CN108893434B - Microbial treatment agent for degrading aflatoxin B1 as well as preparation method and application thereof - Google Patents
Microbial treatment agent for degrading aflatoxin B1 as well as preparation method and application thereof Download PDFInfo
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- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 title claims abstract description 271
- 229930020125 aflatoxin-B1 Natural products 0.000 title claims abstract description 271
- 230000000593 degrading effect Effects 0.000 title claims abstract description 204
- 239000002115 aflatoxin B1 Substances 0.000 title claims abstract description 119
- 239000003795 chemical substances by application Substances 0.000 title claims abstract description 72
- 230000000813 microbial effect Effects 0.000 title claims abstract description 70
- 238000011282 treatment Methods 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 41
- 108090000790 Enzymes Proteins 0.000 claims abstract description 153
- 102000004190 Enzymes Human genes 0.000 claims abstract description 153
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 claims abstract description 152
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 claims abstract description 152
- 238000002156 mixing Methods 0.000 claims abstract description 83
- 239000006041 probiotic Substances 0.000 claims abstract description 52
- 235000018291 probiotics Nutrition 0.000 claims abstract description 52
- 230000000529 probiotic effect Effects 0.000 claims abstract description 46
- 150000001875 compounds Chemical class 0.000 claims abstract description 45
- 241000287828 Gallus gallus Species 0.000 claims abstract description 34
- 230000000694 effects Effects 0.000 claims abstract description 17
- 238000000855 fermentation Methods 0.000 claims description 141
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- 238000001914 filtration Methods 0.000 claims description 86
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 79
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 79
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 71
- 239000007787 solid Substances 0.000 claims description 66
- 238000012258 culturing Methods 0.000 claims description 62
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- 241000194108 Bacillus licheniformis Species 0.000 claims description 60
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- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 44
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- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 22
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 11
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- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 description 2
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- IJAPPYDYQCXOEF-UHFFFAOYSA-N phthalazin-1(2H)-one Chemical compound C1=CC=C2C(=O)NN=CC2=C1 IJAPPYDYQCXOEF-UHFFFAOYSA-N 0.000 description 2
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
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- A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
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Abstract
The invention discloses a microbial treatment agent for degrading aflatoxin B1, and a preparation method and application thereof, wherein the microbial treatment agent comprises a probiotic compound and an aflatoxin B1 degrading enzyme crude enzyme product in a volume ratio of 1:3-3: 1. The preparation method comprises the following steps: preparing a probiotic compound; preparing crude enzyme of aflatoxin B1 degradation enzyme; and mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 1:3-3:1 to prepare the microorganism treating agent for degrading aflatoxin B1. The microbial treatment agent can obviously relieve the adverse effect of AFB1 on the production performance of chickens and pigs.
Description
Technical Field
The invention belongs to the technical field of animal feeding and breeding, and particularly relates to a microbial treatment agent for degrading aflatoxin B1, and a preparation method and application thereof.
Background
Aflatoxin B1(Aflatoxin B1, abbreviated as AFB1) is a derivative of dihydrofuran phthalazone containing one difuranic ring and one phthalazone (coumarin). Among natural foods for humans and animals, aft (aflatoxin) is widely available, most commonly AFB1, and is most hazardous, and AFB1 is highly toxic to humans and several animals, including: the harmfulness to poultry is mainly: the feed intake, weight gain and feed conversion rate are reduced, and infertility, abortion, pulmonary edema, epidemic disease susceptibility are increased; the harm to pigs is mainly as follows: the feed intake, weight gain and feed conversion rate are reduced, the infertility and abortion, pulmonary edema, epidemic disease susceptibility are increased, and the like; the harmfulness to the dairy cattle is mainly as follows: decreased feed intake, milk production, weight gain and feed conversion, reproductive performance disorders, embryonic death and abortion, nervous system disorders, spasticity and lack of coordination, increased susceptibility to epidemic diseases, etc. AFB1 is the most carcinogenic of the known chemicals. Therefore, the national quality control agency stipulates that AFB1 is one of the requisite items for most food products. AFB1 is heat resistant and can be cleaved only at 280 ℃, so that it is difficult to destroy the feed at the temperature of cooking and processing.
The pollution of aflatoxin B1 and the like is a global problem, and the traditional detoxification method has the defects of nutrition destruction, incomplete detoxification, high cost and the like, so the actual prevention, control and detoxification effects are not ideal. Therefore, it is a key to solve the problem to detoxify mycotoxins by a more effective and safe method, and the microbial degradation of mycotoxins has brought about extensive attention of researchers due to the advantages of high efficiency, no residue, low cost, simple operation and the like, and especially, the degradation of mycotoxins into non-toxic products by microorganisms or enzymes generated by the microorganisms is a hotspot of current research. Detoxification treatment of food crops and feeds polluted by mycotoxins is an effective method for enlarging feed resources and reducing loss.
Therefore, the microbial treatment agent for degrading mycotoxin is provided, on the basis of probiotics and degrading enzymes which have a degrading effect on AFB1, the probiotics and the AFB1 degrading enzymes are matched and applied to cultivation, and the microbes or metabolites thereof which have the function of inhibiting the growth of mold are added into feed raw materials in proportion to inhibit the growth of mold by utilizing the competition effect among the microbes, so that the generation of toxin is inhibited, the purpose of preventing mycotoxin poisoning of livestock and poultry is achieved, and the microbial treatment agent becomes an important direction for degrading AFB 1.
Disclosure of Invention
In view of the above, the invention provides a microbial treatment agent for degrading aflatoxin B1 and a preparation method and application thereof, aiming at the problems of nutrition damage, incomplete detoxification and high cost of an aflatoxin B1 detoxification method.
In order to solve the technical problem, the invention discloses a microbial treatment agent for degrading aflatoxin B1, which comprises a probiotic compound and aflatoxin B1 degrading enzyme crude enzyme in a volume ratio of 1:3-3: 1.
