Bacillus subtilis MLF-1 and its composite bacterial agent and application

Abstract

The present invention relates to the field of microbial technology, and in particular to Bacillus grosvenoris MLF-1, a composite bacterial agent and an application thereof. The Bacillus grosvenoris strain MLF-1 of the present invention is isolated by a research group from the rectal contents of healthy cattle. Testing shows that the strain has good β-1,4-glucanase production ability and has antibacterial effects on Enterobacter aerogenes, Staphylococcus aureus, Salmonella typhimurium and Escherichia coli. The Bacillus grosvenoris MLF-1 has good fermentation ability for monk fruit residue. After the Bacillus grosvenoris MLF-1 is mixed with Bacillus licheniformis strain JSF-9 and Bacillus saffron strain MLL-5 to prepare a composite bacterial agent, it is found that the nutritional value and disease resistance of the monk fruit residue fermented with the composite bacterial agent are significantly improved. Adding 10%-15% of the composite bacterial agent to the basal diet can reduce the cost and increase the efficiency of feeding Jersey cattle.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, in particular to Bacillus subtilis MLF-1 and a composite bacterial agent and application thereof. Background Art

[0002] Momordica grosvenori, commonly known as "god fruit," is a perennial vine in the Cucurbitaceae family. Its fruit is used as medicine and contains medicinal ingredients such as mogrosides, various amino acids, and vitamins. Guangxi is a major production area for Momordica grosvenori. After being squeezed, Momordica grosvenori is processed into preparations, lozenges, beverages, and other products. The remaining waste pomace has been less studied and utilized. Currently, Momordica grosvenori pomace is typically incinerated as fuel, and most of the pomace is discarded, polluting the environment and wasting precious resources. There are also some existing methods for fermenting Momordica grosvenori pomace into fermented feed. However, in our actual work, we found that due to the high content of mogrosides, flavonoids, and other components in the substrate, some probiotics have a certain inhibitory effect. Some microorganisms reported to have good fermentation effects cannot effectively ferment the Momordica grosvenori pomace when used in Momordica grosvenori, resulting in low substrate utilization. Direct composting, however, may breed some intestinal pathogens due to the uncertain microbial composition in nature, thus affecting the utilization rate of the Momordica grosvenori pomace.

[0003] Therefore, we need to conduct a large amount of screening and verification of microorganisms, and develop probiotics suitable for the fermentation of monk fruit residue substrates, produce monk fruit 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 amount of screening and verification of microorganisms, and to develop probiotics suitable for the fermentation of monk fruit residue substrates, produce monk fruit residue fermented feed, and verify the feasibility of this feed in feeding animals.

[0005] In order to achieve the above purpose, the present invention screened out a new strain: Bacillus subtilis ( Bacillus height )MLF-1, its classification is named as: Altitude Bacillus MLF-1, Chinese classification name: Bacillus subtilis MLF-1, preservation number: GDMCC NO: 65789; the strain is deposited in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025.

[0006] The present invention also includes the highland Bacillus ( Altitude Bacillus ) MLF-1 composite bacterial agent.

[0007] Furthermore, the composite bacterial agent also includes Bacillus licheniformis ( Bacillus paralicheniformiss) JSF-9 and / or Bacillus saffron ( Bacillus safensis )MLL-5; the Bacillus licheniformis ( Bacillus paralicheniform s) JSF-9, with its classification designation: Bacillus paralicheniformis JSF-9, Chinese classification name: Bacillus licheniformis JSF-9, deposit number: GDMCC NO: 65786; the strain is deposited in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the deposit date is January 14, 2025; the Bacillus safovici ( Bacillus safensis )MLL-5, its classification is named as: Bacillus safensis MLL-5, Chinese classification name: Bacillus shaforniensis MLL-5, preservation number is GDMCC NO: 65790; the strain is deposited in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is February 10, 2025.

[0008] Furthermore, the composite bacterial agent is composed of Bacillus subtilis ( Altitude Bacillus )MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis s)JSF-9 and Bacillus saffron ( Bacillus safensis )MLL-5 was prepared by mixing in a volume ratio of (4-6): (1-3): (1-3).

[0009] Furthermore, the composite bacterial agent is composed of Bacillus subtilis ( Altitude Bacillus )MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis s)JSF-9 and Bacillus saffron ( Bacillus safensis )MLL-5 was mixed in a volume ratio of 4:3:1.

[0010] The present invention also includes the highland Bacillus ( Altitude Bacillus ) Application of MLF-1 or the composite bacterial agent in the preparation of fermented feed from monk fruit residue.

