Bacillus safensis MLL-5 as well as complex microbial inoculant and application thereof

Abstract

The invention relates to the technical field of microorganisms, in particular to bacillus safensis MLL-5 and a complex microbial inoculant and application thereof.The strain MLL-5 is separated from rumen contents of healthy cattle by a research group, and it is detected that the strain has the good beta-1, 4-glucanase producing capacity and can be used for producing beta-1, 4-glucanase. The antibacterial effect is achieved on escherichia coli, yersinia enterocolitica, shigella flexneri and salmonella typhimurium; the bacillus safensis MLL-5 has relatively good fermentation capability on the cassava residues, can effectively reduce the cyanide content of the cassava residue fermented feed and improve the protein content of the cassava residue fermented feed, is a good biological fermentation agent aiming at the cassava residues, and has good application prospects. After Jersey cattle are fed with manioc waste fermented feed prepared from the strain and the complex microbial inoculant, it is found that addition of 10%-15% of fermented manioc waste into daily ration does not have adverse effects on the growth performance and health conditions of the cattle, the number of diarrhea times is obviously reduced, and the manioc waste fermented feed is safe.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to Bacillus sabdariffa MLL-5 and a composite bacterial agent and application thereof. Background Art

[0002] Cassava is one of the three major tubers in the world, and is known as the "underground granary" and the "king of starch". Cassava residue is a major agricultural and sideline product produced by the production of starch and alcohol using cassava. Its main component is cellulose, and it also contains a small amount of protein. Due to the high crude fiber content, poor palatability, and difficulty in digestion of cassava residue, it is easy to cause problems such as low feed intake of livestock and poultry, and even serious problems such as animal refusal to eat, so it is not suitable for direct feeding. In recent years, feed biological fermentation technology has been continuously innovated, and biological treatment technology has been used to change the physical and chemical properties of cassava residue, making it a new type of feed resource with broad development prospects. In addition, cassava residue also contains a certain amount of cyanide, which can cause animal poisoning and affect the application of cassava residue in biological feed; the selection and matching of strains for fermenting cassava residue using microbial fermentation technology is very critical. There are many varieties of microorganisms that can be used for cassava residue fermentation, but the varieties and matching of microorganisms currently studied are very limited. Therefore, selecting strains suitable for cassava residue fermentation to screen and verify microorganisms is an important and indispensable research for the preparation of cassava residue into biological feed. In addition, seeking strain combinations to prepare composite bacterial agents can further effectively improve the utilization rate of cassava residue.

[0003] Therefore, we need to develop microorganisms suitable for the fermentation of cassava residue substrates, produce cassava 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 screening and verification of microorganisms, and develop microorganisms suitable for fermentation of cassava residue substrates. The microorganisms have good functions of reducing cyanide in cassava residue and reducing the toxic defects of cassava residue in preparing biological feed. In addition, it is necessary to study the composite strains for the production of cassava residue fermented feed and verify the feasibility of the feed in feeding animals.

[0005] In order to achieve the above purpose, the present invention screened out a new strain: Bacillus saffron ( Bacillus safensis )MLL-5, whose classification is named as: Bacillus safensis MLL-5, Chinese classification name: Bacillus shafusus MLL-5, preservation number is GDMCC NO: 65790; 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 February 10, 2025.

[0006] The present invention also includes a compound microbial agent containing the Bacillus safensis ( Bacillus safensis ). MLL-5.

[0007] Furthermore, the compound microbial agent further includes Bacillus altitudinis ( Bacillus altitudinis ). MLF-1 and / or Bacillus licheniformis ( Bacillus paralicheniformis ). JSF-9; the Bacillus altitudinis ( Bacillus altitudinis ). MLF-1, its taxonomic name is: Bacillus altitudinis MLF-1, the Chinese taxonomic name is: Bacillus altitudinis MLF-1, and the deposit number is GDMCC NO: 65789; this strain is deposited in the Guangdong Provincial Culture Collection Center of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit date is January 14, 2025; the Bacillus licheniformis Bacillus paralicheniformis JSF-9, the Chinese taxonomic name is: Bacillus licheniformis JSF-9, and the deposit number is GDMCC NO: 65786; this strain is deposited in the Guangdong Provincial Culture Collection Center of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit date is January 14, 2025.