Further, the probiotic compound comprises 1-3 parts by mass: 0.5-1.5: 0.5-1.5 of Saccharomyces cerevisiae culture solution, Acetobacter pasteurianus culture solution and Bacillus licheniformis culture solution.
Further, the contents of the Saccharomyces cerevisiae culture solution, the Acetobacter pasteurianus culture solution and the Bacillus licheniformis culture solution are respectively 2 × 1091 x 10 per g91 x 10 per g9Per gram.
Further, the crude enzyme material of the aflatoxin B1 degrading enzyme is a mixture of crude enzyme liquid which is produced by aspergillus niger and is used for degrading AFB1 and crude enzyme liquid which is produced by rhizopus and is used for degrading AFB1, wherein the volume ratio of the crude enzyme material of the aflatoxin B1 degrading enzyme is 1:1, and the enzyme activity of the crude enzyme material is 150U/g.
The invention also discloses a preparation method of the microbial treatment agent for degrading aflatoxin B1, which comprises the following steps:
step 1, preparing a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 1:3-3:1 to prepare the microorganism treating agent for degrading the aflatoxin B1.
Further, the preparation of the probiotic compound in the step 1 specifically comprises:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in the Saccharomyces cerevisiae fermentation medium according to the inoculum size of 4% -8% (V/V), shake culturing at 25-30 deg.C and 155-9CFU/ml；
Step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 4% -8% (V/V), performing shake culture at 25-35 deg.C and 95-105r/min, harvesting when the culture is for 36h, and regulating the content of bacteria to 1 × 109CFU/ml；
Step 1.3, culturing the bacillus licheniformisPreparing nutrient solution: inoculating Bacillus licheniformis in Bacillus licheniformis fermentation medium according to the inoculation amount of 3-7% (V/V), performing shake culture at 32-38 deg.C and 155-165r/min, and harvesting after 24 hr culture, wherein the content of the regulating bacteria is 1 × 109CFU/ml；
Step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 1-3: 0.5-1.5: mixing at a ratio of 0.5-1.5, adding cereal shell powder or crude fiber powder, and drying at 40-45 deg.C to obtain probiotic compound.
Further, the composition of the saccharomyces cerevisiae fermentation medium: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural; the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5; the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and pH 6.5.
Further, the crude enzyme product for preparing the aflatoxin B1 degradation enzyme in the step 2 specifically comprises the following steps:
step 2.1, coating the degraded AFB1 Aspergillus niger strains on a PDA solid plate, and culturing at the constant temperature of 26-30 ℃ for 144 hours until a large amount of spores are produced; adding sterilized normal saline into the dish, scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residue, and adjusting spore concentration to 1 × 109Inoculating CFU/ml into solid fermentation culture medium according to 5ml per bottle, mixing well, culturing in constant temperature incubator at 26-30 deg.C for 5d, and harvesting; according to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1 hour at room temperature; filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, culturing at the constant temperature of 15-35 ℃ for 96 hours until a large amount of spores are produced, and harvesting the strains on the plateAdding appropriate amount of sterilized normal saline, scraping off spores on a plate with a coating rod, filtering with four layers of gauze to remove mycelium residue, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 15-35 ℃ for 5d, and then harvesting. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, soaking at room temperature for 1h, filtering the fermentation culture by using 8 layers of gauze after soaking is finished, centrifuging the filtrate for 5min under the condition of 5000r/min, filtering by using qualitative filter paper, filtering by using a filter membrane of 0.20 mu m for sterilization, and storing in a refrigerator at 4 ℃ to obtain crude enzyme liquid for degrading AFB1 produced by rhizopus;
and 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare the crude enzyme product of the aflatoxin B1 degrading enzyme.
Further, the sterilized normal saline contains tween 80 in a volume fraction of 0.05%; the degraded AFB1 Aspergillus niger strains and the degraded AFB1 Rhizopus strains are provided by a strain preservation center of the research and design institute of food fermentation industry in Sichuan province, wherein the degraded AFB1 Aspergillus niger strains (Aspergillus niger 20180420) are preserved in the China center for type culture collection in 2018, 05 and 02, and the preservation number is CCTCC NO: 2018245; the degraded AFB1 rhizopus strain is preserved in the China center for type culture Collection in 2018, 05 and 28, with the preservation number of CCTCC NO: 2018313.
The invention also discloses an application of the microbial treatment agent for degrading the aflatoxin B1 in preparing chicken feed or pig feed, wherein the addition amount of the microbial treatment agent for degrading the aflatoxin B1 accounts for 0.10-0.15% of the chicken feed or pig feed.
Compared with the prior art, the invention can obtain the following technical effects:
1) the invention separates and purifies AFB1 degrading enzyme, then combines probiotics and AFB1 degrading enzyme to be applied to cultivation, observes the degrading effect of the probiotics on AFB1 in feed, and lays a foundation for the application of AFB1 microorganism treatment in animal production and taking measures to relieve the harm of the microorganisms.
2) The method for degrading AFB1 toxin has the advantages of no nutrition damage, thorough detoxification, high efficiency, no residue, low cost, simple and convenient operation and the like, and particularly the method degrades mycotoxin into nontoxic products. Therefore, the detoxification treatment of the grain crops and the feed polluted by the mycotoxin is an effective method for expanding feed resources and reducing loss.
3) The microbial treatment agent can obviously relieve the adverse effect of AFB1 on the production performance of chickens and pigs.
4) On the basis of probiotics and degrading enzymes which have a degrading effect on AFB1, the probiotics and AFB1 degrading enzymes are matched and applied to cultivation, and the microbes or metabolites thereof which can inhibit the growth of the mold are proportionally added into feed raw materials to inhibit the growth of the mold by utilizing the competitive action among the microbes, so that the generation of toxins is inhibited, and the aim of preventing mycotoxicosis of livestock and poultry is fulfilled.
Of course, it is not necessary for any one product in which the invention is practiced to achieve all of the above-described technical effects simultaneously.
