Bacillus saffron MLL-5 and its composite bacterial agent and application

Abstract

The present invention relates to the field of microbial technology, and in particular to Bacillus sabdariffa MLL-5 and its composite bacterial agent and application. The strain MLL-5 of the present invention is isolated by a research team from the rumen contents of healthy cattle. Testing shows that the strain has good β-1,4-glucanase production ability and has antibacterial effects on Escherichia coli, Yersinia enterocolitica, Shigella flexneri and Salmonella typhimurium. The Bacillus sabdariffa MLL-5 has good fermentation ability for cassava residue, can effectively reduce the cyanide content of cassava residue fermented feed and increase the protein content of cassava residue fermented feed, and is a good biological fermentation agent for cassava residue. After feeding Jersey cattle with cassava residue fermented feed prepared by using the strain and the composite bacterial agent, it was found that adding 10%-15% of fermented cassava residue to the diet did not have an adverse effect on the growth performance and health of the cattle, and significantly reduced the number of diarrhea episodes. The Bacillus sabdariffa MLL-5 is a safe fermented feed.

Description

Technical Field

[0001] The present invention relates to the field of microbial technology, in particular to Bacillus saffron MLL-5 and a composite bacterial agent and application thereof. Background Art

[0002] Cassava is one of the world's three major tubers, known as the "underground granary" and the "king of starch." Cassava residue is a major agricultural byproduct produced from the production of starch and alcohol. Its primary component is cellulose, with a small amount of protein. Due to its high crude fiber content, cassava residue has poor palatability and is difficult to digest, which can lead to low feed intake in livestock and poultry, and in severe cases, even cause animals to refuse to eat, making it unsuitable for direct feeding. In recent years, feed biofermentation technology has continued to innovate. Biotreatment techniques have been used to modify the physical and chemical properties of cassava residue, making it a novel feed resource with broad development prospects. However, cassava residue also contains a certain amount of cyanide, which can cause animal poisoning and thus hinder its application in biofeeds. The selection and combination of strains for fermenting cassava residue using microbial fermentation technology is crucial. While numerous microbial species are available for cassava residue fermentation, the currently studied microbial species and combinations are very limited. Therefore, extensive screening and validation of strains suitable for cassava residue fermentation is essential for the preparation of cassava residue into biofeed. In addition, seeking strain combinations to prepare composite microbial 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 amount of screening and verification of microorganisms, and develop microorganisms suitable for the fermentation of cassava residue substrates. These microorganisms have a good function 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 use of composite strains for the production of cassava 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 saffron ( Bacillus wise )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.

[0006] The present invention also includes the Bacillus sabdariffa ( Bacillus safensis ) MLL-5 composite bacterial agent.

[0007] Furthermore, the composite bacterial agent also includes Bacillus subtilis ( Altitude Bacillus ) MLF-1 and / or Bacillus licheniformis ( Bacillus paralicheniformis )JSF-9; the Bacillus subtilis ( Bacillus height )MLF-1, its classification is named as: Altitude Bacillus MLF-1, Chinese classification name: Bacillus licheniformis 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; the Bacillus licheniformis 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.

[0008] Furthermore, the composite bacterial agent is composed of Bacillus subtilis ( Altitude Bacillus )MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis )JSF-9 and Bacillus sabdariffa ( Bacillus safensis )MLL-5 was prepared by mixing in a volume ratio of (1-2): (2-3): (2-6).

[0009] Furthermore, the composite bacterial agent is composed of Bacillus subtilis ( Altitude Bacillus )MLF-1, Bacillus licheniformis ( Bacillus paralicheniformis )JSF-9 and Bacillus sabdariffa ( Bacillus safensis )MLL-5 was mixed in a volume ratio of 1:1:2.

[0010] The present invention also includes the Bacillus sabdariffa ( Bacillus safensis ) Application of MLL-5 or the composite bacterial agent in the preparation of cassava residue fermented feed.

