Bacillus amyloliquefaciens and application thereof in biological fermentation feed

Abstract

The invention discloses bacillus amyloliquefaciens with the characteristic of inhibiting mould growth and application of the bacillus amyloliquefaciens, and belongs to the field of microorganisms and fermentation engineering, the bacillus amyloliquefaciens is preserved in China General Microbiological Culture Collection Center on August 5, 2024, and the preservation number is CGMCC No.31538. The strain is bacillus amyloliquefaciens obtained by screening, separating and purifying from peasant pickles, and the strain can inhibit the growth of escherichia coli, clostridium perfringens and mould. The bacillus amyloliquefaciens disclosed by the invention solves the problem of insufficient effect of a microorganism mildew-proof method of the existing fermented feed, and the bacillus amyloliquefaciens is added into the biological fermented feed, so that the quantity of harmful microorganisms in the biological fermented feed can be effectively reduced, the generation of mycotoxin is inhibited, the feed is prevented from mildewing and going bad, and the shelf life of the feed is prolonged.

Description

Technical Field

[0001] The invention relates to the field of microorganisms and fermentation engineering, and in particular to Bacillus amyloliquefaciens capable of inhibiting the growth of Escherichia coli, Clostridium perfringens and mold, and application of the bacillus amyloliquefaciens in biological fermentation feed. Background Art

[0002] Bio-fermented feed uses composite microbial agents to re-decompose and synthesize the nutritional components in feed. In this process, a variety of organic acids, biological enzymes, vitamins, amino acids and other unknown growth factors will be produced, which can enhance the disease resistance of livestock and poultry, improve feed utilization, reduce feed costs and increase economic benefits. The production of biological feed in my country is mainly based on solid-state fermentation technology. Common fermentation methods include pool fermentation, tank fermentation, belt fermentation, box fermentation, etc. These production equipment are suitable for large-scale production of bio-fermented feed. Due to the large size of these equipment and many dead corners, they cannot avoid contact with the outside world during the fermentation process, and local mold cannot be avoided, and the stability of the product is relatively poor. The key to the fermentation of this type of equipment is to do a good job of flushing and disinfection before fermentation, minimize the contact between the material and the external environment during the fermentation process, reduce the chance of contamination, and dry the fermented mature materials as soon as possible to ensure the stability of the product. At present, many group feed companies’ production workshops are equipped with bio-fermentation feed production lines. The fermented materials are not dried, but directly added to the complete feed in a certain proportion. This not only reduces the drying cost, but also better exerts the probiotic properties of bio-fermented feed and its metabolites. However, if the fermented wet materials are not stored properly, there is a risk of mold, which will lead to a shortened shelf life of the product and excessive toxin content, affecting feed costs and breeding costs.

[0003] The existing technology can effectively extend the shelf life by adding mildew inhibitors to biological feeds, but mildew inhibitors not only inhibit the growth of mold, but also have certain restrictions on the growth of other microorganisms. As the amount of mildew inhibitors increases, the inhibitory effect on microorganisms gradually increases. Therefore, the ideal solution for mildew prevention in biological feeds is to inhibit the growth of mold through the metabolites and competitive characteristics of the microorganisms themselves, destroy the growth conditions of mold, and extend the shelf life of the material. However, in the existing literature and patent reports, there are few technologies that prevent biological feeds from mildewing by simply adding microorganisms.

[0004] The above contents are only used to assist in understanding the technical solution of the present invention and do not constitute an admission that the above contents are prior art. Summary of the invention

[0005] In view of the shortcomings of the prior art, the technical problem to be solved by the present invention is to obtain a Bacillus amyloliquefaciens with antibacterial and antifungal properties through strain screening, and to obtain a biological fermented feed that inhibits mold growth and has a longer shelf life.

[0006] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0007] The present invention provides a Bacillus amyloliquefaciens with antibacterial and antifungal effects, which was deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration on August 5, 2024. The address of the depository is No. 3, Yard No. 1, Chaoyang Road, Beijing. The deposit number is CGMCC No. 31538, and the classification name is: Bacillus amyloliquefaciens.

[0008] The present invention also provides a method for preparing a biological fermentation feed, which comprises adding the above-mentioned Bacillus amyloliquefaciens.

