CN113005065A - Microbial preparation for reducing aflatoxin content of corn silage - Google Patents
Microbial preparation for reducing aflatoxin content of corn silage Download PDFInfo
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- CN113005065A CN113005065A CN202110337721.1A CN202110337721A CN113005065A CN 113005065 A CN113005065 A CN 113005065A CN 202110337721 A CN202110337721 A CN 202110337721A CN 113005065 A CN113005065 A CN 113005065A
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- 239000004460 silage Substances 0.000 title claims abstract description 41
- 229930195730 Aflatoxin Natural products 0.000 title claims abstract description 40
- 239000005409 aflatoxin Substances 0.000 title claims abstract description 40
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- 238000002360 preparation method Methods 0.000 title claims abstract description 16
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- 229940002008 bifidobacterium bifidum Drugs 0.000 claims abstract description 32
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- 241000218588 Lactobacillus rhamnosus Species 0.000 claims abstract description 21
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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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
- A23K—FODDER
- A23K30/00—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs
- A23K30/10—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder
- A23K30/15—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging
- A23K30/18—Processes specially adapted for preservation of materials in order to produce animal feeding-stuffs of green fodder using chemicals or microorganisms for ensilaging using microorganisms or enzymes
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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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- C12N1/14—Fungi; Culture media therefor
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/175—Rhamnosus
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/51—Bifidobacterium
- A23V2400/517—Bifidum
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Abstract
The microbial preparation comprises microorganisms selected from lactobacillus plantarum, bifidobacterium bifidum, lactobacillus rhamnosus and botrytis cinerea, not only reduces the aflatoxin content in silage products, but also can inhibit the rising speed of the aflatoxin content in the aerobic exposure process.
Description
Technical Field
The application belongs to the field of microorganisms, and particularly provides a microbial preparation for reducing aflatoxin content of corn silage, which comprises microorganisms selected from lactobacillus plantarum, bifidobacterium bifidum, lactobacillus rhamnosus and botrytis cinerea.
Background
Ensiling refers to a process of converting carbohydrates into organic acids mainly comprising lactic acid by anaerobic fermentation of lactic acid bacteria and the like attached to ensiling raw materials under a sealed condition, lowering the pH, and inhibiting the growth of harmful bacteria, thereby enabling the feed to be stored for a long time and optimizing the nutritional value thereof. Silage is one of the main sources of livestock feed in cold regions since ancient times, the silage mainly comprises corn, alfalfa, barley, oat, sorghum, milk vetch, beet, potato and the like, and the corn is the main silage variety in northern China.
The quality of corn silage is determined by indexes such as sensory quality, pH, organic acid content and the like, and on the other hand, mycotoxin represented by aflatoxin is also an index which cannot be ignored. Although the influence of aflatoxin on ruminants is small, on one hand, rumen microorganisms in the ruminants are sensitive to the aflatoxin, and the digestion and absorption capacity of the ruminants is reduced when a large amount of feed containing the aflatoxin is fed for a long time, and on the other hand, the aflatoxin in meat and milk products is easy to exceed the standard when the feed containing the aflatoxin is fed for a high amount, and the situation that food supervision is increasingly strict is very unfavorable. Therefore, it is necessary to find a green, convenient and low-cost method for controlling aflatoxin.
Disclosure of Invention
The applicant tries to solve the problems in a microbial additive mode, and after a large number of microbial cultures and screening researches, the addition of lactobacillus rhamnosus is found to obviously reduce the aflatoxin content of the silage product and inhibit the rising speed of the aflatoxin content of the silage product after opening; in addition, the applicant also unexpectedly finds that the botrytis cinerea which causes plant gray mold or produces noble rot wine can also reduce the aflatoxin content of the silage product, and the effect of inhibiting the rising speed of the aflatoxin content of the silage product after opening is more obvious.
In one aspect, the present application provides a microbial preparation for reducing aflatoxin content of corn silage comprising a microorganism selected from the group consisting of lactobacillus plantarum, bifidobacterium bifidum, lactobacillus rhamnosus, and botrytis cinerea.
Further, the microorganism contained in the strain is Lactobacillus plantarum, Bifidobacterium bifidum, or Lactobacillus rhamnosus.
Further, the microorganism contained therein is Lactobacillus plantarum, Bifidobacterium bifidum, Botrytis cinerea.