Detailed Description
The following embodiments are described in detail with reference to the accompanying drawings, so that how to implement the technical features of the present invention to solve the technical problems and achieve the technical effects can be fully understood and implemented.
The Saccharomyces Cerevisiae (Saccharomyces Cerevisiae) CGMCC2.3866, Acetobacter pasteurianus (Acetobacter pasteurianus) CGMCC1.2269 and Bacillus licheniformis (Bacillus licheniformis) CGMCC1.7461 used in the invention are purchased from China general microbiological culture collection center; aflatoxin B1 was purchased from Zhongji and assayed by Aflatoxin B1(Aflatoxin B1) enzyme-linked immunosorbent assay (ELISA), i.e., by ELISA kit and its supporting instrument of Shanghai Haalin Biotech Co., Ltd., the specific method was by national standard (GB/T5009.221996 second method).
The invention discloses a microbial treatment agent for degrading aflatoxin B1, which comprises a probiotic compound and aflatoxin B1 degrading enzyme crude enzyme in a volume ratio of 1:3-3: 1.
Wherein the probiotic compound comprises the following components in a mass ratio of 1-3: 0.5-1.5: 0.5-1.5 of Saccharomyces cerevisiae culture solution, Acetobacter pasteurianus culture solution and Bacillus licheniformis culture solution.
The contents of Saccharomyces cerevisiae culture solution, Acetobacter pasteurianus culture solution and Bacillus licheniformis culture solution are respectively 2 × 1091 x 10 per g91 x 10 per g9Per gram.
The crude enzyme of the aflatoxin B1 degrading enzyme is crude enzyme liquid produced by Aspergillus niger and used for degrading AFB1 or crude enzyme liquid produced by Rhizopus and used for degrading AFB1, and the enzyme activity of the crude enzyme of the aflatoxin B1 degrading enzyme is 150U/g.
The enzyme activity of AFB1 degrading enzyme is defined as that 1ng (nanogram) of AFB1 is degraded into one enzyme activity unit (U) per minute at the temperature of 37 ℃.
The invention also discloses a preparation method of the microbial treatment agent for degrading aflatoxin B1, which comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in the Saccharomyces cerevisiae fermentation medium according to the inoculum size of 4% -8% (V/V), shake culturing at 25-30 deg.C and 155-9CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 4% -8% (V/V), performing shake culture at 25-35 deg.C and 95-105r/min, harvesting when the culture is for 36h, and regulating the content of bacteria to 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating bacillus licheniformis in a bacillus licheniformis fermentation culture medium according to the inoculation amount of 3-7% (V/V)The bacteria are subjected to shake cultivation at the temperature of 32-38 ℃ and the speed of 155-. Regulating bacteria content to 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 1-3: 0.5-1.5: mixing the mixture according to the proportion of 0.5-1.5, and drying the mixture at low temperature by using a loading body to prepare a probiotic compound;
wherein the carrier is cereal shell powder or crude fiber powder, and the low temperature is 40-45 ℃;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, screening and separating aspergillus niger:
aspergillus niger strains which degrade AFB1 were picked, plated on PDA solid plates, and harvested at a constant temperature of 26-30 ℃ until spores are produced in large quantities (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 26-30 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by Sichuan province food fermentation industry research and design institute; aspergillus niger strain Aspergillus niger 20180420 (Aspergillus niger) degraded AFB1 was deposited in the China center for type culture Collection (same below) at 2018, 05 and 02.
The Aspergillus niger strain for degrading AFB1 is prepared by the following method:
a. separation: mashing 1-2g of old wine Daqu, diluting with 100ml of sterile water, adding 15ml of PDA culture medium with the temperature of about 50 ℃, culturing at the constant temperature of 26-30 ℃ for 72 hours, and selecting single bacteria to inoculate on the slant of a test tube for later use.
b. Screening: preparing 10g of corn flour infected by aflatoxin (measuring the content of aflatoxin before use), inoculating the selected bacteria on a culture medium, culturing for 6 days, dissolving with sterile water, mixing with corn flour, standing for 1 day, measuring the content of aflatoxin in the corn flour, and obtaining the strain when the degradation value reaches more than 80%.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 15-35 ℃ until spores are produced in large quantity, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 15-35 ℃ for 5d, and then harvesting. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 produced by Rhizopus;
wherein, the degraded AFB1 Rhizopus strain is provided by Sichuan province food fermentation industry research design institute, and the degraded AFB1 Rhizopus strain (Rhizopus sp.20180520) is preserved in the China center for type culture Collection in 2018, 05 and 28 months, with the preservation number of CCTCC NO:2018313 (the same below).
The degraded AFB1 rhizopus strain is prepared by the following method:
a. separation: mashing 1-2g of old wine Daqu, diluting with 100ml of sterile water, adding 15ml of PDA culture medium with the temperature of about 50 ℃, culturing at constant temperature of 15-35 ℃ for 4 days, and selecting single bacteria to inoculate on the slant of a test tube for later use.
b. Screening: preparing 10g of corn flour infected by aflatoxin (measuring the content of aflatoxin before use), inoculating the selected bacteria on a culture medium, culturing for 4 days, dissolving with sterile water, mixing with corn flour, standing for 1 day, measuring the content of aflatoxin in the corn flour, and obtaining the strain when the degradation value reaches more than 80%.
Step 2.3, degrading the AFB1 by the crude enzyme solution:
selecting 3ml of each of the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus (taking another 6ml of the crude enzyme liquid inactivated at high temperature as a control group), adding AFB1 to make the concentration of the crude enzyme liquid be about 100ug/L, uniformly mixing, placing at 30 ℃ for constant temperature culture, and sampling after 48h to determine the content of AFB 1. The degradation rate of the crude enzyme solution on AFB1 was determined to be 89.04%. The crude enzyme liquid of AFB1 degrading enzyme is proved to be protein by heat denaturation test.
