CN113462589A - Leaven for degrading aflatoxin in feed and preparation method thereof - Google Patents
Leaven for degrading aflatoxin in feed and preparation method thereof Download PDFInfo
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- 230000000593 degrading effect Effects 0.000 title claims abstract description 29
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- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 2
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
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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 COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
- A23L5/28—Removal of unwanted matter, e.g. deodorisation or detoxification using microorganisms
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Abstract
The invention relates to the technical field of toxin degradation, and particularly discloses a leavening agent for degrading aflatoxin in feed and a preparation method thereof. The fermentation inoculum can rapidly and effectively degrade aflatoxin in the feed, has high degradation rate on aflatoxin, is safe and environment-friendly, and can be directly added into the feed as a feed additive.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to a starter culture for degrading aflatoxin in feed and a preparation method thereof.
Background
Aflatoxins (AFT) are secondary metabolites produced by Aspergillus species such as Aspergillus flavus, Aspergillus parasiticus, and Aspergillus terreus, and currently known aflatoxin molecules include aflatoxin B1、B2、G1、G2、M1Etc. 20 of various types, wherein aflatoxin B1(AFB1) The most and the most toxic. It is reported that aflatoxin B is the cause of 28% of primary liver cancer patients worldwide1Poisoning and aflatoxin B1Has been classified as the first carcinogen by the international agency for research on cancer (IARC). The generation of aflatoxin is influenced by temperature, moisture, humidity and the like, and proper temperature and humidity are main factors for inducing the generation of aflatoxin. Therefore, it can be naturally produced in agricultural and sideline products and foods, and thus widely exists in grains (corn, wheat, peanut, rice, etc.), feeds, milk, bread and spices. The intake of the feed polluted by AFT by livestock and poultry can cause liver injury, immunologic dysfunction, production performance reduction and the like. In addition, AFT can also remain in animal products such as meat, eggs, milk, and the like, and poses a great threat to human health through the food chain.
The traditional aflatoxin detoxification method comprises a physical method and a chemical method, such as a high-temperature method, an alkaline treatment method, an oxidation resistance treatment method and the like. However, the methods have the defects of incomplete detoxification, low efficiency, high cost, nutrient loss, difficulty in large-scale production and the like, and are not widely applied. The microbial detoxification method mainly comprises an adsorption method and a degradation method, wherein the microbial adsorption method mainly utilizes microbial cells to adsorb AFB1, mainly related to cell wall components, can reduce the content of aflatoxin to a certain extent, but cannot decompose the aflatoxin, causes secondary pollution after being discharged out of an animal body, and some adsorbents can adsorb nutrient substances in the feed to reduce the nutritional value of the feed. The microbial degradation method mostly utilizes the enzyme generated in the metabolic process to degrade the aflatoxin, and the method is safe, effective, thorough in detoxification, strong in specificity and is concerned by researchers. Although many studies have been reported, some bacteria, fungi and their metabolitesDegradable AFB1On one hand, the degradation activity is low, the separation and purification of degrading enzymes are difficult, on the other hand, some degrading strains have high degradation activity on aflatoxin standard products, but have low degradation activity on aflatoxin in feed, and some degrading strains can not be directly added into the feed for utilization, so that the degrading strains are still difficult to be used in actual production.
Therefore, to solve the problem of AFB in the feed and raw materials1The pollution problem is solved, the problems that the feed aflatoxin has low degradation efficiency and can not be directly used as a feed additive are solved, and the research and development of the method for efficiently degrading the AFB in the feed are needed1And the microbial preparation can be directly added into feed.
Disclosure of Invention
The invention aims to provide a leaven for degrading aflatoxin in feed, and the leaven can efficiently degrade AFB in the feed1Is safe and harmless, and can be directly added into feed for use.
The invention also aims to provide a preparation method of the leaven for degrading the aflatoxin in the feed, so as to obtain the leaven capable of effectively degrading the aflatoxin in the feed, and the preparation method is simple and has lower cost.