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

[0009] Furthermore, the compound microbial agent is prepared by mixing Bacillus altitudinis ( Bacillus altitudinis ). MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis ). JSF-9 and Bacillus safensis ( Bacillus safensis ). MLL-5 in a volume ratio of 1:1:2.

[0010] The present invention also includes the application of the Bacillus safensis ( Bacillus safensis ). MLL-5 or the compound microbial agent in the preparation of cassava residue fermented feed.

[0011] The present invention also includes the application of the Bacillus safensis ( Bacillus safensis ). MLL-5 or the compound microbial agent in reducing the cyanide content of cassava residue fermented feed.

[0012] The present invention also includes the application of the Bacillus safensis ( Bacillus safensis ). MLL-5 in the production of β-1,4-glucanase.

[0013] The present invention also includes the application of the Bacillus safensis ( Bacillus safensis ) MLL-5 in the preparation of an intestinal pathogen bacteriostatic agent, wherein the intestinal pathogens are Escherichia coli ( Escherichia coli ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ) and / or Salmonella typhimurium ( Salmonella typhimurium. ).

[0014] The present invention also includes a fermented feed containing the Bacillus safensis ( Bacillus safensis ) MLL-5 or the composite bacterial agent as described above. The preparation method of the fermented feed is as follows: inoculate the Bacillus safensis ( Bacillus safensis ) MLL-5 or the composite bacterial agent into cassava residue at an inoculation amount of 100 ml / kg and ferment for 30 days to obtain the fermented feed.

[0015] The present invention also includes the application of the fermented feed in feeding Jersey cows.

[0016] The present invention also includes the application of the fermented feed in the preparation of the basic diet for Jersey cows.

[0017] The present invention also includes the application of the fermented feed in reducing the diarrhea frequency of Jersey cows.

[0018] The present invention also includes the application of the Bacillus safensis ( Bacillus safensis ) MLL-5 or the composite bacterial agent as described above in reducing the cyanide content of the cassava residue fermented feed and / or increasing the protein content of the cassava residue fermented feed.

[0019] The present invention has the following beneficial effects: 1. The strain MLL-5 of the present invention is isolated from the rumen content of healthy cows by the research group. After detection, this strain has good β-1,4-glucanase production ability and has antibacterial effects on Escherichia coli, Yersinia enterocolitica, Shigella flexneri and / or Salmonella typhimurium; Bacillus safensis MLL-5 has good fermentation ability for cassava residue, can effectively reduce the cyanide content of the cassava residue fermented feed, increase the protein content of the cassava residue fermented feed, and effectively overcome the technical defect that excessive cyanide content in cassava residue may cause animal poisoning. It is a good biological ferment for cassava residue. After feeding Jersey cows with the cassava residue fermented feed prepared by using this strain and the composite bacterial agent, it is found that: for the cassava residue fermented feed prepared by using the composite bacterial agent, adding 15% to the basic diet has no adverse effects on the growth performance and health status of cows, and the diarrhea frequency is significantly reduced. This shows that the nutritional value of the fermented cassava residue is improved after being fermented by the strain, the toxicity of the fermented feed can be reduced, the disease resistance and immunity of Jersey cows can be improved, and it is a safe and good fermented feed.

[0020] 2. This 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. Through the adjustment of the cassava residue fermentation experiment, the optimal volume ratio of the compound microbial agent is obtained. The fermented feed prepared by fermenting cassava 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 cassava residue waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 It is a colony morphology diagram of strain MLF-1.

[0022] Figure 2 It is a colony morphology diagram of strain JSF-9.

[0023] Figure 3 It is a colony morphology diagram of strain MLL-5.