[0009] Furthermore, the method for preparing the biological fermentation feed comprises:

[0010] Strain activation: inoculate the Bacillus amyloliquefaciens stored in the freeze-dried tube onto the sterilized LB slant medium to obtain activated Bacillus amyloliquefaciens;

[0011] Preparation of seed solution: Pick a single colony of activated Bacillus amyloliquefaciens and inoculate it into a sterile LB broth medium, culture it aerobically at a temperature of 35-37°C for 10 hours and a shaking speed of 200 r / min to prepare a seed solution;

[0012] Preparation of fermentation agent: adding liquid fermentation medium and organosilicon defoamer in a mass ratio of 100:0.05 into a fermentation tank for high pressure steam sterilization to obtain a sterile fermentation medium; inoculating the seed liquid in the sterile fermentation medium at an inoculation amount of 1% of the volume of the fermentation medium, adding saccharomyces cerevisiae after the cultivation is completed, canning and sealing, and obtaining a fermentation agent;

[0013] Preparation of biological fermentation feed: mixing the fermentation agent and the fermentation matrix at a weight ratio of 3:10 to 5:10 to obtain a mixed fermentation matrix, adjusting the initial water content of the mixed fermentation matrix to 35% to 45%, sealing and fermenting, and obtaining the biological fermentation feed.

[0014] Further, in the preparation of the fermentation bacteria agent, the culture is carried out in a fermentation medium in a fermentation tank, and the culture conditions include: setting the fermentation tank pressure to 0.05Mpa, the air flow to 15L / min, the rotation speed to 225r / min, the fermentation temperature to 35-37°C, and the fermentation time to 48-72h.

[0015] Furthermore, before the preparation of the fermentation agent, the steps include:

[0016] A liquid fermentation medium was prepared, wherein the liquid fermentation medium contained the following components: 10 g / L corn flour, 5 g / L soybean protein concentrate, 6 g / L peptone, 8 g / L glucose, 0.1 g / L magnesium sulfate, and 4 g / L sodium chloride.

[0017] Furthermore, the pH of the liquid fermentation medium is between 6.5 and 7.0.

[0018] Furthermore, before the preparation of the biological fermentation feed, the process includes:

[0019] A fermentation substrate is prepared, wherein the fermentation substrate comprises the following components: 10% to 30% corn, 5% to 30% soybean meal and 40% to 60% bran.

[0020] Furthermore, the preparation of the biological fermentation feed is as follows: the fermentation agent and the fermentation matrix are mixed at a weight ratio of 3:10 to 5:10 to obtain a mixed fermentation matrix, wherein the number of effective live bacteria per gram of the mixed fermentation matrix is ​​5.0×10 8 ~1.0×10 10 CFU.

[0021] Furthermore, the amount of brewer's yeast added is 5.0×10 6 ~5.0×10 7 CFU / g.

[0022] Furthermore, the sealed fermentation in the preparation of the biological fermentation feed to obtain the biological fermentation feed, the conditions of the sealed fermentation are:

[0023] The fermentation temperature is 35-38°C and the fermentation time is ≥3 days.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] 1. The present invention adopts a strain selection method to obtain a Bacillus amyloliquefaciens that has the ability to inhibit the growth of Escherichia coli, Clostridium perfringens and mold.

[0026] 2. The antibacterial and antifungal properties of this strain can effectively reduce the number of harmful microorganisms in biological fermentation feed, inhibit the production of mycotoxins, prevent feed from mold and deterioration, and extend the shelf life of materials.

[0027] 3. The biological fermented feed obtained by the present invention does not add mildew inhibitors, but produces beneficial substances through the cooperation of microorganisms to antagonize molds, thereby reducing the destructiveness of mildew inhibitors to microorganisms to the greatest extent and achieving the best microbial fermentation effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1A schematic diagram of the antibacterial effect of Bacillus amyloliquefaciens (CGMCC No. 31538) provided in an embodiment of the present invention on a culture medium in which the quality control bacteria is Escherichia coli;

[0029] Figure 2 A schematic diagram of the antibacterial effect of Bacillus amyloliquefaciens (CGMCC No. 31538) provided in an embodiment of the present invention on a culture medium containing Clostridium perfringens as the quality control bacteria;

[0030] Figure 3 A schematic diagram of the antibacterial effect of Bacillus amyloliquefaciens (CGMCC No. 31538) provided in an embodiment of the present invention on a culture medium containing Aspergillus flavus as the quality control bacteria;

[0031] Figure 4 A schematic diagram of the colony morphology of Bacillus amyloliquefaciens (CGMCC No. 31538) on a culture medium provided in an embodiment of the present invention;

[0032] Figure 5 A schematic diagram of Gram staining microscopy of Bacillus amyloliquefaciens (CGMCC No. 31538) provided in an embodiment of the present invention;

[0033] Figure 6 This is a PCR nucleic acid electrophoresis diagram of Bacillus amyloliquefaciens (CGMCC No. 31538) provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0034] The present invention is further described in detail below in conjunction with specific examples, which are not intended to limit the present invention but are only intended to illustrate the present invention. In this article, unless otherwise specified, when expressing the content of a substance, it refers to the percentage of the mass of the substance in the total content of the substance.

