CN110777099A - Streptomyces albus for preventing and controlling aspergillus flavus and aflatoxin and application thereof - Google Patents
Streptomyces albus for preventing and controlling aspergillus flavus and aflatoxin and application thereof Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- 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
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
- C12R2001/47—Streptomyces albus
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B9/00—Preservation of edible seeds, e.g. cereals
- A23B9/16—Preserving with chemicals
- A23B9/24—Preserving with chemicals in the form of liquids or solids
- A23B9/26—Organic compounds; Microorganisms; Enzymes
- A23B9/28—Microorganisms; Enzymes; Antibiotics
-
- 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
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The invention relates to a streptomyces albus for preventing and controlling aspergillus flavus and aflatoxin and application thereof, wherein oligotrophic culture conditions only containing agar powder in a culture medium are utilized to separate microorganisms from sea cucumber intestinal tracts; and the streptomyces albus is separated from the healthy sea cucumber intestinal tract for the first time. After fermentation, the streptomyces produces a large amount of active secondary metabolites which can inhibit the growth of pathogenic fungi aspergillus flavus and the synthesis of aflatoxin. Moreover, the streptomyces can completely inhibit the pollution of fungi and mycotoxin in the storage process of the grains such as peanuts and the like, and has wide application prospect in the field of grain safety.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to the field of control of harmful microorganisms, wherein a streptomyces albus HSB5 is obtained by separating a seawater culture medium from a sea cucumber intestinal tract. The streptomycete HSB5 and the extract thereof are used for inhibiting the growth of pathogenic fungi aspergillus flavus and pollution caused by aflatoxin, and the LC-MS technology is used for identifying active ingredients in the crude extract.
Background
The aflatoxin is a secondary metabolite produced by fungi such as aspergillus flavus, aspergillus parasiticus and the like, and is a difuranoxanaphthalenone compound. According to the difference of chemical structures, aflatoxin is mainly divided into subtypes B1, B2, G1, G2, M1 and M26, wherein B1 is the most toxic. Aflatoxin B1 (Aflatoxin, AFB 1) is by far the most recognized natural toxicant with the highest carcinogenic potential in the world and is recognized by the food and agriculture organization and the world health organization of the united nations as a class 1A carcinogen. The long-time and low-dose intake of aflatoxin AFB1 is most likely to induce liver cancer, kidney cancer, lung cancer and the like; and one-time excessive intake of AFB1 can cause severe acute liver poisoning. More seriously, aflatoxin has very stable physicochemical properties, and industrial processing, daily cooking and pasteurization can not effectively remove the aflatoxin, so that the aflatoxin continuously exists in a food chain and threatens the health of the public for a long time.
Aspergillus flavus can infect important economic crops before harvest such as peanut, corn, walnut and the like, and can cause pollution in a plurality of links such as storage, transportation and processing after harvest. On the one hand, infection with Aspergillus flavus can cause a reduction in yield of important commercial crops. On the other hand, a large amount of aflatoxin is generated due to the infection of aspergillus flavus, so that the quality of agricultural products is seriously reduced, and finally, huge economic loss is caused. According to the world food and agricultural organization (FOA), about 25% of crops worldwide are contaminated with fungi and mycotoxins every year, causing economic losses of several billion dollars. Among the most predominant mycotoxins are aflatoxins produced by fungi of the genus aspergillus. The economic losses due to aflatoxin contamination are approximately $ 10 billion per year in the united states alone, and it is estimated that national economic losses are far greater in asia and africa.
Measures for controlling and reducing aspergillus flavus and aflatoxin are always hot spots of research, and the main measures comprise plant genetic breeding, physical and chemical treatment and biological prevention and control. The corn and the peanut have some resistant varieties which resist aspergillus flavus and reduce toxin generation, however, the breeding of the resistant varieties has the defects of long time consumption, insufficient resistance, low yield potential, narrow adaptation range and the like, and the strategy can only play a role in the growth period of the plants and is ineffective in the storage and processing periods of crops. The use of physical or chemical methods not only destroys the quality of agricultural products and introduces secondary pollution, but also may induce drug resistance or enhance the toxic-producing ability of pathogenic bacteria. Therefore, biological control becomes a new direction for effectively controlling and reducing the pollution of aspergillus flavus and mycotoxin in the future. Bacteria with biocontrol effect have attracted wide attention of foreign and domestic scholars in the aspect of prevention and control of aspergillus flavus. Bacteria derived from Bacillus, Burkholderia, Ralstonia, Streptomyces, Pseudomonas and the like easily exhibit inhibitory activity against the growth or toxin production of Aspergillus flavus. The bacillus, the ralstonia solanacearum and the pseudomonas which are separated from the almond can inhibit the growth of aspergillus flavus, and the bacillus which is separated from the rice phyllosphere can inhibit the growth or the toxin production of the aspergillus flavus by secreting lipopeptide compounds. The burkholderia can inhibit the growth of aspergillus flavus by synthesizing terpene compounds. It has been reported that a strain of pseudomonas fluorescens reduces the synthesis of aflatoxin by three ways of inhibiting growth, inhibiting gene expression and biodegradation. The ocean is a huge microbial resource library which needs to be developed, and unique physical and chemical conditions create unique microbial resources. Particularly, the bacillus megaterium for inhibiting aspergillus flavus, which is separated from the water in the east sea of China, can specifically and underground regulate the expression of an aflatoxin gene cluster. The Shewanella alga selected from marine sediments can completely inhibit the growth and toxin synthesis of aspergillus flavus during storage of peanuts and corns, and deep analysis finds that the strain produces sulfur-containing aromatic compounds, and has strong bacteriostatic activity and toxin production inhibiting effect.