Further, the microbial preparation is bacterial powder.
Further, the content of Lactobacillus plantarum, Bifidobacterium bifidum, and Lactobacillus rhamnosus is 5 × 109cfu/g、1×109cfu/g、4×108cfu/g。
Further, the content of Lactobacillus plantarum, Bifidobacterium bifidum, and Botrytis cinerea is 5 × 109cfu/g、1×109cfu/g、6×107cfu/g。
In another aspect, the present application provides the use of lactobacillus plantarum and bifidobacterium bifidum, and a third bacterium selected from lactobacillus rhamnosus or botrytis cinerea for reducing the aflatoxin content of silage.
Further, the method for reducing the aflatoxin content of the silage is to reduce the aflatoxin level of the silage after opening and sealing.
Further, the silage is silage corn.
Furthermore, the application uses bacterial powder preparation, and the content of Lactobacillus plantarum, Bifidobacterium bifidum and Lactobacillus rhamnosus in the bacterial powder preparation is 5 × 109cfu/g、1×109cfu/g、4×108cfu/g, or the content of Lactobacillus plantarum, Bifidobacterium bifidum, and Botrytis cinerea is 5 × 109cfu/g、1×109cfu/g、6×107cfu/g。
The strains in the application are not limited in source, and the strains which are self-separated, presented and purchased and are identified to meet the requirements can be used in the invention.
The corn variety in the present application is not limited to the variety used in the examples, and various kinds of corn for silage and for both food and feed, which are developed in the present and future, can be used.
The use of other additive components, including but not limited to enzymatic additives and chemical additives, is not excluded from the additive product of the present application.
Examples
Materials, instruments, reagents and methods
Strain: the strains of lactobacillus plantarum, bifidobacterium bifidum, lactobacillus rhamnosus and botrytis cinerea are all preserved and cultured by the enterprises of the applicant; wherein original strains of botrytis cinerea and bifidobacterium bifidum are presented by the grape wine institute of Ningxia university, and strains of lactobacillus plantarum and lactobacillus rhamnosus are separated and identified by the applicant.
The instrument comprises the following steps: a pH meter: CT-6020A portable pH meter (manufactured by Kodak electronics).
Ion chromatography: ICS-2500 ion chromatograph (manufactured by DIONEX).
A fiber analyzer: a2000i fiber Analyzer (manufactured by ANKOM).
A nitrogen determination instrument: KDN-08A Kai type azotometer (Hangzhou Hui Er manufacturing)
Aflatoxin immunoaffinity column RBRP07 (Shanghai Bi-high Biotech Co., Ltd.)
Mycotoxin tester HED-IG-ZD (Shandong Hold electronics technologies, Ltd., detection B1 aflatoxin)
Other reagents and instruments are all in conventional domestic models.
Ensiling corn varieties and basic processing:
the Danyu 217 commonly used in the Nemeng-northeast region is selected, and the corns used in the experimental process are from the same batch of corns in a fixed planting base, and have basically consistent quality (the average plant height is about 250cm, the average fruit head length is about 20cm, and the planting density is 3500 plants/mu). The harvesting period of the corn is 38 th to 41 th day of the silking period, the height of the remained stubble is 30cm, and the cutting length is about 2 cm.
Experimental ensiling method and sampling method:
adding additives into the cut corns, uniformly mixing, pressing the mixture into a 1L glass bottle according to about 500g/L, sealing the joint of the bottle cap by using glass cement, and storing the mixture at the room temperature of 20-25 ℃ in a dark place; at the desired measurement time (90 days in the usual test), 3 parallel silage tanks were taken and silage from 2 to 10cm above was taken and mixed for testing.
Sensory evaluation:
according to the German agriculture Association (DLG) scoring method, the smell, the structure and the color are graded into four grades of excellent, good, medium and low according to the score.
And (3) pH measurement:
weighing and taking 10g of silage, adding the silage into 100mL of deionized water, stirring for 3min, filtering by two layers of gauze, and immediately measuring the pH value of the leachate by using the pH meter after filtering.
Aerobic stability:
after ensiling for 90 days, 100g of ensilage is placed in a 500ml flask, the flask is placed in a closed incubator, a thermometer is inserted into the flask to measure the temperature change, the room temperature is monitored, and the time (h) from the time when the sample contacts the air to the time when the sample temperature is 2 ℃ higher than the room temperature during sampling is the time for aerobic stabilization.