In order to achieve the above object, the present invention provides a method for preparing a starter for degrading aflatoxin in a feed, comprising:
respectively culturing the bacillus aryabhattai and the flavobacterium brevifibrum to obtain a bacillus aryabhattai bacterial solution and a flavobacterium brevifibrum bacterial solution;
mixing the bacillus aspolis bacterial liquid and the flavobacterium brevibacterium bacterial liquid to obtain a composite bacterial liquid;
putting the compound bacterium liquid into a compound bacterium culture medium for mixed fermentation;
the cell ratio of the Bacillus aryabhattai bacterium liquid to the Flavobacterium parvum bacterium liquid in the composite bacterium liquid is 11: 15-11: 20;
further, in the above technical scheme, the mixed fermentation is carried out by filling the composite bacteria culture medium into a fermentation tank, and then filling the composite bacteria culture medium into the fermentation tankInoculating the bacterial liquid into the composite bacterial culture medium, and culturing for 27-33 hours at the temperature of 28-33 ℃; the charging coefficient of the composite bacteria culture medium in the fermentation tank is 0.6; the composite bacteria culture medium comprises 6-9 g/L of corn flour, 10-13 g/L of glucose, (NH4)240.6-0.8 g/L of SO and 7-9 g/L of corn steep liquor.
Furthermore, the Bacillus aryabhattai is from the strain preservation number of CGMCC14949, and the Flavobacterium parvum is from the strain preservation number of CCTCC No. M2017329.
Further, in the above technical solution, the step of culturing the bacillus aryabhattai is to inoculate the bacillus aryabhattai into a BYP liquid culture medium for culturing to obtain a first activated seed solution, and then continuously inoculate the first activated seed solution into a fermentation culture medium for culturing to obtain a bacillus aryabhattai bacterial solution; the step of culturing the flavobacterium brevibacterium is to inoculate the flavobacterium brevibacterium into a BYP liquid culture medium for culturing to obtain a first activated seed solution, and then continuously inoculate the first activated seed solution into a fermentation culture medium for culturing to obtain a flavobacterium liquid.
The BYP liquid culture medium comprises 10 parts of peptone, 5 parts of glucose, 5 parts of yeast extract, 5 parts of sodium chloride and 1000 parts of distilled water by mass.
Further, in the technical scheme, the fermentation medium comprises 10-12 parts of corn flour, 18-20 parts of bean cake powder and CaCO by weight35-6 parts of glucose, 8-10 parts of (NH)4)2SO41-1.2 parts of corn steep liquor, 5-6 parts of MgSO 42-2.5 parts of MnSO40.5-0.6 parts of distilled water.
Further, in the above technical solution, the step of culturing the bacillus aryabhattai is to insert the bacillus aryabhattai into a BYP liquid culture medium, culture the bacillus aryabhattai at 29-32 ℃ for 15-18h at 200-220 r to obtain a first bacillus aryabhattai activation seed solution, then continuously inoculate the first bacillus aryabhattai activation seed solution into a fermentation culture medium, and culture the bacillus aryabhattai at 29-31 ℃ for 24-26 h at 200-220 r to obtain a bacillus aryabhattai bacterial solution.
Further, in the technical scheme, the step of culturing the flavobacterium brevifibrum is to inoculate the flavobacterium brevifibrum into a BYP liquid culture medium, culture the flavobacterium brevifibrum for 15-18 hours at 29-32 ℃ and 200-220 r to obtain a first flavobacterium brevifibrum activation seed liquid, continuously inoculate the first flavobacterium brevifibrum activation seed liquid into a fermentation culture medium, and culture the flavobacterium brevifibrum for 24-26 hours at 29-31 ℃ and 200-220 r to obtain a flavobacterium liquid.
Further, the mass ratio of the composite bacteria liquid to the composite bacteria culture medium in the technical scheme is 1-1.5%.
Meanwhile, the invention provides a composite microbial inoculum for degrading zearalenone, which is prepared by the preparation method of the fermentation microbial inoculum for degrading zearalenone in the technical scheme.
Compared with the prior art, the invention has the following beneficial effects:
the method comprises the steps of respectively culturing the bacillus aryabhattai and the flavobacterium brevifibrum to obtain a bacillus aryabhattai bacterial solution and a flavobacterium brevifibrum bacterial solution, then mixing the bacillus aryabhattai bacterial solution and the flavobacterium brevifibrum bacterial solution to obtain a composite bacterial solution, and finally putting the composite bacterial solution into a composite bacterial culture medium for mixed fermentation to obtain a zymogen agent for degrading aflatoxin in the feed1The degradation rate can reach more than 90% after 16h reaction, and the prepared microbial inoculum is safe and effective and can be directly added into feed as a feed additive.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments.