[0024] Figure 4 It is the decolorization result of strain MLL-5 on Congo red-stained carboxymethyl cellulose sodium screening medium.

[0025] BIOLOGICAL MATERIAL DEPOSIT INFORMATION

[0026] The strain information deposited in this application is: Bacillus altitudinis ( Bacillus altitudinis ) MLF-1, its taxonomic name is: Bacillus altitudinis MLF-1, the Chinese taxonomic name is: Bacillus altitudinis MLF-1, the deposit number is GDMCC NO: 65789; this strain is deposited in the Guangdong Provincial Culture Collection of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit date is January 14, 2025.

[0027] The strain information deposited in this application is: Bacillus licheniformis ( Bacillus paralicheniformis ) JSF-9, its taxonomic name is: Bacillus paralicheniformis JSF-9, the Chinese taxonomic name is: Bacillus licheniformis JSF-9, the deposit number is GDMCC NO: 65786; this strain is deposited in the Guangdong Provincial Culture Collection of Microorganisms, address: 5th Floor, Building 59, No. 100 Compound, Xianlie Middle Road, Guangzhou, and the deposit date is January 14, 2025.

[0028] The strain information deposited in this application is: Bacillus safensis ( Bacillus safensis ) MLL-5, its taxonomic name is: Bacillus safensisMLL-5, named as Bacillus safensis MLL-5 in Chinese classification, with the preservation number of GDMCC NO: 65790; this strain is preserved in the Guangdong Provincial Culture Collection Center of Microorganisms, address: 5th Floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, and the preservation date is February 10, 2025. Detailed implementation manners

[0029] All the features disclosed in this specification, or all the steps in the disclosed methods or processes, except for mutually exclusive features and / or steps, can be combined in any way.

[0030] Any feature disclosed in this specification (including any additional claims, abstract), unless specifically stated, each feature is only an example among a series of equivalent or similar features.

[0031] Example 1

[0032] This example is for the isolation and identification of the strain.

[0033] Isolation and purification of the strain: 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 taken back to the laboratory in a liquid nitrogen tank for storage at -80 °C for later use. The rumen contents stored at -80 °C were thawed at low temperature in a sterile operating bench 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 a 10-fold increasing sample homogenate was made according to the above operation sequence. Each time the dilution increased, a new sterile pipette or tip was used. 100 μl of 3 - 4 appropriate gradient dilutions were respectively taken and spread on the surface of NA agar medium. After spreading, the plate was left standing to allow the inoculum to be completely absorbed by the medium, then the petri dish was 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 in 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 plate 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. The cellulase-producing strains were determined according to the ratio of the diameter of the clear zone (D) to the diameter of the colony (d) (D / d), and each strain was repeated 3 times. A total of 15 cellulase-producing strains were screened out, and the strains with a larger ratio of the clear zone size to the colony size were selected through the ratio screening, and the enzyme production conditions were optimized. The specific results are shown in Table 1.

[0034]

[0035] Three strains with a large D / d ratio were selected from Table 1 for strain identification.

[0036] (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 detection of housekeeping genes, 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 .

[0037] (2)Identification of strain JSF-9: ① Morphological identification is as Figure 2 shown: The JSF-9 strain was inoculated on an LB plate medium and cultured for 24 h. The colonies were 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 detection of housekeeping genes, it was determined that the strain was closely related to Bacillus paralicheniformis ... and, combined with morphological identification, the strain was classified and named as Bacillus paralicheniformis .

[0038] (3)Identification of strain MLL-5: ① Morphological identification is as Figure 3 shown: The MLL-5 strain was inoculated on an LB plate medium and cultured for 24 h. The colonies were 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 detection of housekeeping genes, it was determined that the strain was closely related toBacillus safensis With a relatively close genetic relationship and combined with morphological identification, this strain was classified and named as Bacillus safensis .

[0039] Example 2

[0040] This example is for the determination of the enzyme production of strain MLL-5 and its optimal enzyme production conditions.