Step 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 1:3-3:1 to prepare a microbial treating agent for degrading aflatoxin B1;
wherein the crude enzyme for degrading the enzyme by the aflatoxin B1 is a mixed solution of crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and crude enzyme liquid for degrading AFB1 produced by Rhizopus in a volume ratio of 1: 1.
Example 1
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in 6% (V/V) of fermentation medium, shake culturing at 29 deg.C and 160r/min, harvesting when culturing for 27h, regulating bacteria content to 1 × 109CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium at 6% (V/V), and culturing at 30 deg.C and 100%r/min shake culture, harvesting after 36 hr, regulating bacteria content to 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating Bacillus licheniformis in a Bacillus licheniformis fermentation culture medium according to the inoculation amount of 5% (V/V), performing shake culture at 35 deg.C and 160r/min, and harvesting after 24 hr of culture; regulating bacteria content to 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 2: 1:1, adding grain shell powder, and drying at 40 ℃ to prepare a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, the Aspergillus niger strains capable of degrading AFB1 are picked, spread on a PDA solid plate, and cultured at the constant temperature of 28 ℃ until spores are produced in a large amount, and then harvested (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 28 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 25 ℃ until spores are produced in a large amount, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant temperature incubator at 25 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 produced by Rhizopus;
wherein, the strain for degrading AFB1 rhizopus is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 1:1 to prepare the microbial treating agent for degrading aflatoxin B1.
The prepared microbial treatment agent for degrading aflatoxin B1 is added into pig feed, wherein the addition amount of the microbial treatment agent for degrading aflatoxin B1 accounts for 0.10% of the pig feed.
Example 2
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in the fermentation culture medium of Saccharomyces cerevisiae at an inoculation amount of 4% (V/V), shake culturing at 30 deg.C and 155r/min, and standingHarvesting after 27h, regulating the content of bacteria to 1 × 109CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 4% (V/V), performing shake culture at 35 deg.C and 95r/min, harvesting when the culture is for 36h, and regulating the bacteria content to 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating Bacillus licheniformis into a Bacillus licheniformis fermentation medium according to the inoculation amount of 3% (V/V), performing shake culture at 38 deg.C and 155r/min, and harvesting after 24 hr of culture. Regulating bacteria content to 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution according to a mass ratio of 1: 1.5: 0.5, adding coarse fiber powder, and drying at the temperature of 45 ℃ to prepare a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, the Aspergillus niger strain degraded AFB1 is picked, coated on a PDA solid plate, and cultured at the constant temperature of 26 ℃ until the spores are produced in large quantity, and then harvested (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant temperature incubator at 30 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2 ratio of solid fermentation culture to biomassMixing with saline water, and soaking at room temperature for 1 hr (stirring intermittently during soaking). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 15 ℃ until spores are produced in a large amount, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (5) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a 15 ℃ constant temperature incubator for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 produced by Rhizopus;
wherein, the strain for degrading AFB1 rhizopus is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 1:2 to prepare the microbial treating agent for degrading aflatoxin B1.
Adding the prepared microbial treatment agent for degrading aflatoxin B1 into chicken feed, wherein the addition amount of the microbial treatment agent for degrading aflatoxin B1 accounts for 0.15% of the chicken feed.
Example 3
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in fermentation medium of Saccharomyces cerevisiae in an amount of 8% (V/V), shake culturing at 25 deg.C and 165r/min, harvesting when culturing for 27h, and regulating bacteria content to 1 × 109CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 8% (V/V), performing shake culture at 25 deg.C and 105r/min, harvesting when the culture is for 36h, and regulating the content of bacteria to 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating Bacillus licheniformis into a Bacillus licheniformis fermentation medium according to the inoculation amount of 7% (V/V), performing shake culture at 32 ℃ at 165r/min, and harvesting after 24h of culture. Regulating bacteria content to 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 3: 0.5: 1.5, adding coarse fiber powder, and drying at the temperature of 42 ℃ to prepare a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, selecting and degrading AFB1 Aspergillus niger strains, coating the strains on a PDA solid plate, and culturing at constant temperature of 30 ℃ until spores are largeHarvest (about 144 hours) when the amount was produced. Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant temperature incubator at 26 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 35 ℃ until spores are produced in a large amount, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 35 ℃ for 5 d. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 produced by Rhizopus;
wherein, the strain for degrading AFB1 rhizopus is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 1:3 to prepare the microbial treating agent for degrading aflatoxin B1.
Adding the prepared microbial treatment agent for degrading aflatoxin B1 into chicken feed, wherein the addition amount of the microbial treatment agent for degrading aflatoxin B1 accounts for 0.15% of the chicken feed.
Example 4
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in fermentation medium of Saccharomyces cerevisiae at 5% (V/V), shake culturing at 26 deg.C and 162r/min, harvesting when culturing for 27 hr, regulating bacteria content to 1 × 109CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 5% (V/V), performing shake culture at 28 deg.C for 102r/min, harvesting when the culture is for 36h, and regulating the bacteria content to 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating Bacillus licheniformis into a Bacillus licheniformis fermentation medium according to the inoculation amount of 4% (V/V), performing shake culture at 34 deg.C and 162r/min, and harvesting after 24h of culture. Regulating bacteria content to 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution according to a mass ratio of 1.8: 1.2: 0.8, adding the cereal shell powder, and drying at the temperature of 40 ℃ to prepare a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, the Aspergillus niger strains capable of degrading AFB1 are picked, spread on a PDA solid plate, and cultured at the constant temperature of 27 ℃ until spores are produced in a large amount, and then harvested (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 27 ℃ for 5 d. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 32 ℃ until spores are produced in a large amount, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 32 ℃ for 5 d. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate at 5000r/min for 5min, filtering with qualitative filter paper, and filtering with the final productFiltering with 0.20 μm filter membrane, sterilizing, and storing in refrigerator at 4 deg.C to obtain crude enzyme solution produced by Rhizopus for degrading AFB 1;
wherein, the strain for degrading AFB1 rhizopus is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 2:1 to prepare the microbial treating agent for degrading aflatoxin B1.