[0035] The present invention obtains a Bacillus amyloliquefaciens strain that can inhibit the growth of Escherichia coli, Clostridium perfringens and mold through a large amount of screening work. The Bacillus amyloliquefaciens was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on August 5, 2024, with a deposit number of CGMCC No.31538. It has been verified that after adding the Bacillus amyloliquefaciens to the bio-fermented feed, it can effectively reduce the number of harmful microorganisms in the bio-fermented feed, inhibit the production of mycotoxins, prevent the feed from moldy and deteriorating, and extend the shelf life of the feed. Therefore, the Bacillus amyloliquefaciens of the present invention has a wide range of application prospects. The provided Bacillus amyloliquefaciens CGMCC No.31538 can be made into a fermentation agent with Saccharomyces cerevisiae and used as a composite agent. To this end, in one aspect of the present invention, the present invention provides a fermentation agent, the fermentation agent comprising Bacillus amyloliquefaciens CGMCC No.31538. The provided fermentation agent can be added to biological fermentation feed for fermentation, thereby reducing the number of harmful microorganisms in the biological fermentation feed, inhibiting the production of mycotoxins, preventing the feed from becoming moldy and deteriorating, and extending the shelf life of the feed. The provided fermentation agent can be obtained by fermentation. To this end, in another aspect of the present invention, the present invention provides a method for preparing the above-mentioned microbial agent, comprising: fermenting Bacillus amyloliquefaciens CGMCC No.31538; and obtaining the microbial agent based on the product of the fermentation treatment. In at least some embodiments of the present invention, the fermentation treatment comprises: subjecting the Bacillus amyloliquefaciens to liquid fermentation culture to obtain seed liquid; and inoculating the seed liquid into a fermentation culture liquid for fermentation culture to obtain a fermentation agent.

[0036] The embodiment of the present invention also provides a preparation process of Bacillus amyloliquefaciens for fermented feed, specifically: inoculate the Bacillus amyloliquefaciens stored in a freeze-drying tube on a sterilized LB slant medium to obtain activated Bacillus amyloliquefaciens, pick a single colony after activation and inoculate it into a sterilized liquid LB medium, culture temperature 35-37°C, aerobic conditions, constant temperature shaking table culture 10h, speed 200r / min, to prepare seed liquid; inoculate the prepared seed liquid into a sterilized fermentation tank containing liquid culture medium and organosilicon defoamer at an inoculation amount of 1% (volume ratio of seed liquid to culture medium) for fermentation, and the mass ratio of organosilicon defoamer to culture medium is 100:0.05. Fermentation conditions: tank pressure 0.05Mpa, air volume 15L / min, speed 225r / min, temperature 35-37°C, add saccharomyces cerevisiae after fermentation culture for 48h-72h to end fermentation, can, seal, and obtain fermentation agent. The fermentation bacteria agent and the fermentation matrix are mixed at a weight ratio of 3:10 to 5:10 to obtain a mixed fermentation matrix, the initial water content of the mixed fermentation matrix is ​​adjusted to 35% to 45%, and the mixed fermentation matrix is ​​sealed and fermented to obtain the target biological fermentation feed.

[0037] Preferably, the fermentation bacteria culture temperature is 37° C., the culture conditions are aerobic culture, and the culture time is 60 hours.

[0038] Preferably, the weight ratio of the fermentation bacteria agent to the fermentation substrate is 1:2.5.

[0039] Preferably, the total number of bacterial colonies in the mixed fermentation matrix is ​​1.0×10 9 ~5.0×10 9 CFU / g.

[0040] Preferably, the fermentation medium is 10 g / L corn flour, 5 g / L soy protein concentrate, 6 g / L peptone, 8 g / L glucose, 0.1 g / L magnesium sulfate, 4 g / L sodium chloride, and pH 7.0.

[0041] Preferably, the fermentation substrate comprises 25% corn, 20% soybean meal and 55% bran.

[0042] Preferably, the amount of brewer's yeast added is 1×10 7 ~5.0×10 7 CFU / g fermentation substrate.

[0043] Preferably, the initial water content of the mixed fermentation substrate is adjusted to 38% to 40%, the fermentation temperature is 37° C., and the fermentation time is 3 days.