[0041] (1) Seed liquid medium: 10.0 g of peptone, 5.0 g of yeast extract powder, 10.0 g of sodium chloride, 1 L of distilled water, natural pH, sterilized at 121 °C for 20 min.

[0042] (2) Liquid fermentation medium: 50 g of bagasse, 10.0 g of peptone, 10.0 g of sodium chloride, 1 L of distilled water, natural pH, sterilized at 121 °C for 20 min.

[0043] (3) Inoculate strain MLL-5 into the seed liquid medium and shake culture at 39 °C and 160 r / min until the OD 600 is 0.6 to obtain the seed liquid.

[0044] (4) Inoculate the seed liquid 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. Using bagasse as the inducer, induce the strain to secrete cellulase. The shaking speed of the shaker is 160 r / min, and ferment at 39 °C for 2 d, 3 d, 4 d, and 5 d. Centrifuge the fermentation broth at 4 °C and 6000 r / min for 10 min, and take the supernatant, which is the crude enzyme solution. Finally, the enzyme production of strain MLL-5 is as shown in Figure 4 shown: The figure shows that strain MLL-5 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 is shown in Table 2.

[0045]

[0046] As can be seen from Table 2, for strain MLL-5, when the inoculation amount is 3% and the shake flask fermentation time is 3 d, the best enzyme production effect of endo-β-1,4-glucanase is 976.55 U / ml; when the inoculation amount is 9% and the shake flask fermentation time is 3 d, the best enzyme production effect of exo-β-1,4-glucanase is 581.08 U / ml.

[0047] Example 3

[0048] This example is for the antibacterial effect of strain MLL-5 against common intestinal pathogenic bacteria.

[0049] The agar diffusion method with punching was used to determine the antibacterial effect of strain MLL-5 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 ) antibacterial activity, each sample was repeated 3 times; the obtained results are shown in Table 3.

[0050]

[0051] Note: “—” in the table indicates no inhibitory effect, a, b, c: indicate significant differences in the data of the same column (p < 0.05).

[0052] As can be seen from Table 3, the strain MLL-5 has antibacterial effects on Escherichia coli ( Escherichia coli ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ) and / or Salmonella typhimurium ( Salmonella typhimurium. ), and has no antibacterial effect on Staphylococcus aureus ( Staphylococcus aureus ) and Enterobacter aerogenes ( Enterobacter aerogenes ); the inhibition zone of Escherichia coli ( Escherichia coli ) > Yersinia enterocolitica ( Yersinia enterocolitica ) > Shigella flexneri ( Shigella flexneri ) > Salmonella typhimurium ( Salmonella typhimurium. ) and reaches a significant level (p < 0.05), indicating that the strain MLL-5 has the best antibacterial effect on Escherichia coli ( Escherichia coli ), followed by Yersinia enterocolitica ( Yersinia enterocolitica ), then Shigella flexneri ( Shigella flexneri ) and / or Salmonella typhimurium ( Salmonella typhimurium. ).

[0053] Example 4

[0054] This example is about the fermentation effects of different bacterial agents screened on cassava residue fermented feed.

[0055] Experimental design: Four treatment groups were set up in the experiment: CK group: Without adding any bacterial agents, directly ferment the cassava residue naturally; MLF-1 group: Add strain MLF-1 and inoculate it into the cassava residue at an inoculation amount of 100 ml / kg for fermentation for 30 days; JSF-9 group: Add strain JSF-9 and inoculate it into the cassava residue at an inoculation amount of 100 ml / kg for fermentation for 30 days; MLL-5 group: Add strain MLL-5 and inoculate it into the cassava residue at an inoculation amount of 100 ml / kg for fermentation for 30 days; The viable bacteria count of each group of bacterial agents was 10 6 -10 8 cfu / ml; After fermentation was completed, discard the upper 10 cm of the sample, and the remaining samples were sampled by the quartering method, and the nutritional components and fermentation quality of the fermented samples were detected. The indicators for the determination of nutritional components included dry matter, crude protein, crude fiber, neutral detergent fiber (NDF), and acid detergent fiber (ADF) contents. The results are shown in Table 4; The indicators for the determination of fermentation quality included lactic acid, acetic acid, propionic acid, butyric acid, and cyanide contents (among them, the determination method for cyanide was: pyridine barbituric acid method). The results are shown in Table 5.

