safensis ( Bacillus safensis ) MLL-5 is GDMCC NO: 65790.

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

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

1.

6. The application of Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 as claimed in claim 1 or the compound microbial agent as claimed in claim 2 in the preparation of fermented feed from Momordica grosvenori residue.

7. The application of Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 in the production of β-1,4-glucanase.

8. The application of Bacillus altitudinis ( Bacillus altitudinis ) MLF-1 in the preparation of an intestinal pathogen bacteriostatic agent, characterized in that The intestinal pathogen is Enterobacter aerogenes( Enterobacter aerogenes ), Staphylococcus aureus( Staphylococcus aureus ), Salmonella typhimurium( Salmonella typhimurium. ) and / or Escherichia coli( Escherichia coli ).

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

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

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