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

[0012] The present invention also includes the highland Bacillus ( Altitude Bacillus ) Application of MLF-1 in the preparation of an antibacterial agent for intestinal pathogens, wherein 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 the highland Bacillus ( Altitude Bacillus ) MLF-1 or the fermented feed of the composite microbial agent, the preparation method of the fermented feed is: Bacillus subtilis ( Bacillus height ) MLF-1 or composite bacterial agent is inoculated into the monk fruit residue at an inoculation rate of 100 ml / kg and fermented for 30 days.

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

[0015] The present invention has the following beneficial effects: 1. The strain MLF-1 of the present invention is isolated by the research team from the contents of healthy rectum. After testing, the strain has good β-1,4-glucanase production ability and has an antibacterial effect on Enterobacter aerogenes, Staphylococcus aureus, Salmonella typhimurium and / or Escherichia coli; Bacillus subtilis MLF-1 has good fermentation ability for monk fruit residue. After feeding the prepared monk fruit residue fermented feed to Jersey cattle, it was found that the nutritional value and disease resistance of the monk fruit residue fermented with the composite bacterial agent were significantly improved. Adding 10%-15% to the basic diet has the effect of reducing costs and increasing efficiency in feeding Jersey cattle.

[0016] 2. This application also mixes Bacillus subtilis MLF-1 with Bacillus licheniformis strain JSF-9 and Bacillus saffron strain MLL-5 to prepare a composite bacterial agent. The optimal volume ratio of the composite bacterial agent is obtained by adjusting the fermentation experiment of monk fruit residue. The fermented feed prepared by fermenting monk fruit residue with the composite bacterial agent can effectively improve the utilization rate of the feed. The research on the strain, composite bacterial agent and fermentation method provides good support and technical means for solving the waste problem of monk fruit processing enterprises. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0018] Figure 2 The colony morphology of strain JSF-9.

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

[0020] Figure 4 The decolorization results of strain MLF-1 on Congo red stained sodium carboxymethylcellulose screening medium.

[0021] Biomaterial deposit information

[0022] The strain information deposited in this application is: Bacillus subtilis ( Altitude Bacillus )MLF-1, its classification is named as: Altitude Bacillus MLF-1, Chinese classification name: Bacillus subtilis MLF-1, preservation number: GDMCC NO: 65789; the strain is deposited in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is January 14, 2025.

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

[0024] The strain information deposited in this application is: Bacillus saffron ( Bacillus safensis )MLL-5, its classification is named as: Bacillus safensis MLL-5, Chinese classification name: Bacillus shaforniensis MLL-5, preservation number: GDMCCNO: 65790; the strain is deposited in Guangdong Provincial Microbial Culture Collection Center, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, and the preservation date is February 10, 2025. DETAILED DESCRIPTION

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

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

[0027] Example 1

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

[0029] Isolation and Purification of the Strain: 67 samples of rumen and rectal contents from healthy cattle were collected and placed into sterile cryovials containing glycerol. The vials were sealed with parafilm, placed in liquid nitrogen tanks, and brought back to the laboratory for storage at -80°C until use. The rumen contents, stored at -80°C, were cryothed in a sterile operating table and diluted 10-fold in sterile PBS. The samples were shaken thoroughly (using a vortex to mix) to form a 1:10 homogenous solution. Using a separate sterile pipette or micropipette tip, the sample was diluted 10-fold in increments, following the above procedure. The sterile pipette or tip was replaced with a new one for each incremental dilution. 100 μl of each dilution from three to four appropriate gradients was spread onto the surface of NA agar. After spreading, the plates were allowed to stand to allow the inoculum to be completely absorbed by the medium. The plates were then inverted and incubated at 39°C. After colonies grew, colonies of varying morphology were selected and purified by streaking (2-3 times). These colonies were then stored in magnetic bead culture tubes. A total of 256 strains were purified. The purified strains were inoculated onto sodium carboxymethylcellulose plates and incubated at 39°C for 18-24 hours. The plates were then stained with 0.1% Congo red solution for 15 minutes, the stain discarded, and decolorized with 1 mol / L NaCl solution for 30 minutes. Cellulase-producing strains were identified based on the ratio of clearing zone diameter (D) to colony diameter (d). Three replicates were performed for each strain. A total of 15 cellulase-producing strains were identified. Strains with a high ratio of clearing zone size to colony size were selected, and enzyme production conditions were optimized. The results are shown in Table 1.