[0056]

[0057] 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.

[0058] As can be seen from Table 4, after the cassava residue was fermented by strains MLF-1, JSF-9, and MLL-5, except that the dry matter content in the JSF-9 group was significantly reduced (p < 0.05), the dry matter contents in the experimental groups of other strains had no significant differences compared with CK (p > 0.05); After the cassava residue was fermented by strains MLF-1, JSF-9, and MLL-5, the crude protein content was significantly increased (p < 0.05), and the strain MLL-5 had the most obvious effect on the improvement of crude protein; After the cassava residue was fermented by strain MLL-5, strain MLF-1, and strain JS-9, the crude fiber, neutral detergent fiber (NDF), and acid detergent fiber (ADF) contents were significantly reduced (p < 0.05), and among them, the strain MLL-5 had the most obvious reduction effect; It shows that for the cassava residue substrate, the strain MLL-5 has the most significant cellulose degradation effect and protein improvement effect, indicating that the strain MLL-5 has the best fermentation effect on the cassava residue substrate.

[0059]

[0060] As can be seen from Table 5, after the fermentation of cassava residues by strains MLF-1, JSF-9 and MLL-5, the amounts of lactic acid and acetic acid increased significantly (p<0.05). In terms of the improvement effect, for lactic acid, strain MLF-1 > strain JSF-9 > strain MLL-5; for acetic acid, strain MLL-5 > strain MLF-1 > strain JSF-9. The content of propionic acid in the fermentation group of strain MLF-1 was not significantly different from that in the control group (p>0.05), while the content of propionic acid in the fermentation groups of strains JSF-9 and MLL-5 decreased significantly (p<0.05). The content of butyric acid in the fermentation group of strain MLF-1 increased significantly. The difference in the butyric acid content between the experimental group of strain MLL-5 and the control group was not significant (p>0.05), and the content of butyric acid in the experimental group of strain JSF-9 decreased significantly compared with the control group (p<0.05). For cyanide, no strains were added to the CK group for fermentation. The cyanide content of the cassava residues in this application was as high as 59.23 mg / kg, which did not meet the requirements of feed safety. After fermentation with the bacterial agent in this application, the reduction of cyanide by strain MLF-1 was not obvious, and there was no significant difference from the CK group (p>0.05), which could not meet the requirements of feed safety. The content of cyanide in the strain JSF-9 decreased significantly (p<0.05), reaching below 35 mg / kg, less than 50 mg / kg, meeting the requirements of GB 13078-2017 and NY5072-2002 for other compound feeds. The strain MLL-5 had a more obvious decrease compared with the other two strains, reaching 26.80 mg / kg, and it was the best strain among the three strains for decomposing cyanide.

[0061] Considering the nutritional components and quality of the fermented feed, the fermentation effect of using strain MLL-5 on cassava residues was the best, especially its ability to decompose cyanide, with the best reduction effect. Strains JSF-9 and MLF-1 had a certain fermentation effect on cassava residues, but their fermentation effects and the reduction effect of cyanide were not as good as those of strain MLL-5, indicating that the most suitable fermentation strain in the cassava residue substrate was MLL-5.

[0062] Example 5

[0063] This example was about the fermentation effect of the compound bacterial agent on cassava residue fermented feed.