The prepared microbial treatment agent for degrading the aflatoxin B1 is added into chicken feed or pig feed, wherein the addition amount of the microbial treatment agent for degrading the aflatoxin B1 accounts for 0.10-0.15% of the chicken feed or pig feed.
Example 5
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in 6% (V/V) of fermentation medium, shake culturing at 29 deg.C and 160r/min, harvesting when culturing for 27h, regulating bacteria content to 1 × 109CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 6% (V/V), performing shake culture at 30 ℃ at 100r/min, harvesting when the culture is carried out for 36h, and adjusting the content of bacteria to be 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3 lichen budPreparation of a bacillus culture solution: inoculating Bacillus licheniformis in Bacillus licheniformis fermentation medium according to the inoculation amount of 5% (V/V), shake culturing at 35 deg.C and 160r/min, and harvesting after 24 hr, wherein the content of regulating bacteria is 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 2: 1:1, adding grain shell powder, and drying at the temperature of 45 ℃ to prepare a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, the Aspergillus niger strains capable of degrading AFB1 are picked, spread on a PDA solid plate, and cultured at the constant temperature of 28 ℃ until spores are produced in a large amount, and then harvested (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 28 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 25 ℃ until spores are produced in a large amount, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the plate with a coating rod, and passing through four layers of gauzeFiltering to remove mycelium residue, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant temperature incubator at 25 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 produced by Rhizopus;
wherein, the strain for degrading AFB1 rhizopus is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude aflatoxin B1 degrading enzyme according to the volume ratio of 2:3 to prepare the microbial treating agent for degrading aflatoxin B1.
Adding the prepared microbial treatment agent for degrading aflatoxin B1 into chicken feed, wherein the addition amount of the microbial treatment agent for degrading aflatoxin B1 accounts for 0.15% of the chicken feed.
Example 6
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in fermentation medium of Saccharomyces cerevisiae in an amount of 7% (V/V), shake culturing at 29 deg.C and 158r/min, harvesting when culturing for 27h, and regulating bacteria content to 1 × 109CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution:inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 7% (V/V), performing shake culture at 28 deg.C for 102r/min, harvesting after culturing for 36h, and regulating the bacteria content to 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating Bacillus licheniformis into a Bacillus licheniformis fermentation medium according to the inoculation amount of 6% (V/V), performing shake culture at 36 deg.C and 158r/min, and harvesting after 24 hr of culture. Regulating bacteria content to 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution according to a mass ratio of 2.8: 0.8: 1.2, adding coarse fiber powder, and drying at the temperature of 40 ℃ to prepare a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, the Aspergillus niger strain degraded AFB1 is picked, coated on a PDA solid plate, and cultured at the constant temperature of 29 ℃ until the spores are produced in large quantity, and then harvested (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 29 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 28 ℃ until spores are produced in a large amount, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 28 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 produced by Rhizopus;
wherein, the strain for degrading AFB1 rhizopus is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 3:1 to prepare the microbial treating agent for degrading aflatoxin B1.
The prepared microbial treatment agent for degrading aflatoxin B1 is added into pig feed, wherein the addition amount of the microbial treatment agent for degrading aflatoxin B1 accounts for 0.10% of the pig feed.
Example 7
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculation according to 6% (V/V)Inoculating Saccharomyces cerevisiae into Saccharomyces cerevisiae fermentation medium, shake culturing at 29 deg.C and 160r/min, harvesting when culturing for 27 hr, regulating bacteria content to 1 × 109CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 6% (V/V), performing shake culture at 30 ℃ at 100r/min, harvesting when the culture is carried out for 36h, and adjusting the content of bacteria to be 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating Bacillus licheniformis in Bacillus licheniformis fermentation medium according to the inoculation amount of 5% (V/V), shake culturing at 35 deg.C and 160r/min, and harvesting after 24 hr, wherein the content of regulating bacteria is 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 2: 1:1, adding grain shell powder, and drying at 40 ℃ to prepare a probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme:
step 2.1, the Aspergillus niger strains capable of degrading AFB1 are picked, spread on a PDA solid plate, and cultured at the constant temperature of 28 ℃ until spores are produced in a large amount, and then harvested (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109CFU/ml, inoculating 5ml of the mixture into solid fermentation medium per bottle, mixing, and standing at 28 deg.CAnd (5) culturing in a warm incubator for 5d and then harvesting. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 25 ℃ until spores are produced in a large amount, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant temperature incubator at 25 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 produced by Rhizopus;
wherein, the strain for degrading AFB1 rhizopus is provided by the research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
and 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 3:2 to prepare the microbial treating agent for degrading aflatoxin B1.
The prepared microbial treatment agent for degrading the aflatoxin B1 is added into chicken feed or pig feed, wherein the addition amount of the microbial treatment agent for degrading the aflatoxin B1 accounts for 0.10-0.15% of the chicken feed or pig feed.
Comparative example 1
A preparation method of a microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound:
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in the Saccharomyces cerevisiae fermentation medium according to the inoculum size of 4% -8% (V/V), shake culturing at 25-30 deg.C and 155-9CFU/ml；
Wherein, the saccharomyces cerevisiae fermentation medium comprises the following components: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural;
step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 4% -8% (V/V), performing shake culture at 25-35 deg.C and 95-105r/min, harvesting when the culture is for 36h, and regulating the content of bacteria to 1 × 109CFU/ml；
Wherein the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5;
step 1.3, preparing a bacillus licheniformis culture solution: inoculating bacillus licheniformis in a bacillus licheniformis fermentation culture medium according to the inoculation amount of 3-7% (V/V), performing shake culture at 32-38 ℃ and 165r/min, and harvesting after 24h of culture. Regulating bacteria content to 1 × 109CFU/ml；
Wherein the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and 6.5 of PH;
step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 1-3: 0.5-1.5: mixing at a ratio of 0.5-1.5, adding cereal shell powder, and drying at 40 deg.C to obtain probiotic compound.