[0030]

[0031] Three bacterial strains with large D / d ratios were selected from Table 1 for species identification.

[0032] (1) Identification of strain MLF-1: ① Morphological identification such as Figure 1 As shown: The MLF-1 strain was inoculated on LB plate culture medium and cultured for 24 hours. The colonies were round, white, 1.5 to 3.5 mm in diameter, with irregular edges, thick and moist, and easy to pick. ② After DNA sequencing of the strain was extracted and molecularly identified, the DNA sequence of the strain was amplified and sequenced. The 16S region was amplified and sequenced by PCR. The primers used were forward sequence 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse sequence 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. After sequencing the PCR product, the 16S sequence obtained was shown in the sequence listing SEQ ID NO: 1. After BLAST comparison and addition of housekeeping genes, it was confirmed that the strain was the same as Altitude Bacillus The strain is closely related and is classified and named based on morphological identification. Altitude Bacillus .

[0033] (2) Identification of strain JSF-9: ① Morphological identification such as Figure 2 As shown: The JSF-9 strain was inoculated on LB plate culture medium and cultured for 24 hours. The colonies were round, milky white, smooth on the surface, and with regular edges. ② After DNA was extracted and sequenced, molecular identification was performed to amplify the DNA sequence of the strain. The 16S region was amplified and sequenced by PCR. The primers used were forward sequence 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse sequence 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. After sequencing the PCR product, the 16S sequence obtained was shown in the sequence listing SEQ ID NO: 2. After BLAST comparison and addition of housekeeping genes, it was confirmed that the strain was the same as Bacillus paralicheniformis The strain is closely related and is classified and named based on morphological identification. Bacillus paralicheniformis .

[0034] (3) Identification of strain MLL-5: ① Morphological identification such as Figure 3 As shown: The MLL-5 strain was inoculated on LB plate culture medium and cultured for 24 hours. The colonies were round, yellow-white, smooth on the surface, and with regular edges. ② After DNA was extracted and sequenced, molecular identification was performed to amplify the DNA sequence of the strain. The 16S region was amplified and sequenced by PCR. The primers used were forward sequence 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and reverse sequence 1492R: 5'-TACGGCTACCTTGTTACGACTT-3'. After sequencing the PCR product, the 16S sequence obtained was shown in the sequence listing SEQ ID NO: 3. After BLAST comparison and addition of housekeeping genes, it was confirmed that the strain was the same as Bacillus safensis The strain is closely related and is classified and named based on morphological identification. Bacillus safensis .

[0035] Example 2

[0036] This example describes the enzyme production of strain MLF-1 and the determination of the optimal enzyme production conditions.

[0037] (1) Seed liquid culture 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: 50 g bagasse, 10.0 g peptone, 10.0 g sodium chloride, 1 L distilled water, natural pH, sterilized at 121°C for 20 min.

[0039] (3) Inoculate strain MLF-1 into seed liquid culture medium and culture at 39°C and 160 rpm until OD 600is 0.6, and seed liquid is obtained.

[0040] (4) The seed liquid was inoculated into a 250ml triangular flask containing 100ml liquid fermentation medium at an inoculum rate of 3%, 5%, 7%, and 9% (V / V) for shake flask fermentation. Sugarcane bagasse was used as an inducer to induce the strain to secrete cellulase. The shaker speed was 160r / min and the fermentation culture was carried out at 39℃ for 2d, 3d, 4d, and 5d. The fermentation liquid was centrifuged at 4℃ and 6000r / min for 10min, and the supernatant was taken as the crude enzyme liquid. Altitude Bacillus The enzyme production of strain MLF-1 is as follows Figure 4 As shown in the figure: The strain MLF-1 can produce a transparent circle on the Congo red-stained sodium carboxymethyl cellulose screening medium, indicating that the strain can produce β-1,4-glucanase. The specific enzyme production situation is shown in Table 2.

[0041]

[0042] As shown in Table 2, the optimal enzyme production conditions for strain MLF-1 are: 9% inoculation amount, shake flask fermentation for 4 days, the optimal endo-β-1, 4-glucanase production effect is 687.14 U / ml; 7% inoculation amount, shake flask fermentation for 4 days, the optimal exo-β-1, 4-glucanase production effect is 805.33 U / ml.

[0043] Example 3

[0044] This example shows the antibacterial effect of strain MLF-1 on common intestinal pathogens.