[0064] According to the experimental results of Example 4, we found that: Strain MLL-5 can increase the crude protein content, reduce the crude fiber, neutral detergent fiber, and acid detergent fiber contents of cassava residue, increase the lactic acid and acetic acid contents, and reduce the cyanide content; Strain JSF-9 can increase the crude protein content, lactic acid, and acetic acid contents; Strain MLF-1 can increase the crude protein content, reduce the crude fiber, neutral detergent fiber, and acid detergent fiber contents of cassava residue, increase the lactic acid and acetic acid contents, and reduce the cyanide content. In addition, in our previous studies, it was found that these three strains have different antibacterial characteristics against different intestinal pathogens. In order to effectively improve the quality of fermented cassava residue feed and increase the strain diversity to enhance the inhibitory function against animal diarrhea pathogens, the research group considered preparing a compound bacterium agent from strains MLF-1, JSF-9, and MLL-5 and then fermenting it with cassava residue, and using an orthogonal experiment to optimize the strain ratio of the compound bacterium agent. The specific method is as follows: Mix strains MLF-1, JSF-9, and MLL-5 to prepare a compound bacterium agent, and then inoculate the compound bacterium agent into cassava residue at an inoculation amount of 100 ml / kg and ferment for 30 days. 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 viable bacteria of each strain were used to select the optimal experimental group with the crude protein and cyanide contents as the optimization indexes, as shown in Tables 6 and 7 specifically.

[0065]

[0066] As can be seen from Table 7, in terms of crude protein, the improvement effects of Tests 1, 3, 5, and 6 are better than those of single-strain MLL-5 fermentation, and the improvement effect of Test 1 is the most obvious; in terms of cyanide content, the reduction effects of Tests 1, 3, 5, 6, and 9 are better than those of single-strain MLL-5 fermentation, and the reduction effects of other experimental groups are not as good as those of single-strain MLL-5 fermentation; Therefore, overall, selecting the strain volume ratios of Tests 1, 3, 5, and 6 to prepare the compound bacterium agent has a good fermentation effect on cassava residue, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 is (1-2):(2-3):(2-6); The optimal experimental group is Test 1, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 is 1:1:2.

[0067] In terms of the range, the strain MLL-5 has the greatest impact on the crude protein of fermented cassava residue feed, followed by the strain MLF-1, and the strain JSF-9 has the least impact; the strain MLL-5 has the greatest impact on the cyanide in fermented cassava residue feed, followed by the strain JSF-9, and the strain MLF-1 has the least impact.

[0068] Example 6

[0069] This example is a feeding experiment of cassava residue fermented feed on Jersey cows.

[0070] Forty 7-month-old Jersey cows with good body conditions 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 basal diet; Experimental group 1: 10% fermented cassava residue (fermented without a fungicide) was added to the basal diet; Experimental group 2: 10% fermented cassava residue (the compound fungicide was added according to the addition ratio of Test 1 in Example 5) was added to the basal diet; Experimental group 3: 15% fermented cassava residue (the compound fungicide was added according to the addition ratio of Test 1 in Example 5) was added to the basal diet; The pre-feeding period was 7 days, the test period was 28 days, and 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, 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.

[0071]

[0072] As can be seen from Table 8, in terms of average daily gain, there was no significant difference among Experimental Group 2, Experimental Group 3 and the control group (CK) (p>0.05). The daily gains of Experimental Group 2, Experimental Group 3 and the control group (CK) were significantly higher than that of Experimental Group 1 (p<0.05), indicating that after fermenting cassava residue with the compound microbial agent (Experimental Group 2, Experimental Group 3), the feeding level of the basal diet could be achieved, part of the basal diet could be saved, and the feeding cost of farmers was effectively reduced. However, the average daily gain of Experimental Group 1 was lower than that of the control group and reached a significant level (p<0.05), which indicated that the conversion effect of the cassava residue without adding the microbial agent was not good, and the feeding effect decreased significantly after substituting part of the basal diet. In terms of feed-to-gain ratio: the feed-to-gain ratio of Experimental Group 1 was significantly higher than that of Experimental Group 2, Experimental Group 3 and the control group (p<0.05), and there was no significant difference in the feed-to-gain ratio among Experimental Group 2, Experimental Group 3 and the control group (p>0.05), which indicated that the feed utilization rate of the cassava residue without microbial agent fermentation (Experimental Group 1) could not reach the effect of the basal diet, while the feed utilization rate of the cassava residue fermented with the microbial agent was equivalent to that of the basal diet. In terms of the number of diarrhea: the number of diarrhea in the control group reached 5 times, the number of diarrhea in Experimental Group 1 reached 8 times, which was higher than that in the control group, and the number of diarrhea in Experimental Group 2 and Experimental Group 3 was 0 times. This indicated that after preparing the compound microbial agent from several microbial agents with the function of inhibiting intestinal pathogenic bacteria, the number of diarrhea in Jersey cows was effectively reduced. The number of diarrhea in Experimental Group 1 of Jersey cows fed with the cassava residue without microbial agent fermentation was the highest, indicating that the cassava residue feed without compound microbial agent fermentation might breed other pathogenic bacteria, resulting in a higher number of diarrhea than that in the control group. Based on the above conclusions, adding 10%-15% of the cassava residue fermented with the compound microbial agent to the basal diet would not have an adverse impact on the growth performance and health status of cows, and the number of diarrhea decreased significantly, indicating that the nutritional value of the fermented cassava residue in this application was improved after being fermented by the strain, the toxicity of the fermented feed could be reduced, and the disease resistance function and immunity of Jersey cows could be improved.