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

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

[0013] The present invention also includes the Bacillus sabdariffa ( Bacillus safensis ) Application of MLL-5 in the preparation of an antibacterial agent for enteric pathogens, wherein the enteric 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 the Bacillus sabdariffa ( Bacillus safensis ) MLL-5 or the composite microbial agent fermented feed, the fermented feed preparation method is: Bacillus sabdariffa ( Bacillus safensis ) MLL-5 or composite bacterial agent is inoculated into cassava residue at an inoculum rate of 100 ml / kg and fermented for 30 days.

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

[0016] The present invention also includes the use of the fermented feed in preparing a basic diet for Jersey cattle.

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

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

[0019] The present invention has the following beneficial effects: 1. The strain MLL-5 of the present invention was isolated by the research team from the rumen contents of healthy cattle. After testing, the strain has good β-1,4-glucanase production ability and has an antibacterial effect on Escherichia coli, Yersinia enterocolitica, Shigella flexneri and / or Salmonella typhimurium; Bacillus flexneri MLL-5 has good fermentation ability on cassava residue, can effectively reduce the cyanide content of cassava residue fermented feed, increase the protein content of cassava residue fermented feed, and effectively reduce the cyanide content of cassava residue. The technical defect that too high may cause animal poisoning is a good biological fermentation agent for cassava residue. After feeding Jersey cattle with cassava residue fermented feed prepared by this strain and compound microbial agent, it was found that: adding 15% of cassava residue fermented feed prepared by compound microbial agent to the basic daily diet had no adverse effect on the growth performance and health status of cattle, and the number of diarrhea episodes was significantly reduced, indicating that the nutritional value of fermented cassava residue was improved after fermentation by the strain, which can reduce the toxicity of fermented feed and improve the disease resistance and immunity of Jersey cattle. It is a safe and good fermented feed.

[0020] 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 through adjustment of the cassava residue fermentation experiment. The fermented feed prepared by fermenting cassava residue with the composite bacterial agent can effectively improve the utilization rate of the feed. The research on this strain, composite bacterial agent and fermentation method provides good support and technical means for solving cassava residue waste. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the colony morphology of strain MLF-1.

[0022] Figure 2 The colony morphology of strain JSF-9.

[0023] Figure 3 This is the colony morphology of strain MLL-5.

[0024] Figure 4 The decolorization results of strain MLL-5 on Congo red-stained sodium carboxymethylcellulose screening medium.

[0025] Biomaterial deposit information

[0026] 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.

[0027] The strain information deposited in this application is: Bacillus licheniformis ( Bacillus paralicheniformis )JSF-9, its classification is named: 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.

[0028] The strain information deposited in this application is: Bacillus saffron ( Bacillus safensis )MLL-5, its classification is named as: Bacillus safensisMLL-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

[0029] 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.

[0030] 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.

[0031] Example 1

[0032] This example is about the isolation and identification of strains.

[0033] 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.

[0034]

[0035] Three bacterial strains with large D / d ratios were selected from Table 1 for species identification.

[0036] (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 .

[0037] (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 .

[0038] (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 .

[0039] Example 2

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

[0041] (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.

[0042] (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.

[0043] (3) Inoculate strain MLL-5 into seed liquid culture medium and culture at 39°C and 160 rpm until OD 600 is 0.6, and seed liquid is obtained.

[0044] (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. The enzyme production of strain MLL-5 was finally obtained as follows: Figure 4 As shown in the figure: The strain MLL-5 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.

[0045]

[0046] As shown in Table 2, the strain MLL-5 had the best endo-β-1,4-glucanase production of 976.55 U / ml at a 3% inoculation and 3-day shake flask fermentation; and the best exo-β-1,4-glucanase production of 581.08 U / ml at a 9% inoculation and 3-day shake flask fermentation.

[0047] Example 3

[0048] This example shows the antibacterial effect of strain MLL-5 on common intestinal pathogens.

[0049] The punch agar diffusion method was used to determine the activity of strain MLL-5 against Staphylococcus aureus ( Staphylococcus golden) 、Escherichia coli ( Escherichia coli ), Salmonella typhimurium ( Salmonella typhoid fever ), 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.