[0045] The punch agar diffusion method was used to determine the activity of strain MLF-1 against Staphylococcus aureus ( Staphylococcus golden ) 、Escherichia coli ( Escherichia coli ), Salmonella typhimurium ( Salmonella typhimurium. ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ) 、Enterobacter aerogenes ( Enterobacter aerogenes ) were tested for their antibacterial activity, and the results were shown in Table 3.

[0046]

[0047] Note: “—” in the table indicates no inhibitory effect; a, b, c: indicate significant differences in the data in the same column (p<0.05).

[0048] As shown in Table 3, the strain MLF-1 has a strong Enterobacter aerogenes ), Staphylococcus aureus ( Staphylococcus aureus ), Salmonella typhimurium ( Salmonella typhimurium. ) and / or Escherichia coli ( Escherichia coli ) have antibacterial effects on Yersinia enterocolitica ( Yersinia enterocolitica ) and Shigella flexneri ( Shigella flexneri ) No antibacterial effect; inhibition zone Enterobacter aerogenes ( Enterobacter aerogenes )>Staphylococcus aureus( Staphylococcus aureus ) > Salmonella typhimurium ( Salmonella typhimurium. ) >Escherichia coli( Escherichia coli ) indicates that the strain MLF-1 is effective against Enterobacter aerogenes ( Enterobacter aerogenes ) had the best antibacterial effect, followed by Staphylococcus aureus ( Staphylococcus aureus ), followed by Salmonella typhimurium ( Salmonella typhimurium. ) and / or Escherichia coli ( Escherichia coli ).

[0049] Example 4

[0050] This example shows the fermentation effects of different screened bacterial agents on fermented feed made from monk fruit residue.

[0051] Experimental design: Four treatment groups were set up in the experiment: CK group: no bacterial agent was added, and the monk fruit residue was directly fermented naturally; MLF-1 group: strain MLF-1 was added to the monk fruit residue at an inoculation volume of 100 ml / kg and fermented for 30 days; JSF-9 group: strain JSF-9 was added to the monk fruit residue at an inoculation volume of 100 ml / kg and fermented for 30 days; MLL-5 group: strain MLL-5 was added to the monk fruit residue at an inoculation volume of 100 ml / kg and fermented for 30 days; the number of viable bacteria in each group was 10 7 -10 8 cfu / ml; after fermentation, the upper 10 cm of the sample was discarded, and the remaining samples were sampled according to the 4-point method. The nutritional components and fermentation quality after fermentation were tested. The indicators for nutritional component determination included dry matter, crude protein, crude fiber, neutral detergent fiber (NDF) and acid detergent fiber (ADF) content, and the results are shown in Table 4; the indicators for fermentation quality determination included lactic acid, acetic acid, propionic acid, butyric acid and ammonia nitrogen, and the results are shown in Table 5.

[0052]

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

[0054] As shown in Table 4, after the strains MLF-1, JSF-9 and MLL-5 fermented the monk fruit residue, the dry matter content was significantly reduced (p<0.05), among which the strain MLF-1 had the lowest dry matter content; after the strain MLF-1 fermented the monk fruit residue, the crude protein content was significantly increased (p<0.05), and there was no significant difference in crude protein between the strains JSF-9 and MLL-5 (p>0.05); after the strain MLF-1 fermented the monk fruit residue, the crude fiber, neutral detergent fiber (NDF) and acid detergent fiber (ADF) contents were significantly reduced (p<0.05), and there was no significant difference in crude fiber, neutral detergent fiber (NDF) and acid detergent fiber (ADF) between the strains JSF-9 and MLL-5 (p>0.05); this shows that for the monk fruit residue substrate, the strain MLF-1 has the most significant cellulose degradation effect and protein enhancement effect, indicating that the strain MLF-1 has the best fermentation effect on the monk fruit residue substrate.

[0055]

[0056] As shown in Table 5, after the strains MLF-1, JSF-9 and MLL-5 fermented the monk fruit residue, the amount of lactic acid was significantly increased (p<0.05). In terms of the increase effect, strain MLF-1> strain MLL-5> strain JSF-9; the acetic acid content of the strain MLF-1 fermentation group was significantly increased (p<0.05), and the acetic acid content of the other strains JSF-9 and MLL-5 fermentation groups was not significantly different from that of the control group (p>0.05); the three strains had no obvious effect on increasing the propionic acid and butyric acid content in the monk fruit residue feed (p>0.05); the three strains had a significant effect on reducing the ammonia nitrogen content in the monk fruit residue feed (p<0.05), and in terms of the reduction effect, strain MLF-1=strain MLL-5<strain JSF-9; this shows that strains MLF-1 and strain MLL-5 have the most obvious effect on reducing ammonia nitrogen in monk fruit residue, and strain JSF-9 has the second best effect on reducing ammonia nitrogen.