And adding the prepared probiotic compound into chicken feed or pig feed, wherein the addition amount of the probiotic compound accounts for 0.15% of that of the chicken feed or pig feed.
Comparative example 2
A preparation method of crude aflatoxin B1 degradation enzyme comprises the following steps:
step 1, preparing crude aflatoxin B1 degrading enzyme:
step 1.1, an Aspergillus niger strain capable of degrading AFB1 is picked, coated on a PDA solid plate, and cultured at the constant temperature of 26-30 ℃ until spores are produced in a large quantity, and then harvested (about 144 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 26-30 ℃ for 5 days to obtain the CFU/ml. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). Filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
wherein, the Aspergillus niger strain for degrading AFB1 is provided by research and design institute of food fermentation industry in Sichuan province.
Step 1.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, and culturing at the constant temperature of 15-35 ℃ until spores are produced in large quantity, and harvesting (about 96 hours). Adding appropriate amount of sterilized normal saline (containing Tween 80 with volume fraction of 0.05%), scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residues, and adjusting spore concentration to 1 × 109And (3) inoculating the CFU/ml into a solid fermentation culture medium according to 5ml per bottle, uniformly mixing, and culturing in a constant-temperature incubator at 15-35 ℃ for 5d, and then harvesting. According to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1h at room temperature (intermittently stirring in the soaking process). After soaking is finishedFiltering the fermentation culture with 8 layers of gauze, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution produced by Rhizopus for degrading AFB 1;
wherein, the strain for degrading AFB1 rhizopus is provided by research and design institute of food fermentation industry in Sichuan province.
Step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme;
adding the prepared crude aflatoxin B1 degrading enzyme into chicken feed, wherein the addition amount of the crude aflatoxin B1 degrading enzyme accounts for 0.15% of the chicken feed.
The following is described with specific experimental data:
firstly, AFB1 was added to 60ml of the aflatoxin B1-degrading microbial treatment agent obtained in examples 1-7 and comparative examples 1-2 to a final concentration of about 50ug/L in the aflatoxin B1-degrading microbial treatment agent, and the mixture was subjected to shaking culture in a constant temperature gas bath at 37 ℃. The same volume of PBS buffer (phosphate buffer saline) plus an equivalent dose of AFB1 was used as a blank.
Mixing the culture solution uniformly, sucking 1ml by a pipette, centrifuging for 5min under the condition of 13000 r/min, and taking the centrifuged supernatant for AFB1 content determination; the degradation of AFB1 by examples 1-7 and comparative examples 1-2 is shown in Table 1.
TABLE 1 degradation of examples 1-7 and comparative examples 1-2 on AFB1
As can be seen from table 1, the degradation rate of example 5 to AFB1 is the highest, and reaches 90.02%, which indicates that the ratio of the crude enzyme of the probiotic complex and the aflatoxin B1 degrading enzyme is 2: the proportion of 3 is the best to degrade AFB 1. The degradation rates of the crude enzyme pair AFB1 of the separate probiotic compound and aflatoxin B1 degradation enzyme are 68.32% and 69.31% respectively. Fully indicates that the probiotic compound has good compatibility with crude enzyme of the aflatoxin B1 degrading enzyme.
Secondly, 0.15 percent of the microbial treatment agent for degrading aflatoxin B1 is added into chicken feed with 200ug/kg of AFB1 to influence the laying hens
1. Purpose of the experiment
The experiment verifies that 0.15 percent of the microbial treatment agent for degrading the aflatoxin B1 is added into chicken feed with the AFB1 content of 200ug/kg, the influence on various production indexes of laying hens is realized, and the experiment mainly considers relevant indexes such as laying rate, feed intake, morbidity and the like.
2. Test site
The test site is arranged in a chicken raising experimental base of Luzhou Zhengtai bioengineering Co., Ltd in Lucounty, Sichuan province.
3. Materials and methods
3.1 test materials
AFB1 (purchased from mid-test): the addition amount of the chicken feed is 200 ug/kg. The experimental group of laying hens are treated with the microbial treatment agent for degrading the aflatoxin B1, and the microbial treatment agent comprises the following components: 0.15 percent of chicken feed is added; the yeast feed additive produced by a certain feed enterprise in Sichuan for the control group of laying hens: 0.15 percent of chicken feed is added.
3.2 test animals and test design
2000 feathers of the same variety of laying hens in the same batch are selected, the feathers are divided into 4 test units according to stall conditions, the 4 test units are randomly divided into 2 treatments (groups), each treatment comprises 2 repetitions, and the number of the repeated chickens is 500.
Before the test, according to the daily egg laying condition, the production performance of each group is not greatly different and has no significant difference statistically when the groups are grouped. The pre-test period is 11 days, and the test period is 60 days. The same feed is fed in the pre-test period and the test period, and the feeding management is carried out according to the convention. The laying rate, feed intake and morbidity of each group are counted during the test period,
the experimental design is shown in table 2:
table 2 experimental design protocol
3.3 Experimental daily ration and feed management
The test daily ration is shown in table 3;
TABLE 3 test daily ration table
| Control group ratio (%) | Test group ratio (%) |
| Corn 35 | Corn 35 |
| Wheat 35 | Wheat 35 |
| Bean pulp 24 | Bean pulp 24 |
| Stone powder 5.85 | Stone powder 5.85 |
| 0.15 of degraded AFB1 agent | Yeast 0.15 |
| Total 100 | Total 100 |
4. Data processing
Data results are expressed as "mean ± sem", and data statistics were performed using SPSS 17.0.