The invention patent application about aflatoxin control at home and abroad is searched, and the invention patent application of biocontrol bacteria from the intestinal tracts of marine animals is not found.
Disclosure of Invention
The invention mainly aims to obtain a biocontrol strain which can inhibit the growth of aspergillus flavus and reduce the synthesis of aflatoxin from the intestinal tract of marine animals: the invention adopts the following technical method:
a strain of streptomyces albus, and the strain is named as streptomyces albus (A)
Streptomyces albus) HSB5, which has been deposited at the China general microbiological Culture Collection Center (CGMCC) on 28.10.2019, with the addresses of: xilu No.1 Hospital No. 3, Beijing, Chaoyang, with the deposit number: CGMCC No. 18754.
Further, the streptomyces albus is applied to prevention and control of aspergillus flavus and aflatoxin.
The invention has the secondary purpose of identifying the bacteriostatic components of the biocontrol bacteria:
1. strain screening: the method comprises the steps of purchasing large and healthy sea cucumbers from an aquatic product market, disinfecting the body surfaces of the sea cucumbers by using 75v/v% alcohol, cutting the abdomen of the sea cucumbers by using a scalpel, taking out intestinal contents, immediately placing the sea cucumbers in sterile water, and performing vortex oscillation to obtain uniform suspension. Each plate was coated uniformly in an amount of 100. mu.l per plate on a seawater oligotrophic screening medium (formulation: tryptone 0.1%, yeast extract 0.05%, sodium chloride 0.1%, sea salt 30 g/L), incubated at 28 ℃ for 10 days, and single colonies were selected for bacteriostatic experiments.
2. Streptomyces albus HSB5 bacteriostasis experiment
The bacteriostasis experiment is carried out on a PDA plate (a preparation method of the PDA comprises the steps of cleaning, peeling, weighing 200g of potatoes, cutting into small blocks, boiling for 30 minutes, filtering by eight layers of gauze, heating, adding 15g of agar, continuously heating, stirring and uniformly mixing, adding 20g of cane sugar after the agar is dissolved, uniformly stirring, slightly cooling, supplementing water to 1000 milliliters, subpackaging into a test tube or a conical flask, plugging and binding, sterilizing at 115 ℃ for about 20 minutes, pouring the plate, simultaneously inoculating aspergillus flavus and biocontrol bacteria, evaluating the bacteriostasis activity of different bacteria by measuring a bacteriostasis zone, and finally determining that the streptomyces albus HSB5 obviously inhibits the growth and sporulation of the aspergillus flavus.
3. Experiment for inhibiting aspergillus flavus from producing toxin
MTSB culture medium (formula: tryptone 1.5%, soybean peptone 0.5%, sodium chloride 0.5%, sea salt 3%) is used for fermenting streptomyces albus HSB5, and the fermentation conditions are as follows: the temperature is 28 ℃, the rotating speed is 150 rpm, and the fermentation days are 10 days. After the fermentation is finished, the thalli and the fermentation liquor are centrifugally separated, and equal volume of ethyl acetate is respectively added for extraction for 2 times to obtain a crude substance.
Five groups are set in the toxin production experiment, and aspergillus flavus spores (the final concentration is 10)
5And/ml) in YES liquid medium, adding 0, 1ug/ml, 5ug/ml, 10ug/ml and 20 ug/ml of crude streptomyces albus, standing at 28 deg.C for 6 days, and collecting the culture. Adding dichloromethane with the same volume to extract aflatoxin, and determining the content of aflatoxin in different groups by using a thin layer chromatography.
4. LC-MS/MS identification of biocontrol bacteria active coarse substance
Weighing 1mg of HSB5 crude substance, adding 1ml of methanol for dissolving, filtering the crude substance solution with a 0.22 micron filter membrane to remove impurities, and analyzing with a high performance liquid chromatography-mass spectrometer.
The invention has the advantages that: the invention separates the microorganism from the intestinal tract of the sea cucumber by using the oligotrophic culture condition that the culture medium only contains agar powder; and the streptomyces albus is separated from the healthy sea cucumber intestinal tract for the first time. The streptomyces grows rapidly, tolerates oligotrophic conditions, and produces a large number of spores in the late growth phase. After fermentation, the streptomyces produces a large amount of active secondary metabolites which can inhibit the growth of pathogenic fungi aspergillus flavus and the synthesis of aflatoxin. Moreover, the streptomyces can completely inhibit the pollution of fungi and mycotoxin in the storage process of the grains such as peanuts and the like, and has wide application prospect in the field of grain safety.