Example 1
(1) Inoculating Bacillus aryabhattai strain with the preservation number of CGMCC14949 to BYP liquid culture medium (the inoculation amount is 1 percent), and culturing at 30 ℃ and 200r for 17h to obtain the once-activated Bacillus aryabhattai strain liquid. And continuously inoculating the primary activated seed solution into a fermentation culture medium (the inoculation amount is 1 percent), and culturing at 30 ℃ and 200r for 24 hours to obtain the bacillus aryabhattai bacterium solution.
Wherein BYP liquid culture medium: 10g of peptone, 5g of glucose, 5g of yeast extract, 5g of sodium chloride and 1000mL of distilled water, and sterilizing the mixture at 121 ℃ for 15min by high-pressure steam.
Wherein the fermentation medium: corn flour 12g, bean cake powder 18g, CaCO35g, glucose 8g, (NH)4)2SO41g, 5g of corn steep liquor, MgSO42g, MnSO40.5g, 1000mL of distilled water, 121 ℃, and 15min high-pressure steam sterilization.
(2) The Brevibacterium strain with the preservation number of CCTCC No. M2017329 is inoculated into a BYP liquid culture medium (the inoculum size is 1 percent), and the culture is carried out at 30 ℃ and 200r for 15-18h to obtain the once activated Brevibacterium strain liquid. Continuously inoculating the primary activated seed liquid into a fermentation culture medium (the inoculation amount is 1 percent), and culturing at 30 ℃ and 200r for 24h to obtain the flavobacterium brevibacterium liquid.
BYP liquid medium: 10g of peptone, 5g of glucose, 5g of yeast extract, 5g of sodium chloride and 1000mL of distilled water, and sterilizing the mixture at 121 ℃ for 15min by high-pressure steam.
Fermentation medium: corn flour 12g, bean cake powder 18g, CaCO35g, glucose 8g, (NH)4)2SO41g, 5g of corn steep liquor, MgSO42g, MnSO40.5g, 1000mL of distilled water, 121 ℃, and 15min high-pressure steam sterilization.
(3) A certain amount of the obtained Bacillus aryabhattai bacterial liquid and Flavobacterium parvum bacterial liquid are mixed according to the proportion of the cell number of 11:17 to obtain a composite bacterial liquid.
(4) Inoculating the compound bacterial liquid into a sterilized fermentation medium (adopting a 500L fermentation tank), wherein the inoculation amount is 1%, and fermenting to obtain the liquid leaven.
The fermentation conditions were: the charging coefficient is 0.6, the temperature is 30 ℃, the pressure is 0.05MPa, the stirring speed is 0-8 hours and 200r/min, after 8 hours, the stirring speed is 250r/min, the ventilation rate is 15-40L/min, the pH value is natural, and the fermentation period is 30 hours.
The composite bacteria liquid culture medium is 8g/L corn flour, 12g/L glucose, (NH4)2SO40.8 g/L and corn steep liquor 7 g/L.
(5) Respectively carrying out degradation tests on the aflatoxin in the feed by using the prepared liquid of the Bacillus aryabhattai and the prepared liquid of the Flavobacterium parvum and the obtained leavening agent for degrading the aflatoxin in the feed.
The test method comprises the following steps: uniformly spraying equal amount of the fungus solution of each group on the AFB1Contaminated feed (among others AFB)1The content is more than 20 mu g/ml), and the culture solution sprayed without inoculation is taken as a control group. Adding 50% distilled water into the above mixed feed, stirring, culturing at 30 deg.C for 8 hr, 16 hr, and 24 hr, oven drying, pulverizing, and detecting AFB in feed sample with rapid mycotoxin detection equipment1Concentration and calculating the degradation rate. Degradation rate of bacterial liquid to AFB1 in feed (AFB in initial feed)1concentration-AFB at sample time1concentration)/AFB in initial feed1Concentration X100%.