[0044] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. The experimental methods used in the following examples are generally based on conventional conditions unless otherwise specified, and the experimental methods in which specific conditions are not specified in the examples. If specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. The reagents or instruments used that do not specify the manufacturer are all conventional products that can be obtained commercially.

[0045] Example 1: Isolation and screening of Bacillus amyloliquefaciens

[0046] The Bacillus amyloliquefaciens (CGMCC No. 31538) in this experiment was isolated from kimchi by plate spreading method and plate streaking method, which included the following steps:

[0047] (1) Prepare experimental materials

[0048] Enrichment medium: 1% peptone, 0.3% beef extract, 0.5% NaCl, pH 6.0-6.5, sterilized at 121°C for 20 minutes.

[0049] Slant preservation medium (Escherichia coli antibacterial screening medium): 1% tryptone, 0.5% yeast extract powder, 1% NaCl, 1.5% agar powder, sterilized at 121°C for 15 minutes.

[0050] LB medium, Clostridium perfringens assay medium and potato culture medium were purchased from Qingdao Hibo Biological.

[0051] (2) Screening, separation and purification of Bacillus

[0052] Weigh 1g of homemade kimchi from a farmer in Hubei, add it to a 50mL / 250mL conical flask filled with sterile water containing glass beads, place it in a shaking incubator at 150rpm and 37℃ for 30min, take 5ml of the bacterial suspension and add it to a 45ml broth medium / 250ml conical flask, heat it in a water bath at 75℃~80℃ for 20min, cool it to 35℃~37℃, shake it in a shaking incubator at 150r / min for 24h, aspirate the supernatant, and dilute it stepwise by 10 -1 ~10 -7 , select three gradient mixed bacteria and spread them on LB medium, with 3 parallels for each gradient, and place the plate at 35℃~37℃ for 24~48h. Select the colonies with fast growth, large colonies, dry, rough and opaque surface and transfer them to the inclined surface for later use, pick a small amount of bacterial moss smear, and do spore staining to determine whether it is Bacillus.

[0053] (3) Screening of antibacterial strains

[0054] The isolated and purified single strain was inoculated into LB broth medium and cultured at 37°C for 24 h, which was regarded as the test bacterial liquid.

[0055] Take about 5 ml of LB agar medium (C. perfringens assay medium) and quickly spread it on the bottom of the plate. After solidification, place Oxford cups on the bottom of the plate in sequence, with a maximum of 4 cups placed on one plate. Add 10 7 cfu / mL LB agar medium (clostridium perfringens assay medium) of quality control bacteria 12.5ml (pay attention to the temperature of the medium), wait for solidification, take out the Oxford cup, and mark the wells. Add 150μL of the test bacteria solution to each Oxford cup well, and 75ppm chlortetracycline as a positive control; each plate requires a positive control test group. The plate is placed in a 4℃ refrigerator for diffusion for 16h. After the sample is absorbed, it is placed in a 37℃ biochemical incubator and cultured for 6h until a useful quality control bacterial moss appears. Use a vernier caliper to measure the diameter of the inhibition zone, compare the size and inhibition rate between the inhibition zones, and judge the antibacterial effect based on the size of the inhibition zone.

[0056] The following formula is used to determine the antibacterial rate:

[0057] η=φtest / φcontrol×100%

[0058] in,

[0059] η: antibacterial rate (%);

[0060] φTo be tested: the diameter of the inhibition zone produced by the sample to be tested on the plate (mm);

[0061] φ control: The diameter of the inhibition zone produced by the positive control on the same plate (mm).

[0062] (4) Screening of fungal inhibitory strains

[0063] Initial screening: ferment the strain in culture medium, obtain the culture solution, melt the culture medium, and when the temperature drops to 55°C, add the Aspergillus flavus spore suspension to make the concentration of Aspergillus flavus spores 2×10 4 ~4×10 4 CFU / mL, pour the spore suspension of the culture medium into the culture dish, 20mL / culture dish, to make a mixed bacterial plate. The biological activity of antibacterial substances was detected by the Oxford cup method, and the diameter of the inhibition zone was measured by the cross method. The strains with high anti-aflatoxin activity were selected according to the size of the inhibition zone diameter.

[0064] Rescreening: Through multiple enrichment and screening separation, a microbial strain that has an inhibitory effect on the growth of Escherichia coli, Clostridium perfringens and mold was obtained. The antibacterial effect test of the screened strain showed that the diameter of the antibacterial zone of the strain for Escherichia coli was about 15.51±0.21mm, and the antibacterial rate was about 77.1±0.34%; the diameter of the antibacterial zone for Clostridium perfringens was 32.35±0.87mm, and the antibacterial rate was about 107.8±0.58%, indicating that the strain has an antibacterial effect on both Escherichia coli and Clostridium perfringens. Figure 1 and Figure 2 ,in Figure 1 and Figure 2 A and B above are Bacillus amyloliquefaciens, and C and D are positive controls.