5. Test results
0.15 percent of the microbial treatment agent for degrading aflatoxin B1 is added into chicken feed with 200ug/kg of AFB1, and 0.15 percent of yeast feed additive is added into chicken feed with 200ug/kg of AFB1, so that the influence on various production indexes of laying hens is caused. Results of egg production, feed intake and morbidity for each group during the trial period are shown in table 4:
TABLE 4 laying rate, feed intake and morbidity for each group
| Group of | Feed intake (g/day) | Laying rate (%) | Incidence (%) |
| Control group | 126.20±0.72 | 71.33±4.28 | 5.86±0.69 |
| Test group | 132.60±0.43 | 75.69±3.42 | 3.12±0.61 |
As can be seen from Table 4 above, the feed intake test group was 5.07% higher than the control group, the difference was significant ((p < 0.05), the laying rate test group was 6.11% higher than the control group, the difference was significant ((p < 0.05), the incidence test group was 46.76% lower than the control group, and the difference was very significant (p < 0.01).
6. Conclusion
Compared with the yeast feed additive, the 0.15 percent of the microbial treatment agent for degrading aflatoxin B1 is added into the chicken feed with the AFB1 content of 200ug/kg, which is favorable for preventing the feed intake and the laying rate of laying hens from being reduced, preventing the incidence of the laying hens from being improved, thereby obviously relieving the adverse effect of AFB1 on the production performance of the laying hens and improving the production economic value.
Thirdly, 0.10 percent of the microbial treatment agent for degrading aflatoxin B1 is added into chicken feed with 200ug/kg of AFB1 to influence the laying hens
1. Purpose of the experiment
The experiment verifies that the influence of 0.10 percent of the microbial treatment agent for degrading the aflatoxin B1 on various production indexes of fattening pigs is realized by adding 0.10 percent of the microbial treatment agent for degrading the aflatoxin B1 into the pig feed with the AFB1 content of 200ug/kg, and the experiment mainly inspects the relevant indexes including daily gain, morbidity and the like.
2. Test site
The test site is arranged in a pig raising experiment base of Luzhou Zhengtai bioengineering Co., Ltd. of Luzhou province, Luzhou province.
3. Materials and methods
3.1 test materials
AFB1 (purchased from mid-test): the addition amount of the pig feed is 200 ug/kg. The experimental group fattening pigs treated by the microorganism treating agent for degrading the aflatoxin B1 of the invention: 0.10 percent of pig feed is added; the yeast feed additive produced by a certain feed enterprise in Sichuan for fattening pigs in a control group is as follows: 0.10 percent of pig feed is added.
3.2 test animals and test design
200 fattening pigs of the same breed in the same batch are selected, the fattening pigs are divided into 20 test units according to the stall condition, the 20 test units are randomly divided into 2 treatment (groups), each treatment is 10 repetitions, and the number of each repeated pig is 10.
Before the test, according to the daily egg laying condition, the production performance of each group is not greatly different and has no significant difference statistically when the groups are grouped. The pre-test period is 7 days, and the test period is 60 days. The same feed is fed in the pre-test period and the test period, and the feeding management is carried out according to the convention. The daily gain and the incidence of the disease of each group are counted during the test period,
the experimental design is shown in table 5:
TABLE 5 experimental design protocol with addition of 0.10% of microbial treatment agent to degrade aflatoxin B1
3.3 Experimental daily ration and feed management
The experimental daily ration is shown in table 6:
TABLE 6 Experimental daily ration table with 0.10% addition of microbial treatment to degrade aflatoxin B1
| Control group ratio (%) | Test group ratio (%) |
| Corn 63 | Corn 63 |
| Wheat 12 | Wheat 12 |
| Bean pulp 20 | Bean pulp 20 |
| Calcium carbonate 3 | Calcium carbonate 3 |
| Calcium hydrogen phosphate 1.6 | Calcium hydrogen phosphate 1.6 |
| 0.3 part of common salt | 0.3 part of common salt |
| 0.10 of degraded AFB1 agent | Yeast 0.10 |
| Total 100 | Total 100 |
4. Data processing
Data results are expressed as "mean ± sem", and data statistical work was performed using SPSS 17.0 (data statistical analysis software).
5. Test results
0.10 percent of the microbial treatment agent for degrading aflatoxin B1 is added into the pig feed with the AFB1 content of 200ug/kg, and 0.10 percent of yeast feed additive is added into the pig feed with the AFB1 content of 200ug/kg, so that the influence on various production indexes of fattening pigs is realized. The results of daily gain and incidence for each group during the trial are shown in table 7:
TABLE 7 daily gain and morbidity results for each group with 0.10% addition of a microbial treatment to degrade aflatoxin B1
| Group of | Daily gain (g/day) | Incidence (%) |
| Control group | 612.21±0.71 | 4.7±0.66 |
| Test group | 663.64±0.48 | 2.9±0.50 |
As can be seen from Table 7 above, the daily gain test group was 8.40% higher than the control group, the difference was significant ((p < 0.05), the incidence test group was 38.30% lower than the control group, and the difference was very significant (p < 0.01).
6. Conclusion
Compared with the yeast feed additive, the 0.10 percent of the microbial treatment agent for degrading the aflatoxin B1 is added into the pig feed with the AFB1 content of 200ug/kg, so that the daily gain of the fattening pigs is improved, the incidence of the fattening pigs is prevented from being improved, the adverse effect of the AFB1 on the production performance of the pigs is remarkably relieved, and the production economic value is improved.
The application of the mycotoxin microbial treatment agent in cultivation provided by the invention can be concluded after specific application:
(1) the probiotic compound and crude enzyme of aflatoxin B1 degradation enzyme are mixed according to the weight ratio of 2: the combination ratio of 3 has the best degradation effect on AFB1, and the degradation rate on AFB1 reaches 90.02%.
(2) 0.15 percent of the microbial treatment agent for degrading aflatoxin B1 is added into chicken feed with the AFB1 content of 200ug/kg, so that the adverse effect of AFB1 on the production performance of the chicken can be remarkably relieved.
(3) 0.10 percent of the microbial treatment agent for degrading aflatoxin B1 is added into pig feed with the AFB1 content of 200ug/kg, so that the adverse effect of AFB1 on the production performance of pigs can be remarkably relieved.