The test results are shown in table 1:
TABLE 1
The experimental data in table 1 show that when the ratio of the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid in the embodiment is mixed together and fermented to form the zymocyte liquid, the degradation rate of the zymocyte liquid on the aflatoxin in the feed is the largest, and 24h reaches 92.6%, although the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid have a certain degradation effect on the aflatoxin in the feed, 24h is respectively 61.4% and 67.8%, but the degradation rate is low, and the effect of the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid in the embodiment on the degradation of the aflatoxin in the feed is greatly improved by the zymocyte liquid formed by mixing the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid together and fermenting according to the ratio in the embodiment.
Example 2
(1) Inoculating Bacillus aryabhattai with the preservation number of CGMCC14949 to BYP liquid culture medium (the inoculation amount is 1.3 percent), and culturing at 29 ℃ and 220r for 18h to obtain the once-activated Bacillus aryabhattai bacterial liquid. The primary activated seed solution is continuously inoculated into a fermentation medium (the inoculation amount is 1.3 percent), and the fermentation medium is cultured at 31 ℃ and 220r for 26h to obtain the Bacillus aryabhattai bacterial solution.
Wherein BYP liquid culture medium: 10g of peptone, 5g of glucose, 5g of yeast extract, 5g of sodium chloride and 1000mL of distilled water, and sterilizing the mixture at 121 ℃ for 15min by high-pressure steam.
Wherein the fermentation medium: 10 parts of corn flour, 18 parts of bean cake powder and CaCO36 parts of glucose, 10 parts of (NH)4)2SO41.2 parts of corn steep liquor, 5 parts of MgSO42.5 parts of MnSO40.5 parts of distilled water, and high-pressure steam sterilization at 121 ℃ for 15 min.
(2) The Brevibacterium strain with the preservation number of CCTCC No. M2017329 is inoculated into a BYP liquid culture medium (the inoculation amount is 1.3 percent), and cultured for 15 hours at 32 ℃ and 200r to obtain the once-activated Brevibacterium strain liquid. The primary activated seed liquid is continuously inoculated into a fermentation medium (the inoculation amount is 1.3 percent), and the primary activated seed liquid is cultured for 24 hours at 31 ℃ and 220 r. Obtaining the Brevibacterium flavum bacterial liquid.
BYP liquid medium: 10g of peptone, 5g of glucose, 5g of yeast extract, 5g of sodium chloride and 1000mL of distilled water, and sterilizing the mixture at 121 ℃ for 15min by high-pressure steam. .
Fermentation medium: 12 parts of corn flour, 18 parts of bean cake powder and CaCO35 parts of glucose, 8 parts of (NH)4)2SO41 part, 5 parts of corn steep liquor, MgSO42 parts, MnSO40.6 parts, 1000 parts of distilled water, and high-pressure steam sterilization at 121 ℃ for 15 min.
(3) A certain amount of the obtained Bacillus aryabhattai bacterial liquid and Flavobacterium parvum bacterial liquid are mixed according to the proportion of the cell number of 11:15 to obtain a composite bacterial liquid.
(4) Inoculating the compound bacterial liquid into a sterilized fermentation medium (adopting a 500L fermentation tank), wherein the inoculation amount is 1.3%, and fermenting to obtain the liquid leaven.
The fermentation conditions were: the charging coefficient is 0.6, the temperature is 28 ℃, the pressure is 0.05MPa, the stirring speed is 0-8 hours and 200r/min, after 8 hours, the stirring speed is 250r/min, the ventilation rate is 15-40L/min, the pH value is natural, and the fermentation period is 33 hours.
The composite bacteria liquid culture medium is 6g/L corn flour, 10g/L glucose, (NH4)2SO40.8 g/L and corn steep liquor 7 g/L.
(5) Respectively carrying out degradation tests on the aflatoxin in the feed by using the prepared liquid of the Bacillus aryabhattai and the prepared liquid of the Flavobacterium parvum and the obtained leavening agent for degrading the aflatoxin in the feed.
The test method comprises the following steps: uniformly spraying equal amount of the fungus solution of each group on the AFB1Contaminated feed (among others AFB)1The content is more than 20 mu g/ml), and the culture solution sprayed without inoculation is taken as a control group. Adding 50% distilled water into the above mixed feed, stirring, culturing at 30 deg.C for 8 hr, 16 hr, and 24 hr, oven drying, pulverizing, and detecting AFB in feed sample with rapid mycotoxin detection equipment1Concentration and calculating the degradation rate. Degradation rate of bacterial liquid to AFB1 in feed (AFB in initial feed)1concentration-AFB at sample time1concentration)/AFB in initial feed1Concentration X100%.