[0065] The strain has a significant inhibitory effect on Aspergillus flavus. The Oxford Cup test shows that the strain has a strong inhibitory effect on the growth of Aspergillus flavus hyphae, and the diameter of the antifungal circle reaches 18.10±0.46mm. Figure 3 ,in Figure 3 A and B are 21-fold and 41-fold dilution gradients, respectively, and C is the original culture solution.

[0066] Example 2: Identification of Bacillus amyloliquefaciens

[0067] Example 2 The microbial strain obtained in Example 1 was further identified, and the microbial strain was identified as Bacillus amyloliquefaciens. The microbiological characteristics and molecular biological characteristics were mainly identified, including:

[0068] (1) Microbiological characteristics: Its colony morphology and bacterial morphology under a microscope Figure 4 , Figure 5 As shown; after extracting the strain DNA and performing PCR amplification, agarose gel electrophoresis was used to obtain the PCR nucleic acid electrophoresis diagram of the strain, as shown Figure 6 shown.

[0069] (2) Molecular biological characteristics:

[0070] The 16S rDNA sequence of the strain was compared in the gen bank database and was highly similar to that of Bacillus amyloliquefaciens strain P1-1, with a similarity of 100%. Combined with its microbiological characteristics, the strain in Example 1 of the present invention was determined to be Bacillus amyloliquefaciens, and was named: B18.

[0071] The 16S rRNA sequencing result is shown in SEQ ID NO.1.

[0072]

[0073] The strain obtained in Example 1 of the present invention was identified as Bacillus amyloliquefaciens, and the strain was deposited with a deposit number of CGMCC NO.31538 in the China Microbiological Culture Collection Center (CGMCC).

[0074] Example 3: Preparation of fermentation agent

[0075] S1 strain activation: inoculate the Bacillus amyloliquefaciens in the freeze-dried tube onto a sterilized LB slant medium to obtain activated Bacillus amyloliquefaciens;

[0076] Preparation of S2 seed solution: Pick a single colony of activated Bacillus amyloliquefaciens and inoculate it into a sterile LB medium. The culture temperature is 37°C, and the culture condition is aerobic. The culture is carried out in a constant temperature shaker for 10 hours at a speed of 200 r / min to prepare the seed solution.

[0077] Preparation of S3 fermentation agent: liquid LB culture medium and organosilicon defoamer were added to the fermentation tank for sterilization, and the mass ratio of organosilicon defoamer to culture medium was 100:0.05. The seed liquid prepared in step S2 was inoculated into the sterilized LB culture medium fermentation tank at an inoculation amount of 1% (volume ratio of seed liquid to culture medium) for culture. The fermentation conditions were: tank pressure 0.05 MPa, air flow 15 L / min, speed 225 r / min, temperature 37°C, fermentation culture for 60 hours, and Saccharomyces cerevisiae (purchased from Angel Yeast Co., Ltd., product number: Kelibeijian, 82000657) was added to terminate the fermentation, canned and sealed to obtain the fermentation agent, and the effective viable count was measured to be 8.5×10 9 CFU / g.

[0078] Example 4: Preparation of a biological fermentation feed

[0079] S1: Weigh 25% corn, 20% soybean meal, and 55% wheat bran according to the proportion, mix them evenly, and obtain a fermentation matrix.

[0080] S2: The fermentation agent prepared in Example 3 was added to the fermentation substrate in a ratio of 1:2.5, and the mass ratio of the fermentation agent to the fermentation substrate was ensured to be 37% at the initial fermentation moisture level. The mixture was evenly mixed and sealed in a bag with a breathing valve for fermentation. The fermentation temperature was 37°C. The fermentation was terminated after 3 days to obtain a biological fermentation feed. The effective viable bacteria count of the prepared biological fermentation feed was 4.1×10 8 CFU / g. The bio-fermented feed was sealed and stored in a breathing bag, and samples were taken at regular intervals. The mold, Escherichia coli and Clostridium perfringens were tested according to GB13078-2017 Feed Hygiene Standards, and the contents of mold, Escherichia coli and Clostridium perfringens in the feed at different time periods were obtained, as shown in Table 1.