And (3) measuring aflatoxin:
substantially in accordance with the instructions of the apparatus: 10g of the pulverized sample was taken, and 50mL of methanol: water is 80:20 methanol water solution; stirring and filtering; passing through an aflatoxin immunoaffinity column, eluting with methanol, and collecting eluate; adding color developing solution, and detecting with mycotoxin detector.
Suitable amounts of Lactobacillus plantarum and Bifidobacterium bifidum have been determined in previous experiments and patent applications to be 5X 109cfu/g、1×109cfu/g; the experiments in the following examples were carried out in batches using the same raw materials in the same season, only some of which showed results of typical reference, and other various formulations screened were not shown due to lack of representativeness.
Example 1 Effect of Lactobacillus plantarum, Bifidobacterium bifidum, and Lactobacillus rhamnosus combinations on silage corn feed quality
The microbial inoculum formula comprises:
1, microbial inoculum: lactobacillus plantarum 5 x 109cfu/g, Bifidobacterium bifidum 1X 109cfu/g；
And (2) microbial inoculum: lactobacillus plantarum 5 x 109cfu/g, Bifidobacterium bifidum 1X 109cfu/g, Lactobacillus rhamnosus 1 × 109cfu/g；
And (3) microbial inoculum: lactobacillus plantarum 5 x 109cfu/g, Bifidobacterium bifidum 1X 109cfu/g, lactobacillus rhamnosus 4X 108cfu/g。
Sensory evaluation and PH:
the microbial inoculum 1-3 can help the silage corn feed to reach the excellent level, the pH value is above 3.8, and the quality is superior to that of the silage method without the microbial inoculum.
Aerobic stability:
TABLE 1 Effect of inoculants 1-3 on aerobic stability
Compared with the combination of lactobacillus plantarum and bifidobacterium bifidum, the addition of a proper amount of lactobacillus rhamnosus has certain help to improve aerobic stability, and the help to aerobic stability is reduced when the addition amount is too large.
Aflatoxin content and its variation:
TABLE 2 Effect of various inoculants on aflatoxin (AFB 1) content and its variation (. mu.g/kg)
The addition of lactobacillus rhamnosus contributes to the reduction of aflatoxin content in silage products (effective from unsealing to 3 days of aerobic exposure), which may be related to the effect of lactobacillus rhamnosus in inhibiting mould growth and in itself decomposing aflatoxins (according to general knowledge in the art, aflatoxins are hardly detectable in fresh grass, corn, aflatoxins in silage products are essentially derived from silage, growth of aflatoxins in aerobic exposure).
Example 2 Effect of the combination of Lactobacillus plantarum, Bifidobacterium bifidum and Botrytis cinerea on the quality of silage corn feed
And (4) microbial inoculum: lactobacillus plantarum 5 x 109cfu/g, Bifidobacterium bifidum 1X 109cfu/g；
And (5) microbial inoculum: lactobacillus plantarum 5 x 109cfu/g, Bifidobacterium bifidum 1X 109cfu/g; botrytis cinerea 5X 108cfu/g；
And (6) microbial inoculum: lactobacillus plantarum 5 x 109cfu/g, Bifidobacterium bifidum 1X 109cfu/g; botrytis cinerea (6X 10)7cfu/g；
And (7) microbial inoculum: lactobacillus plantarum 5 x 109cfu/g, Bifidobacterium bifidum 1X 109cfu/g; aspergillus 1X 108cfu/g。
Sensory evaluation:
the microbial inoculum of 4-7 can help the silage corn feed to reach the excellent level, the pH value is above 3.8, and the quality is superior to that of the silage method without the microbial inoculum.
Aerobic stability:
TABLE 3 Effect of inoculants 4-7 on aerobic stability
Compared with the combination of lactobacillus plantarum and bifidobacterium bifidum, the addition of a certain amount of botrytis cinerea and aspergillus hardly affects the aerobic stability, and the addition of an excessive amount of botrytis cinerea and aspergillus adversely affects the aerobic stability.