The test results are shown in table 2:
TABLE 2
The experimental data in table 2 show that when the ratio of the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid in the embodiment is mixed together and fermented to form the zymocyte liquid, the degradation rate of the zymocyte liquid for aflatoxin in the feed is the largest, 24 reaches 93.1%, although the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid have a certain degradation effect on aflatoxin in the feed, 24h is 70.9% and 75.9% respectively, but the degradation rate is low, and the effect of the zymocyte liquid formed by mixing the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid together and fermenting according to the ratio in the embodiment greatly improves the degradation effect on aflatoxin in the feed.
Example 3
(1) Inoculating Bacillus aryabhattai strain with the preservation number of CGMCC14949 to BYP liquid culture medium (the inoculation amount is 1.5 percent), and culturing at 32 ℃ and 200r for 15h to obtain the once activated Bacillus aryabhattai strain liquid. The primary activated seed solution is continuously inoculated into a fermentation culture medium (the inoculation amount is 1.5 percent), and the culture is carried out at 29 ℃ and 200r for 24h to obtain the Bacillus aryabhattai bacterium solution.
Wherein BYP liquid culture medium: 10g of peptone, 5g of glucose, 5g of yeast extract, 5g of sodium chloride and 1000mL of distilled water, and sterilizing the mixture at 121 ℃ for 15min by high-pressure steam. .
Wherein the fermentation medium: 12 parts of corn flour, 20 parts of bean cake powder and CaCO35 parts of glucose, 8 parts of (NH)4)2SO41 part, 6 parts of corn steep liquor, MgSO42.5 parts, MnSO40.5 parts, 1000 parts of distilled water, and high-pressure steam sterilization at 121 ℃ for 15 min.
(2) The Brevibacterium sp strain with the preservation number of CCTCC No. M2017329 is inoculated into a BYP liquid culture medium (the inoculation amount is 1.5 percent), and is cultured for 18 hours at 29 ℃ and 220r to obtain the once-activated Brevibacterium sp strain liquid. The primary activated seed liquid is continuously inoculated into a fermentation medium (the inoculation amount is 1.5 percent), and cultured for 26 hours at 29 ℃ and 200 r. Obtaining the Brevibacterium flavum bacterial liquid.
BYP liquid medium: 10g of peptone, 5g of glucose, 5g of yeast extract, 5g of sodium chloride and 1000mL of distilled water, and sterilizing the mixture at 121 ℃ for 15min by high-pressure steam. .
Fermentation medium: 10 parts of corn flour, 18 parts of bean cake powder and CaCO35 parts of glucose, 8 parts of (NH)4)2SO41.2 parts of corn steep liquor, 6 parts of MgSO42.5 parts of MnSO40.6 parts of distilled water, and high-pressure steam sterilization at 121 ℃ for 15 min.
(3) And mixing a certain amount of the obtained Bacillus aryabhattai bacterial liquid and the obtained Flavobacterium parvum bacterial liquid according to the proportion of the cell number of 11: 15-11: 20 to obtain a composite bacterial liquid.
(4) Inoculating the compound bacterial liquid into a sterilized fermentation medium (adopting a 500L fermentation tank), wherein the inoculation amount is 1.5%, and fermenting to obtain the liquid leaven.
The fermentation conditions were: the charging coefficient is 0.6, the temperature is 33 ℃, the pressure is 0.05MPa, the stirring speed is 0-8 hours and 200r/min, after 8 hours, the stirring speed is 250r/min, the ventilation rate is 15-40L/min, the pH value is natural, and the fermentation period is 27 hours.
The composite bacteria liquid culture medium is 9g/L corn flour, 13g/L glucose, (NH4)2SO40.6 g/L and corn steep liquor 9 g/L.
(5) Respectively carrying out degradation tests on the aflatoxin in the feed by using the prepared liquid of the Bacillus aryabhattai and the prepared liquid of the Flavobacterium parvum and the obtained leavening agent for degrading the aflatoxin in the feed.