[0081] Table 1 Anti-mildew and antibacterial effects of biological fermented feed in Example 4 of the present invention

[0082] Storage time / day 0 30 60 90 120 150 180 210 Mold CFU / g 8 29 44 89 <![CDATA[6.5×10 2 ]]> <![CDATA[2.2×10 3 ]]> <![CDATA[1.0×10 4 ]]> <![CDATA[5.0×10 4 ]]> E. coli CFU / g 0 0 0 10 22 59 87 103 Clostridium perfringens CFU / g 0 0 0 0 0 7 19 30

[0083] According to the requirements of GB13078-2017 Feed Hygiene Standard, the mold content in qualified feed should be less than 4.0×10 4 CFU / g, the bacterial content should be less than 2.0×10 6 CFU / g. It can be seen from Table 1 that the mold index of the biological fermented feed prepared by the strain B18 of the present invention exceeded the national standard when it was stored for 210 days, and it was an unqualified product. The biological fermented feed also had an inhibitory effect on Escherichia coli and Clostridium perfringens, especially on Clostridium perfringens. When stored for 210 days, the number of viable Clostridium perfringens detected was <1.0×10 2 CFU / g. Therefore, considering the two inhibition time indicators of mold and harmful bacteria, the effective shelf life of the feed can reach 180 days. The storage time of general commercially available biological feed in the same season exceeds 120 days, and the content of Escherichia coli and Clostridium perfringens exceeds the standard. It can be seen that the biological fermented feed of the present invention can effectively reduce the number of harmful microorganisms in the feed, inhibit the production of mycotoxins, prevent the feed from mold and deterioration, and extend the shelf life of the feed.

[0084] Example 5

[0085] The fermentation agent of strain B18 prepared in Example 3 was divided into 6 conical flasks, and sterilized potato culture solution was added to 100 mL. The culture solution was diluted to the fermentation agent OD 600nm The values ​​were: 0, 0.06, 0.12, 0.24, 0.36, 0.48, 0.68. 0.1 mL of Aspergillus flavus spore suspension (spore concentration was 10 5 / mL), and cultured in a constant temperature shaker at 30°C and 120r / min for 3 days. The mycelial balls grown in different fermentation agent concentrations in the conical flask were filtered separately, the wet weight of the mycelial balls was weighed, and then the amount of aflatoxin B1 was determined by quantum dot immunofluorescence chromatography. Two groups of parallel experiments were designed, and the average value of the measured values ​​was taken. The experimental results are shown in Table 2.

[0086] Table 2 The experimental results of the fermentation agent of Example 5 of the present invention for inhibiting the growth and toxin production of Aspergillus flavus

[0087] Fermentation agent concentration OD600nm Mycelial pellet wet weight (g) Aflatoxin B1(ppb) 0 (blank) 5.1003 65.78 0.06 4.9875 50.43 0.12 4.1033 34.22 0.24 2.9383 21.85 0.48 1.9961 6.55 0.68 1.6817 <1

[0088] It can be seen from Table 2 that the B18 strain of the present invention and the fermentation agent prepared therefrom have a good inhibitory effect on the growth of Aspergillus flavus and can also effectively reduce the production of aflatoxin.

[0089] Comparative Example 1

[0090] The bio-fermented feed, "Youke Golden Partner", was purchased from the market and stored in a sealed container according to the product specifications. Samples were taken at regular intervals and tested for mold, Escherichia coli and Clostridium perfringens according to GB13078-2017 "Feed Hygiene Standards". The contents of mold, Escherichia coli and Clostridium perfringens in the feed at different time periods were obtained, as shown in Table 3.

[0091] Table 3 Anti-mildew and antibacterial effects of commercially available "Youke Golden Partner" biological fermented feed

[0092] Storage time / day 0 30 60 90 120 150 Mold CFU / g 30 55 103 <![CDATA[2.1×10 2 ]]> <![CDATA[3.5×10 3 ]]> <![CDATA[4.7×10 4 ]]> E. coli CFU / g 58 88 <![CDATA[1.3×10 2 ]]> <![CDATA[4.1×10 2 ]]> <![CDATA[8.9×10 2 ]]> <![CDATA[3.6×10 3 ]]> Clostridium perfringens CFU / g 30 71 <![CDATA[10.1×10 2 ]]> <![CDATA[2.6×10 2 ]]> <![CDATA[5.1×10 2 ]]> <![CDATA[9.2×10 2 ]]>