[0050]

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

[0052] As shown in Table 3, strain MLL-5 has a strong Escherichia coli ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ) and / or Salmonella typhimurium ( Salmonella typhimurium. ) has antibacterial effect on Staphylococcus aureus ( Staphylococcus aureus ) and Enterobacter aerogenes ( Enterobacter aerogenes ) has no antibacterial effect; inhibition zone Escherichia coli ( Escherichia coli ) >Yersinia enterocolitica ( Yersinia enterocolitica ) > Shigella flexneri ( Shigella flexneri ) >Salmonella typhimurium( Salmonella typhimurium. ) and reached a significant level (p<0.05), indicating that strain MLL-5 has an effect on Escherichia coli ( Escherichia coli ) had the best antibacterial effect, followed by Yersinia enterocolitica ( Yersinia enterocolitica ), followed by Shigella flexneri ( Shigella flexneri ) and / or Salmonella typhimurium ( Salmonella typhimurium. ).

[0053] Example 4

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

[0055] Experimental design: Four treatment groups were set up in the experiment: CK group: no bacterial agent was added, and cassava residue was used for natural fermentation; MLF-1 group: strain MLF-1 was added to cassava residue at an inoculum of 100 ml / kg and fermented for 30 days; JSF-9 group: strain JSF-9 was added to cassava residue at an inoculum of 100 ml / kg and fermented for 30 days; MLL-5 group: strain MLL-5 was added to cassava residue at an inoculum of 100 ml / kg and fermented for 30 days; the number of viable bacteria in each group was 10 6 -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 cyanide content (wherein, the determination method of cyanide was: pyridine barbituric acid method), and the results are shown in Table 5.

[0056]

[0057] 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.

[0058] As shown in Table 4, after fermentation of cassava residue by strains MLF-1, JSF-9 and MLL-5, the dry matter content of the strain JSF-9 group was significantly reduced (p<0.05), while the dry matter content of the other strain experimental groups had no significant difference compared with CK (p>0.05); after fermentation of cassava residue 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 increasing crude protein; after fermentation of cassava residue by strains 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), among which the strain MLL-5 had the most obvious reduction effect; this indicates that for cassava residue substrate, the strain MLL-5 has the most significant effect on cellulose degradation and protein improvement, indicating that the strain MLL-5 has the best fermentation effect on cassava residue substrate.

[0059]

[0060] As shown in Table 5, after the fermentation of cassava residue by strains MLF-1, JSF-9 and MLL-5, the amount 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 propionic acid content of the fermentation group of strain MLF-1 was not significantly different from that of the control group (p>0.05), and the propionic acid content of the fermentation groups of strains JSF-9 and MLL-5 was significantly reduced (p<0.05); the butyric acid content of the fermentation group of strain MLF-1 was significantly increased, and the butyric acid content of the experimental group of strain MLL-5 was similar to that of the control group. The difference was not significant (p>0.05), and the butyrate content of the experimental group of strain JSF-9 was significantly lower than that of the control group (p<0.05); for cyanide, the CK group did not add any strains for fermentation, and the cyanide content of the cassava residue of this application was as high as 59.23 mg / kg, which could not meet the requirements of feed safety. After fermentation with the bacterial agent of this application, the strain MLF-1 did not significantly reduce the cyanide content, and the difference with the CK group was not significant (p>0.05), which could not meet the requirements of feed safety. The strain JSF-9 significantly reduced the cyanide content (p<0.05), which could reach below 35 mg / kg and less than 50 mg / kg, meeting the requirements of GB 13078-2017 and NY5072-2002 for other compound feeds, and the strain MLL-5 decreased more significantly than the other two strains, reaching 26.80 mg / kg, which was the best strain for decomposing cyanide among the three strains.