The test method comprises the following steps: uniformly spraying equal amount of the fungus solution of each group on the AFB1Contaminated feed (among others AFB)1The content is more than 20 mu g/ml), and the culture solution sprayed without inoculation is taken as a control group. Adding 50% distilled water into the above mixed feed, stirring, culturing at 30 deg.C for 8 hr, 16 hr, and 24 hr, oven drying, pulverizing, and detecting AFB in feed sample with rapid mycotoxin detection equipment1Concentration and calculating the degradation rate. Degradation rate of bacterial liquid to AFB1 in feed (AFB in initial feed)1concentration-AFB at sample time1concentration)/AFB in initial feed1Concentration X100%.
The test results are shown in table 3:
TABLE 3
The experimental data in table 3 show that when the ratio of the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid in the embodiment is mixed together and fermented to form the zymocyte liquid, the degradation rate of the zymocyte liquid for aflatoxin in the feed is the largest, and 24h reaches 92.9%, although the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid have a certain degradation effect on aflatoxin in the feed, the 24h is 72.3% and 76.3% respectively, but the degradation rate is low, and the effect of the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid in the embodiment when the bacillus aryabhattai bacteria liquid and the flavobacterium brevibacterium liquid are mixed together and fermented together according to the ratio in the embodiment is greatly improved.
In conclusion, the composite bacteria liquid is put into a composite bacteria culture medium for mixed fermentation to obtain the fermentation bacteria agent for degrading aflatoxin in the feed, the fermentation bacteria agent can be used for rapidly and effectively degrading aflatoxin in the feed, the degradation rate can reach more than 90% after 16-hour reaction, the degradation rate can reach more than 92% after 24-hour reaction, and the prepared fermentation bacteria agent is safe and effective and can be directly added into the feed as a feed additive.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
Claims (9)
1. A preparation method of a leaven for degrading aflatoxin in feed is characterized by comprising the following steps:
respectively culturing the bacillus aryabhattai and the flavobacterium brevifibrum to obtain a bacillus aryabhattai bacterial solution and a flavobacterium brevifibrum bacterial solution;
mixing the bacillus aspolis bacterial liquid and the flavobacterium brevibacterium bacterial liquid to obtain a composite bacterial liquid;
putting the compound bacterium liquid into a compound bacterium culture medium for mixed fermentation;
the cell ratio of the Bacillus aryabhattai bacterium liquid to the Flavobacterium parvum bacterium liquid in the composite bacterium liquid is 11: 15-11: 20.
2. The method for preparing the starter culture for degrading aflatoxin in feed according to claim 1, wherein the mixed fermentation is carried out by putting the composite bacteria culture medium into a fermentation tank, inoculating the composite bacteria liquid into the composite bacteria culture medium, and culturing at 28-33 ℃ for 27-33 hours; the charging coefficient of the composite bacteria culture medium in the fermentation tank is 0.6; the composite bacteria culture medium comprises 6-9 g/L of corn flour, 10-13 g/L of glucose, (NH4)240.6-0.8 g/L of SO and 7-9 g/L of corn steep liquor.
3. The method for preparing a starter culture for the degradation of aflatoxin in feed according to claim 1, wherein the Bacillus aryabhattai is from strain preservation number CGMCC14949, and the Flavobacterium brevis is from strain preservation number CCTCC No. M2017329.
4. The method according to claim 1, wherein the step of culturing the Bacillus aryabhattai is to inoculate the Bacillus aryabhattai into BYP liquid culture medium for culturing to obtain a first activated seed solution of Bacillus aryabhattai, and then continuously inoculate the activated seed solution of Bacillus aryabhattai into the fermentation culture medium for culturing to obtain a Bacillus aryabhattai bacterial solution; the step of culturing the brevibacterium is to inoculate the brevibacterium into a BYP liquid culture medium for culturing to obtain a first activated seed solution of the brevibacterium, and then continuously inoculate the first activated seed solution of the brevibacterium into a fermentation culture medium for culturing to obtain a brevibacterium liquid.
5. The preparation method of the leaven for degrading aflatoxin in feed according to claim 4, wherein the fermentation medium comprises 10-12 parts of corn flour, 18-20 parts of bean cake flour and CaCO by weight35-6 parts of glucose, 8-10 parts of (NH)4)2SO41-1.2 parts of corn steep liquor, 5-6 parts of MgSO 42-2.5 parts of MnSO40.5-0.6 parts of distilled water.