[0093] It can be seen from Table 3 that when the commercially available product "Youke Golden Partner" was stored for 150 days, the mold index exceeded the national standard, and the product was already an unqualified product. When the commercially available product "Youke Golden Partner" was stored for 60 days, the number of Escherichia coli and Clostridium perfringens far exceeded the number of the product of the present invention stored for 210 days. According to the data comparison of Table 1 and Table 3, the biological fermentation feed of the present invention shows significant advantages in mildew prevention and inhibition of E. coli growth compared with the commercially available "Youke Golden Partner" biological fermentation feed. In terms of mold inhibition, the feed of the present invention exceeded the national standard when it was stored for 210 days, while the commercially available product was unqualified after being stored for 150 days, indicating that the feed of the present invention has a longer mildew prevention period. In terms of E. coli inhibition, the E. coli content of the feed of the present invention was 10 when it was stored for 210 days. 3 CFU / g, much lower than the 3.6×10 of the commercial product at 150 days. 3 CFU / g, showing stronger antibacterial ability. In general, the bio-fermented feed of the present invention is significantly superior to commercial products in maintaining feed quality and extending shelf life.

[0094] Comparative Example 2: Preparation of fermentation agent

[0095] S1 strain activation: Bacillus subtilis (purchased from Guangdong Microbial Culture Collection Center, No. 1.3819) stored in a freeze-dried tube was inoculated on a sterilized LB slant medium to obtain activated Bacillus subtilis;

[0096] Preparation of S2 seed solution: Pick a single colony of activated Bacillus subtilis and inoculate it into a sterilized slant LB medium. The culture temperature is 37°C, and the aerobic condition is a constant temperature shaker culture for 10 hours at a speed of 200 r / min to prepare the seed solution.

[0097] Preparation of S3 fermentation agent: LB culture medium and silicone defoamer are added to the fermentation tank for sterilization, and the mass ratio of silicone defoamer to culture medium is 100:0.05. The seed liquid prepared in step S2 is inoculated into the sterilized LB culture medium fermentation tank at an inoculation amount of 1% (volume ratio of seed liquid to culture medium) for culture, and the fermentation conditions are: tank pressure 0.05Mpa, air flow 15L / min, speed 225r / min, temperature 37°C, fermentation culture for 60h, and Saccharomyces cerevisiae (purchased from Angel Yeast Co., Ltd., product number: Kelibeijian, 82000657) is added to terminate the fermentation, canned, sealed, and obtain the fermentation agent.

[0098] Comparative Example 3: Preparation of biological fermentation feed

[0099] S1: Weigh 25% corn, 20% soybean meal, and 55% wheat bran according to the proportion, mix them evenly, and obtain a fermentation matrix.

[0100] S2: Add the fermentation agent prepared in Comparative Example 2 to the fermentation substrate at a mass ratio of 1:2.5, while ensuring that the mass ratio of the initial moisture content of the fermentation substrate to the fermentation substrate is 37%, mix evenly, put into a bag with a breathing valve for sealed fermentation, the fermentation temperature is 37°C, and the fermentation is completed after 3 days to obtain biological fermentation feed. The biological fermentation feed is sealed and stored in a breathing bag, and samples are taken at regular intervals. Mold, Escherichia coli and Clostridium perfringens are measured according to GB13078-2017 "Feed Hygiene Standards", and the contents of mold, Escherichia coli and Clostridium perfringens in the feed at each time period are obtained, as shown in Table 4.

[0101] Table 4 Anti-mildew and antibacterial effects of fermented feed from yeast sources

[0102] Storage time / day 0 30 60 90 Mold CFU / g 23 <![CDATA[2.1×10 2 ]]> <![CDATA[3.8×10 4 ]]> <![CDATA[2.7×10 5 ]]> Escherichia coli CFU / g 0 41 86 <![CDATA[3.3×10 2 ]]> Clostridium perfringens CFU / g 0 67 90 <![CDATA[5.1×10 2 ]]>

[0103] It can be seen from Table 4 that when the biological fermentation feed of Comparative Example 3 was stored for 90 days, the mold index exceeded the national standard, and the product was already an unqualified product. According to the data comparison of Table 1 and Table 4, the biological fermentation feed of the present invention performs well in mold prevention and inhibition of harmful bacteria growth. Compared with the biological fermentation feed of Comparative Example 3, the feed of the present invention has a more obvious advantage in the inhibition of mold, Escherichia coli and Clostridium perfringens. Specifically, the mold content of the feed of the present invention exceeded the national standard when it was stored for 210 days, while the mold content of the feed of Comparative Example 3 exceeded the standard when it was stored for 90 days; in terms of the inhibition of Escherichia coli and Clostridium perfringens, the number of the feed of the present invention when it was stored for 210 days was much lower than the number of the feed of Comparative Example 3 when it was stored for 60 days. These results show that the biological fermentation feed of the present invention can effectively reduce the number of harmful microorganisms in the feed and inhibit the production of mycotoxins, thereby extending the shelf life of the feed. Compared with commercially available products, the feed of the present invention has significant advantages in maintaining feed quality and safety.