[0057] Considering the nutritional composition and quality of fermented feed, strain MLF-1 had the best fermentation effect on monk fruit residue. Strain JSF-9 and strain MLL-5 had certain fermentation effects on monk fruit residue, but the fermentation effect was not as good as that of strain MLF-1, indicating that among the monk fruit residue substrates, MLF-1 was the most suitable fermentation strain.

[0058] Example 5

[0059] This example shows the fermentation effect of the composite bacterial agent on the fermented feed of monk fruit residue.

[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 monk fruit residue, increase the lactic acid and acetic acid contents, and reduce the ammonia nitrogen content; strain JSF-9 can increase lactic acid and reduce ammonia nitrogen content; strain MLL-5 can increase lactic acid and reduce ammonia nitrogen content. In order to effectively improve the quality of monk fruit residue fermented feed and increase strain diversity, the research team considered preparing a composite bacterial agent with strains MLF-1, JSF-9, and MLL-5 and then fermenting it with monk fruit residue. The orthogonal experiment was used to optimize the strain ratio of the composite bacterial agent. The specific method was as follows: strains MLF-1, JSF-9, and MLL-5 were mixed to prepare a composite bacterial agent; the composite bacterial agent was then inoculated into the monk fruit residue at an inoculation rate of 100 ml / kg and fermented for 30 days. Among them, the effective viable count of Bacillus licheniformis JSF-9 was 1.2×10 8 cfu / ml, and the effective viable count of Bacillus subtilis MLF-1 was 3.5×10 7 cfu / ml, and the effective viable count of Bacillus sabdariffa MLL-5 was 7.5×10 7 cfu / ml; crude protein, lactic acid and ammonia nitrogen content were used as optimization indicators to select the optimal experimental group, as shown in Tables 6 and 7.

[0061]

[0062]

[0063] As shown in Table 7, in terms of crude protein, the improvement effects of experiments 1-4 and 9 were not as good as that of single strain MLF-1, and the improvement effect of experiment 6 was the most obvious; in terms of lactic acid content, the improvement effects of experiments 1-4 and 9 were not as good as that of single strain MLF-1, and the improvement effect of experiment 6 was the most obvious; in terms of ammonia nitrogen content, except for experiments 3 and 9, the reduction effects of other experimental groups were close to that of strain MLF-1; therefore, on the whole, the selection of the strain volume ratio of experiments 5-8 to prepare the composite bacterial agent had a better fermentation effect on monk fruit residue, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 was (4-6): (1-3): (1-3); the optimal experimental group was experiment 6, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 was 4:3:1.

[0064] From the perspective of extreme differences, the strain MLL-5 had the greatest impact on crude protein in fermented monk fruit residue feed, followed by strain MLF-1, and the strain JSF-9 had the least impact; the strain MLF-1 had the greatest impact on lactic acid in fermented monk fruit residue feed, followed by strain MLL-5, and the strain JSF-9 had the least impact; the strain JSF-9 had the greatest impact on ammonia nitrogen in fermented monk fruit residue feed, followed by strain MLL-5, and the strain MLF-1 had the least impact.

[0065] Example 6

[0066] This example is a feeding experiment of Jersey cattle with fermented feed made from monk fruit residue.

[0067] Forty seven-month-old Jersey cattle in good condition and of similar weight were randomly divided into four groups of 10 each. The control group (CK) was fed a base diet of whole corn silage; experimental group 1: fermented feed containing 10% monk fruit residue (fermented without sterilizing agents) was added to the base diet; experimental group 2: fermented feed containing 10% monk fruit residue (the compound inoculum in the fermented feed was added according to the proportion of Experiment 6 in Example 5); experimental group 3: fermented feed containing 15% monk fruit residue (the compound inoculum in the fermented feed was added according to the proportion of Experiment 6 in Example 5) was added to the base diet. The pre-feeding period was 7 days, the experimental period was 28 days, and the entire experiment lasted 35 days. The initial and final body weights of the Jersey cattle were measured, and the average daily gain (ADG) 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 episodes was observed and counted (one diarrhea episode was counted as 1, and two diarrhea episodes were counted as 2). The results are shown in Table 8.