6. The method for preparing a starter culture for degrading aflatoxin in feed according to claim 4, wherein the Bacillus aryabhattai is cultured by inoculating the Bacillus aryabhattai into BYP liquid culture medium, culturing at 29-32 ℃ and 200-220 r for 15-18h to obtain first activated seed liquid of Bacillus aryabhattai, then continuously inoculating the first activated seed liquid of Bacillus aryabhattai into the fermentation culture medium, and culturing at 29-31 ℃ and 200-220 r for 24-26 h to obtain Bacillus aryabhattai bacterial liquid.
7. The preparation method of the starter for degrading aflatoxin in feed according to claim 4, wherein the short Flavobacterium is cultured by inoculating the short Flavobacterium into a BYP liquid culture medium, culturing at 29-32 ℃ and 200-220 r for 15-18h to obtain a first short Flavobacterium activated seed solution, then continuously inoculating the first short Flavobacterium activated seed solution into a fermentation culture medium, and culturing at 29-31 ℃ and 200-220 r for 24-26 h to obtain a short Flavobacterium liquid.
8. The method for preparing the leaven for degrading aflatoxin in feed according to claim 1, wherein the mass ratio of the composite bacterial liquid inoculated into the composite bacterial culture medium is 1-1.5%.
9. A composite fermentation inoculant for degrading aflatoxin in feed, which is prepared by the preparation method of the fermentation inoculant for degrading aflatoxin in feed according to any one of claims 1 to 8.
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039197A (en) * | 2015-06-03 | 2015-11-11 | 哈尔滨工业大学（威海） | Deep-sea Bacillus aryabhattai capable of inhibiting aflatoxin |
| CN105925513A (en) * | 2016-07-11 | 2016-09-07 | 湖北省农业科学院农产品加工与核农技术研究所 | Composite microbial inoculant for degrading aflatoxin B1 and preparation method thereof |
| CN106615615A (en) * | 2016-12-22 | 2017-05-10 | 简雪梅 | Mixed microbial fermented feed and production method thereof |
| CN108102963A (en) * | 2017-12-28 | 2018-06-01 | 浙江大学 | The Bacillus strain of degrading aflatoxin B 1 and its screening and application |
| CN109321507A (en) * | 2017-11-29 | 2019-02-12 | 中国农业科学院油料作物研究所 | The flavobacterium breve biocontrol bacterial strain of one high-efficiency degradation aflatoxin and its application |
| CN111154679A (en) * | 2020-01-14 | 2020-05-15 | 浙江大学 | Efficient fermentation method of aflatoxin degradation bacteria |
-
2021
- 2021-04-17 CN CN202110415035.1A patent/CN113462589A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105039197A (en) * | 2015-06-03 | 2015-11-11 | 哈尔滨工业大学（威海） | Deep-sea Bacillus aryabhattai capable of inhibiting aflatoxin |
| CN105925513A (en) * | 2016-07-11 | 2016-09-07 | 湖北省农业科学院农产品加工与核农技术研究所 | Composite microbial inoculant for degrading aflatoxin B1 and preparation method thereof |
| CN106615615A (en) * | 2016-12-22 | 2017-05-10 | 简雪梅 | Mixed microbial fermented feed and production method thereof |
| CN109321507A (en) * | 2017-11-29 | 2019-02-12 | 中国农业科学院油料作物研究所 | The flavobacterium breve biocontrol bacterial strain of one high-efficiency degradation aflatoxin and its application |
| CN108102963A (en) * | 2017-12-28 | 2018-06-01 | 浙江大学 | The Bacillus strain of degrading aflatoxin B 1 and its screening and application |
| CN111154679A (en) * | 2020-01-14 | 2020-05-15 | 浙江大学 | Efficient fermentation method of aflatoxin degradation bacteria |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114196572A (en) * | 2021-11-15 | 2022-03-18 | 中国农业科学院油料作物研究所 | Microbial agent with aflatoxin, toxigenic bacteria prevention and control and crop yield increase promotion and application thereof |
| CN114196572B (en) * | 2021-11-15 | 2023-09-26 | 中国农业科学院油料作物研究所 | Microbial agent with aflatoxin and toxigenic bacteria prevention and control and crop yield increase promotion functions and application thereof |
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