[0073] In summary, the Bacillus safensis MLL-5 self-screened by the applicant of the present invention has good β-1,4-glucanase production ability and has antibacterial effects on Escherichia coli, Yersinia enterocolitica, Shigella flexneri and / or Salmonella typhimurium; Bacillus safensis MLL-5 has good fermentation ability for cassava residue, can effectively improve the protein content of the cassava residue fermented feed and reduce the cyanide content. After feeding the prepared cassava residue fermented feed to Jersey cows, it was found that adding 10%-15% of the fermented cassava residue to the basal diet would not have an adverse impact on the growth performance and health status of cows, and the number of diarrhea decreased significantly, indicating that the nutritional value of the fermented cassava residue in this application was improved after being fermented by the strain, the toxicity of the fermented feed could be reduced, and the disease resistance function and immunity of Jersey cows could be improved. It is a safe and good fermented feed.

[0074] The above-described embodiments merely represent several implementation manners of the present invention. 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 fall within the protection scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. Bacillus safensis ( Bacillus safensis ), MLL-5, characterized in that The Bacillus safensis ( Bacillus safensis ) MLL-5 has a deposit number of GDMCC NO: 65790.

2. A composite microbial agent comprising the Bacillus safensis Bacillus safensis MLL-5 described in claim 1.

3. The composite bacterial agent according to claim 2, characterized in that, The composite bacterium agent further comprises Bacillus altitudinis ( Bacillus altitudinis ), MLF-1 and / or Bacillus licheniformis ( Bacillus paralicheniformis ), JSF-9; the preservation number of the Bacillus altitudinis ( Bacillus altitudinis ), MLF-1 is GDMCC NO: 65789; the preservation number of the Bacillus licheniformis ( Bacillus paralicheniformis ), JSF-9 is GDMCC NO: 65786.

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

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

2.

6. Use of Bacillus safensis ( Bacillus safensis ) MLL-5 or the composite microbial agent according to claim 2 in the preparation of cassava residue fermented feed.

7. The application of Bacillus safensis ( Bacillus safensis ) MLL-5 in reducing the cyanide content of cassava residue fermented feed.

8. The application of Bacillus safensis ( Bacillus safensis ) MLL-5 in the preparation of an intestinal pathogen bacteriostatic agent, characterized in that The intestinal pathogenic bacteria are Escherichia coli ( Escherichia coli ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ) and / or Salmonella typhimurium ( Salmonella typhimurium. ).

9. A fermented feed containing Bacillus safensis Bacillus safensis MLL-5 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 safensis ( Bacillus safensis ) MLL-5 or the compound microbial agent is inoculated into the cassava residue at an inoculation amount of 100 ml / kg and fermented for 30 days to obtain the product.

22. Application of the fermented feed according to claim 9 in feeding Jersey cows.

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