Aflatoxin content and its variation:
TABLE 4 Effect of various inoculants on aflatoxin (AFB 1) content and its variation (. mu.g/kg)
Similar to lactobacillus rhamnosus, the addition of botrytis cinerea also contributes to the reduction of the aflatoxin content in the silage product (the effect is achieved from unsealing to aerobic exposure for 3 days), the inhibition effect is more remarkable, and particularly the aflatoxin content can be kept unchanged within 24 hours (the most common time interval after the silage is unsealed and before the silage is used), so that the control of the aflatoxin content in the milk produced by practically raising livestock, particularly dairy cows, is very favorable. We speculate that the change is caused by the inhibition of a plurality of microorganisms producing aspergillus flavus by the botrytis cinerea and certain bacteria (a mechanism in the fermentation of noble rot wine) which can help the botrytis cinerea to grow (a microbial culture experiment of a sample also proves that the increase of the number of the aspergillus flavus after the opening and the colony number are obviously inhibited, and a specific image is not shown), while the common beneficial mould aspergillus oryzae has no such effect at all.
Claims (10)
1. A microbial preparation for reducing aflatoxin content in corn silage, comprising a microorganism selected from Lactobacillus plantarum, Bifidobacterium bifidum, Lactobacillus rhamnosus, and Botrytis cinerea.
2. The microbial preparation according to claim 1, wherein the microorganism is Lactobacillus plantarum, Bifidobacterium bifidum, Lactobacillus rhamnosus.
3. The microbial preparation according to claim 1, wherein the microorganism is Lactobacillus plantarum, Bifidobacterium bifidum, or Botrytis cinerea.
4. A microbial preparation according to claim 2 or 3 which is a bacterial powder.
5. The microbial preparation according to claim 4, wherein the content of Lactobacillus plantarum, Bifidobacterium bifidum, Lactobacillus rhamnosus is 5X 109cfu/g、1×109cfu/g、4×108cfu/g。
6. The microbial preparation according to claim 4, wherein the content of Lactobacillus plantarum, Bifidobacterium bifidum, Botrytis cinerea is 5X 109cfu/g、1×109cfu/g、6×107cfu/g。
7. The application of lactobacillus plantarum, bifidobacterium bifidum and a third bacterium selected from lactobacillus rhamnosus or botrytis cinerea in reducing the content of aflatoxin in silage.
8. The use according to claim 7 wherein the reduction in the aflatoxin content of the silage is a reduction in the aflatoxin level of the silage after opening.
9. Use according to claim 7 or 8, wherein the silage is silage corn.
10. The use according to claim 7, wherein the bacterial powder preparation is used, and the bacterial powder preparation comprises Lactobacillus plantarum, Bifidobacterium bifidum, and rhamnoseThe lactobacillus content is 5 × 109cfu/g、1×109cfu/g、4×108cfu/g, or the content of Lactobacillus plantarum, Bifidobacterium bifidum, and Botrytis cinerea is 5 × 109cfu/g、1×109cfu/g、6×107cfu/g。
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| US6060429A (en) * | 1994-07-25 | 2000-05-09 | State of Israel--Ministry of Agriculture | Composition and method for controlling plant diseases caused by fungi |
| CN104263573A (en) * | 2014-10-08 | 2015-01-07 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Method for brewing noble rot grape wine |
| CN110591988A (en) * | 2019-11-01 | 2019-12-20 | 四川农业大学 | Lactobacillus rhamnosus753 and application thereof, silage additive and silage |
| CN111996151A (en) * | 2020-09-17 | 2020-11-27 | 李旭业 | Microbial preparation containing multiple lactic acid bacteria and application of microbial preparation in corn silage |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6060429A (en) * | 1994-07-25 | 2000-05-09 | State of Israel--Ministry of Agriculture | Composition and method for controlling plant diseases caused by fungi |
| CN104263573A (en) * | 2014-10-08 | 2015-01-07 | 广西壮族自治区农业科学院葡萄与葡萄酒研究所 | Method for brewing noble rot grape wine |
| CN110591988A (en) * | 2019-11-01 | 2019-12-20 | 四川农业大学 | Lactobacillus rhamnosus753 and application thereof, silage additive and silage |
| CN111996151A (en) * | 2020-09-17 | 2020-11-27 | 李旭业 | Microbial preparation containing multiple lactic acid bacteria and application of microbial preparation in corn silage |
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