[0104] In summary, the present invention separates a kind of Bacillus amyloliquefaciens with the function of inhibiting the growth of Escherichia coli, Clostridium perfringens and mold from kimchi, cooperates with Saccharomyces cerevisiae to effectively ferment the fermentation matrix, and obtains the biological fermented feed by simple mixing, bagging and sealing, and static fermentation for 3 to 7 days, which can inhibit the production of mycotoxins, prevent the feed from being moldy and deteriorating, and prolong the shelf life.

[0105] The above description is only a preferred embodiment of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0106] SEQUENCE LISTING

[0107] Tangrenshen Group Co., Ltd.

[0108] Bacillus amyloliquefaciens and its application in biological fermentation feed 1

[0110] SIPOSequenceListing 1.0 1 1429

[0113] DNA

[0114] Bacillus amyloliquefaciens 1

[0116]

[0117]

Claims

1. A Bacillus amyloliquefaciens, classified as Bacillus amyloliquefaciens, characterized in that: The depository is China General Microbiological Culture Collection Center, and the deposit number is CGMCC No.31538.

2. A method for preparing a bio-fermented feed, comprising adding the Bacillus amyloliquefaciens described in claim 1.

3. The preparation method according to claim 2, characterized in that: The preparation method of the biological fermentation feed comprises: Strain activation: inoculate the Bacillus amyloliquefaciens stored in the freeze-dried tube onto the sterilized LB slant medium to obtain activated Bacillus amyloliquefaciens; Preparation of seed solution: Pick a single colony of activated Bacillus amyloliquefaciens and inoculate it into a sterile LB broth medium, culture it aerobically at a temperature of 35-37°C for 10 hours and a shaking speed of 200 r / min to prepare a seed solution; Preparation of fermentation agent: adding liquid fermentation medium and organosilicon defoamer in a mass ratio of 100:0.05 into a fermentation tank and sterilizing by high pressure steam to obtain a sterile fermentation medium; inoculating the seed liquid in the sterile fermentation medium at an inoculation amount of 1% of the volume of the fermentation medium, adding saccharomyces cerevisiae after the cultivation, canning and sealing, and obtaining a fermentation agent; Preparation of biological fermentation feed: mixing the fermentation agent and the fermentation matrix at a weight ratio of 3:10 to 5:10 to obtain a mixed fermentation matrix, adjusting the initial water content of the mixed fermentation matrix to 35% to 45%, sealing and fermenting, and obtaining the biological fermentation feed.

4. The preparation method according to claim 3, characterized in that: In the preparation of the fermentation agent, the fermentation medium is cultured in a fermentation tank, and the culture conditions include: setting the fermentation tank pressure to 0.05Mpa, the air flow to 15L / min, the rotation speed to 225r / min, the fermentation temperature to 35-37°C, and the fermentation time to 48-72h.

5. The preparation method according to claim 3, characterized in that: Before the preparation of the fermentation agent, the process includes: A liquid fermentation medium was prepared, wherein the liquid fermentation medium contained the following components: 10 g / L corn flour, 5 g / L soybean protein concentrate, 6 g / L peptone, 8 g / L glucose, 0.1 g / L magnesium sulfate, and 4 g / L sodium chloride.

6. The preparation method according to claim 3, characterized in that: The pH of the liquid fermentation medium is between 6.5 and 7.

0.

7. The preparation method according to claim 3, characterized in that: Before the preparation of the biological fermentation feed, the process includes: A fermentation substrate is prepared, wherein the fermentation substrate comprises the following components: 10% to 30% corn, 5% to 30% soybean meal and 40% to 60% bran.

8. The preparation method according to claim 3, characterized in that: Preparation of the biological fermentation feed: The fermentation agent and the fermentation matrix are mixed at a weight ratio of 3:10 to 5:10 to obtain a mixed fermentation matrix, wherein the number of effective live bacteria per gram of the mixed fermentation matrix is ​​5.0×10 8 ~1.0× 10 10 CFU。 9. The preparation method according to claim 3, characterized in that: The amount of brewer's yeast added was 5.0×10 6 ~5.0×10 7 CFU / g.

10. The preparation method according to claim 3, characterized in that: In the preparation of the biological fermentation feed, the sealed fermentation is performed to obtain the biological fermentation feed, and the conditions of the sealed fermentation are: The fermentation temperature is 35-38°C and the fermentation time is ≥3 days.

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