While the foregoing description shows and describes several preferred embodiments of the invention, it is to be understood, as noted above, that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (3)
1. The application of the microbial treatment agent for degrading the aflatoxin B1 in preparing the chicken feed or the pig fattening feed for improving the laying rate of the chicken is characterized in that the addition amount of the microbial treatment agent for degrading the aflatoxin B1 accounts for 0.10-0.15% of the chicken feed or the pig feed;
the preparation method of the microbial treatment agent for degrading aflatoxin B1 comprises the following steps:
step 1, preparing a probiotic compound;
step 1.1, preparation of a saccharomyces cerevisiae culture solution: inoculating Saccharomyces cerevisiae in the Saccharomyces cerevisiae fermentation medium according to the inoculum size of 4% -8% (V/V), shake culturing at 25-30 deg.C and 155-9CFU/ml；
Step 1.2, preparation of a Acetobacter pasteurianus culture solution: inoculating Acetobacter pasteurianus in acetic acid bacteria fermentation medium according to the inoculation amount of 4% -8% (V/V), performing shake culture at 25-35 deg.C and 95-105r/min, harvesting when the culture is for 36h, and regulating the content of bacteria to 1 × 109CFU/ml；
Step 1.3, preparing a bacillus licheniformis culture solution: inoculating Bacillus licheniformis in Bacillus licheniformis fermentation medium according to the inoculation amount of 3-7% (V/V), performing shake culture at 32-38 deg.C and 155-165r/min, and harvesting after 24 hr culture, wherein the content of the regulating bacteria is 1 × 109CFU/ml；
Step 1.4, mixing a saccharomyces cerevisiae culture solution, a pasteurella vinegar culture solution and a bacillus licheniformis culture solution in a mass ratio of 1-3: 0.5-1.5: mixing at a ratio of 0.5-1.5, adding cereal shell powder or crude fiber powder, and drying at 40-45 deg.C to obtain probiotic compound;
step 2, preparing crude aflatoxin B1 degrading enzyme;
step (ii) of2.1, coating the degraded AFB1 Aspergillus niger strains on a PDA solid plate, and culturing at the constant temperature of 26-30 ℃ for 144 hours until a large amount of spores are produced; adding sterilized normal saline into the dish, scraping off spores on the dish with a coating rod, filtering with four layers of gauze to remove mycelium residue, and adjusting spore concentration to 1 × 109Inoculating CFU/ml into solid fermentation culture medium according to 5ml per bottle, mixing well, culturing in constant temperature incubator at 26-30 deg.C for 5d, and harvesting; according to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, and soaking for 1 hour at room temperature; filtering the fermentation culture with 8 layers of gauze after soaking, centrifuging the filtrate for 5min at 5000r/min, filtering with qualitative filter paper, filtering with 0.20 μm filter membrane for sterilization, and storing in a refrigerator at 4 deg.C to obtain crude enzyme solution for degrading AFB1 from Aspergillus niger;
step 2.2, selecting and degrading AFB1 rhizopus strains, coating the strains on a PDA solid plate, culturing at the constant temperature of 15-35 ℃ for 96 hours until a large amount of spores are produced, harvesting, adding a proper amount of sterilized normal saline into the plate, scraping the spores on the plate by using a coating rod, filtering by using four layers of gauze to remove mycelium residues, and adjusting the concentration of the spores to be 1 x 109Inoculating CFU/ml into solid fermentation culture medium according to 5ml per bottle, mixing well, culturing in 15-35 deg.C constant temperature incubator for 5d, and harvesting; according to the solid-liquid ratio of 1:2, uniformly mixing the solid fermentation culture with normal saline, soaking at room temperature for 1h, filtering the fermentation culture by using 8 layers of gauze after soaking is finished, centrifuging the filtrate for 5min under the condition of 5000r/min, filtering by using qualitative filter paper, filtering by using a filter membrane of 0.20 mu m for sterilization, and storing in a refrigerator at 4 ℃ to obtain crude enzyme liquid for degrading AFB1 produced by rhizopus;
step 2.3, mixing the crude enzyme liquid for degrading AFB1 produced by Aspergillus niger and the crude enzyme liquid for degrading AFB1 produced by Rhizopus according to the volume ratio of 1:1 to prepare a crude enzyme product of the aflatoxin B1 degrading enzyme; the enzyme activity is 150U/g;
step 3, mixing the probiotic compound and the crude enzyme of the aflatoxin B1 degrading enzyme according to the volume ratio of 1-3:1-3 to prepare a microbial treating agent for degrading aflatoxin B1;
the preservation number of the saccharomyces cerevisiae is CGMCC 2.3866; the preservation number of the acetobacter pasteurianus is CGMCC 1.2269; the preservation number of the bacillus licheniformis is CGMCC 1.7461;
the degraded AFB1 Aspergillus niger strains and the degraded AFB1 Rhizopus strains are provided by research and design institute of food fermentation industry in Sichuan province, wherein the degraded AFB1 Aspergillus niger strains are preserved in the China center for type culture Collection in 2018, 05 and 02 months, and the preservation number is CCTCC NO: M2018245; the degraded AFB1 rhizopus strain is preserved in China center for type culture Collection in 2018, 05 and 28, with the preservation number of CCTCC NO: M2018313.
2. Use according to claim 1, characterized in that the composition of the saccharomyces cerevisiae fermentation medium is: 20g of glucose, 10g of yeast powder, 1.5g of monopotassium phosphate, 1g of magnesium sulfate and 1.0L of distilled water, wherein the pH value is natural; the composition of the acetic acid bacteria culture medium is as follows: 10.0g of glucose, 10.0g of yeast extract, 0.5g of monopotassium phosphate, 0.5g of magnesium sulfate, 1.0L of distilled water and pH 5.5; the bacillus licheniformis fermentation medium comprises the following components: 10g of sucrose, 10g of peptone, 5g of sodium chloride, 1.0L of distilled water and pH 6.5.
3. The use according to claim 1, wherein the sterilized normal saline contains tween 80 in a volume fraction of 0.05%.
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