[0068]

[0069] As shown in Table 8, in terms of average daily weight 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 weight gain of these three groups was significantly higher than that of experimental group 1 (p<0.05); in terms of feed-to-weight ratio, there was no significant difference between the control group and experimental group 2 and experimental group 3 (p>0.05), but the feed-to-weight ratio of these three groups was significantly lower than that of experimental group 1 (p<0.05); this shows that the fermented monk fruit residue with composite microbial agent (experimental group 2 and experimental group 3) can effectively replace the basic feed, while the fermented monk fruit residue without adding microbial agent has poor conversion effect, and the feeding effect is significantly reduced after replacing part of the basic feed; in terms of the number of diarrhea, the number of diarrhea in experimental group 2 and experimental group 3 was 0, which shows that the intestinal pathogens inhibit After the composite bacterial agent with several bacterial agents with different functions was prepared, it effectively reduced the number of diarrhea in Jersey cattle. The control group had 6 diarrhea times, and the experimental group 1 had 4 diarrhea times, which was slightly lower than the control group. This shows that the monk fruit in the monk fruit residue has certain antibacterial properties and is not easy to breed pathogenic bacteria. However, the single raw material of monk fruit residue cannot completely inhibit all pathogenic bacteria, and thus it will cause diarrhea in Jersey cattle. The above conclusions show that adding 10%-15% of fermented monk fruit residue prepared by the composite bacterial agent to the basal feed will not have an adverse effect on the growth performance and health status of cattle, and the number of diarrhea times is significantly reduced, indicating that the nutritional value of the fermented monk fruit residue of this application is improved after fermentation by the strain, thereby improving the disease resistance and immunity of Jersey cattle.

[0070] In summary, the Bacillus sp. MLF-1 screened by the applicant of the present invention has a good ability to produce β-1,4-glucanase, and has an antibacterial effect on Enterobacter aerogenes, Staphylococcus aureus, Salmonella typhimurium and / or Escherichia coli; Bacillus sp. MLF-1 has a good fermentation ability for monk fruit residue. After feeding the prepared monk fruit residue fermented feed to Jersey cattle, it was found that the feed can effectively replace the basic feed to reduce feed costs, improve the immunity of Jersey cattle and reduce the number of diarrhea episodes.

[0071] The above-described embodiments merely illustrate several embodiments of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Bacillus subtilis ( Bacillus altitudinis )MLF-1, its deposit number is GDMCC NO: 65789.

2. comprising the Bacillus sp. according to claim 1 ( Bacillus altitudinis ) MLF-1 composite bacterial agent.

3. The composite bacterial agent according to claim 2, characterized in that The composite bacterial agent is composed of Bacillus subtilis ( Bacillus altitudinis )MLF-1, Bacillus licheniformis ( Bacillus paralicheniformi s)JSF-9 and Bacillus saffron ( Bacillus safensis )MLL-5 is mixed in a volume ratio of (4-6): (1-3): (1-3); the licheniformis ( Bacillus paralicheniformi s) JSF-9 is deposited with GDMCC NO: 65786; the bacillus saffron ( Bacillus safensis ) The deposit number of MLL-5 is GDMCC NO: 65790.

4. The composite bacterial agent according to claim 3, characterized in that The composite bacterial agent is composed of Bacillus subtilis ( Bacillus altitudinis )MLF-1, Bacillus licheniformis ( Bacillus paralicheniformi s)JSF-9 and Bacillus saffron ( Bacillus safensis )MLL-5 was mixed in a volume ratio of 4:3:

1.

5. The highland Bacillus as claimed in claim 1 ( Bacillus altitudinis ) Use of MLF-1 or the composite bacterial agent as described in claim 2 in the preparation of fermented feed of monk fruit residue.

6. The highland Bacillus as claimed in claim 1 ( Bacillus altitudinis ) Application of MLF-1 in the production of β-1,4-glucanase.

7. The highland Bacillus as claimed in claim 1 ( Bacillus altitudinis ) Application of MLF-1 in the preparation of an antibacterial agent for intestinal pathogens, 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 ).

8. comprising the Bacillus sp. of claim 1 ( Bacillus altitudinis ) MLF-1 or the fermented feed of the composite microbial agent according to claim 2, characterized in that, The preparation method of the fermented feed is as follows: Bacillus altitudinis ) MLF-1 or composite bacterial agent is inoculated into the monk fruit residue at an inoculation rate of 100 ml / kg and fermented for 30 days.

9. Use of the fermented feed according to claim 8 in feeding Jersey cows.

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