[0061] Considering the nutritional composition and quality of fermented feed, strain MLL-5 has the best fermentation effect on cassava residue, especially the ability to decompose cyanide and the best reduction effect. Strain JSF-9 and strain MLF-1 have certain fermentation effects on cassava residue, but the fermentation effect and cyanide reduction effect are not as good as strain MLL-5, indicating that among cassava residue substrates, the most suitable fermentation strain is MLL-5.

[0062] Example 5

[0063] This example shows the fermentation effect of the composite 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 content of cassava residue, increase the lactic acid and acetic acid content, and reduce the cyanide content; strain JSF-9 can increase the crude protein content, lactic acid and acetic acid content; strain MLF-1 can increase the crude protein content, reduce the crude fiber, neutral detergent fiber, and acid detergent fiber content of cassava residue, increase the lactic acid and acetic acid content, and reduce the cyanide content. In addition, in our previous studies, we found that these three strains have different intestinal pathogens. The research team considered preparing a composite microbial agent with strains MLF-1, JSF-9, and MLL-5 and then fermenting it with cassava residue. The strain ratio of the composite microbial agent was optimized by orthogonal experiments. The specific method was as follows: strains MLF-1, JSF-9, and MLL-5 were mixed to prepare a composite microbial agent, and then the composite microbial agent was inoculated into cassava residue at an inoculum rate of 100 ml / kg and fermented for 30 days. Among them, the effective viable bacteria 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 and cyanide content were used as optimization indicators to select the optimal experimental group for each strain, as shown in Tables 6 and 7.

[0065]

[0066] As shown in Table 7, in terms of crude protein, the improvement effects of Experiments 1, 3, 5 and 6 were better than that of the fermentation of single strain MLL-5, and the improvement effect of Experiment 1 was the most obvious; in terms of cyanide content, the reduction effects of Experiments 1, 3, 5, 6 and 9 were better than that of the fermentation of single strain MLL-5, and the reduction effects of other experimental groups were not as good as that of the fermentation of single strain MLL-5; therefore, on the whole, the preparation of the composite bacterial agent with the strain volume ratio of Experiments 1, 3, 5 and 6 had a better fermentation effect on cassava residue, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 was (1-2): (2-3): (2-6); the optimal experimental group was Experiment 1, that is, the volume ratio of strain MLF-1: strain JSF-9: strain MLL-5 was 1:1:2.

[0067] From the perspective of extreme values, the strain MLL-5 had the greatest impact on crude protein in fermented cassava residue feed, followed by strain MLF-1, and the strain JSF-9 had the least impact; the strain MLL-5 had the greatest impact on cyanide in fermented cassava residue feed, followed by strain JSF-9, and the strain MLF-1 had the least impact.

[0068] Example 6

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

[0070] Forty seven-month-old Jersey calves 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: 10% fermented cassava residue (fermented without sterilizing agents) was added to the base diet; experimental group 2: 10% fermented cassava residue (with the compound inoculum added at the same ratio as in Experiment 1 of Example 5) was added to the base diet; and experimental group 3: 15% fermented cassava residue (with the compound inoculum added at the same ratio as in Experiment 1 of 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. Initial and final body weights of the Jersey calves were measured, and average daily gain (ADG) was calculated. Daily feed intake was recorded, and average daily feed intake (ADG) was calculated. Feed-to-gain ratio was calculated based on ADG / ADG. The number of diarrhea episodes was observed and counted (one episode of diarrhea was counted as 1, and two episodes of diarrhea were counted as 2). The results are shown in Table 8.

[0071]

[0072] As shown in Table 8, in terms of average daily weight gain, there was no significant difference between experimental group 2, experimental group 3 and control group (CK) (p>0.05), and the daily weight gain of experimental group 2, experimental group 3 and control group (CK) was significantly higher than that of experimental group 1 (p<0.05), indicating that the use of composite microbial agent to ferment cassava residue (experimental group 2, experimental group 3) can reach the feeding level of basic feed, save part of the basic feed, and effectively reduce the feeding cost of farmers. The average daily weight gain of experimental group 1 was lower than that of the control group, and reached a significant level (p<0.05), which shows that the conversion effect of fermented cassava residue without adding microbial agent is not good, and the feeding effect is significantly reduced after replacing part of the basic feed; in terms of feed-to-weight ratio: the feed-to-weight ratio of experimental group 1 is significantly higher than that of experimental group 2, experimental group 3 and control group (p<0.05), and the feed-to-weight ratio of experimental group 2, experimental group 3 and control group is not significantly different (p>0.05), which shows that the feed utilization rate of cassava residue fermented without microbial agent (experimental group 1) cannot reach The effect of the basic diet was better than that of the fermented cassava residue with added bacterial agents, and the feed utilization rate of the fermented cassava residue with added bacterial agents was equivalent to that of the basic diet; from the perspective of the number of diarrhea: the control group had 5 diarrhea times, the experimental group 1 had 8 diarrhea times, which was higher than the control group, and the number of diarrhea times of the experimental groups 2 and 3 was 0 times. This shows that the composite bacterial agent prepared by several bacterial agents with the function of inhibiting intestinal pathogens effectively reduced the number of diarrhea in Jersey cattle, while the experimental group 1, which did not use bacterial agents to ferment cassava residue, had the highest number of diarrhea in Jersey cattle, indicating that the cassava residue feed not fermented with the composite bacterial agent may have bred other pathogens, and thus the number of diarrhea was higher than that of the control group. The above conclusions show that adding 10%-15% of fermented cassava residue prepared with the composite bacterial agent to the basic feed will not have an adverse effect on the growth performance and health status of cattle, and the number of diarrhea is significantly reduced, indicating that the nutritional value of the fermented cassava residue of the present application is improved after fermentation by the strain, which can reduce the toxicity of the fermented feed and improve the disease resistance and immunity of Jersey cattle.

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

[0074] 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 sabdariffa ( Bacillus safensis ) Application of MLL-5 in the preparation of an antibacterial agent for intestinal pathogens, characterized in that: The intestinal pathogen is Escherichia coli ( Escherichia coli ), Yersinia enterocolitica ( Yersinia enterocolitica ), Shigella flexneri ( Shigella flexneri ) and Salmonella typhimurium ( Salmonella typhimurium. ); the Bacillus sabdariffa ( Bacillus safensis ) The deposit number of MLL-5 is GDMCC NO: 65790.

2. comprising the application of claim 1 Bacillus sabdariffa ( Bacillus safensis ) MLL-5 composite bacterial agent.

3. The composite bacterial agent according to claim 2, characterized in that The composite bacterial agent also includes Bacillus subtilis ( Bacillus altitudinis ) MLF-1 and / or Bacillus paralicheniformis JSF-9; the high ground Bacillus ( Bacillus altitudinis ) MLF-1 is deposited in GDMCC NO: 65789; the Bacillus licheniformis ( Bacillus paralicheniformis ) The JSF-9's deposit number is GDMCC NO: 65786.

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 paralicheniformis ) strain JSF-9 and Bacillus saffron ( Bacillus safensis )MLL-5 is mixed in a volume ratio of (1-2): (2-3): (2-6); the licheniformis ( Bacillus paralicheniformis ) The effective viable count of strain JSF-9 was 1.2×10 8 cfu / ml, Bacillus subtilis ( Bacillus altitudinis ) The effective viable count of MLF-1 was 3.5×10 7 cfu / ml, Bacillus sabdariffa ( Bacillus safensis The effective viable count of MLL-5 was 7.5×10 7 cfu / ml.

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

2.

6. The use of Bacillus sabdariffa ( Bacillus safensis ) Use of MLL-5 or the composite bacterial agent as described in claim 2 in the preparation of cassava residue fermented feed.

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

8. comprising the application of claim 1 Bacillus saffron ( Bacillus safensis ) MLL-5 or the fermented feed of the composite bacterial agent according to claim 2, characterized in that, The preparation method of the fermented feed is as follows: ( Bacillus safensis ) MLL-5 or composite microbial agent was inoculated into cassava residue at an inoculum rate of 100 ml / kg and fermented for 30 days. have to.

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

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