Drawings
FIG. 1 shows the comparison of the growth and sporulation inhibiting effect of Streptomyces albus HSB5 on Aspergillus flavus and its clinical medicine amphotericin.
FIG. 2 is a graph showing the growth of Streptomyces on a plate and the effect of Streptomyces HSB5 in inhibiting the growth of Aspergillus flavus.
FIG. 3 shows the experimental results of the crude extract of Streptomyces albus HSB5 for inhibiting the production of Aspergillus flavus.
Detailed Description
Culture medium:
1. seawater oligotrophic screening culture medium: tryptone 0.1wt%, yeast extract 0.05wt%, sodium chloride 0.1wt%, sea salt 30 g/L.
2. PDA culture medium: the potato is cleaned and peeled, 200g of potato is weighed and cut into small pieces, the small pieces are boiled for 30 minutes, eight layers of gauze are used for filtering, heating is carried out, 15g of agar is added, heating and stirring are carried out continuously, 20g of cane sugar is added after the agar is dissolved, stirring is carried out uniformly, the water is replenished to 1000 ml after slight cooling, test tubes or conical bottles are subpackaged, plugs are added, bandaging is carried out, and the flat plates are poured after sterilization for 20 minutes at the temperature of 115 ℃.
3. MTSB medium: tryptone 1.5wt%, soybean peptone 0.5wt%, sodium chloride 0.5wt%, sea salt 3 wt%.
4. YES liquid medium: 2 wt% of yeast extract, 6 wt% of sucrose and 0.1wt% of magnesium sulfate;
example 1 Strain screening
The method comprises the steps of purchasing large and healthy sea cucumbers from an aquatic product market, disinfecting the body surfaces of the sea cucumbers by using 75v/v% alcohol, cutting the abdomen of the sea cucumbers by using a scalpel, taking out intestinal contents, immediately placing the sea cucumbers in 20ml of sterile water, and performing vortex oscillation to obtain uniform suspension. 100ul of each plate is evenly coated on a seawater oligotrophic screening culture medium, the plate is placed at 28 ℃ for static culture for 10 days, and a single colony is picked for bacteriostasis experiment.
Example 2 Streptomyces albus HSB5 bacteriostasis experiment
The bacteriostatic experiment is carried out on a PDA (personal digital assistant) plate, the experiment for resisting pathogenic fungi aspergillus flavus is carried out by utilizing streptomyces albus HSB5 crude substances and clinical medicine amphotericin, the experiment is divided into 4 groups, and the concentration of the streptomyces albus HSB5 and the amphotericin are set to 3 concentration gradients: 1ug/ml, 5ug/ml, 10ug/ml, and the left-most blank. Experimental results show that the streptomyces albus HSB5 and amphotericin both remarkably inhibit the growth and sporulation of aspergillus flavus, the bacteriostatic activity of the streptomyces albus HSB5 and the amphotericin is equivalent, and the MIC of the streptomyces albus and the amphotericin is 1ug/ml (figure 1). FIG. 2 shows the growth pattern of Streptomyces on a plate and the effect of Streptomyces HSB5 on inhibiting the growth of Aspergillus flavus, and it can be seen from FIG. 2 that Streptomyces ocellatus HSB5 can significantly inhibit the growth and sporulation of Aspergillus flavus.
Example 3 experiment for inhibiting Aspergillus flavus from producing toxin
Fermenting streptomyces albus HSB5 by using an MTSB culture medium, wherein the fermentation conditions are as follows: the temperature is 28 ℃, the rotating speed is 150 rpm, and the fermentation days are 10 days. After the fermentation is finished, the thalli and the fermentation liquor are centrifugally separated, and equal volume of ethyl acetate is respectively added for extraction for 2 times to obtain a crude substance.
Five groups are set in the toxin production experiment, and aspergillus flavus spores (the final concentration is 10)
5And/ml) in YES liquid medium, adding 0, 1ug/ml, 5ug/ml, 10ug/ml and 20 ug/ml of crude streptomyces albus, standing at 28 deg.C for 6 days, and collecting the culture. Adding dichloromethane with the same volume to extract aflatoxin, and determining the content of aflatoxin in different groups by using a thin layer chromatography. The experimental result shows that when the concentration of the HSB5 crude product of the streptomyces albus is more than 1ug/ml, the aflatoxin synthesis is inhibited completely, and when the concentration is more than 10ug/ml, the result is shown in figure 3.
Example 4 LC-MS/MS identification of biocontrol bacteria active crude
Weighing 1mg of HSB5 crude substance, adding 1ml of methanol for dissolving, filtering the crude substance solution with a 0.22 micron filter membrane to remove impurities, and analyzing with a high performance liquid chromatography-mass spectrometer. Table 1 shows the compounds of active ingredients in the crude Streptomyces albus HSB5, and the active compounds in HSB5 are identified by comparing the molecular ion front with the Streptomyces avermitilis secondary metabolism database.
TABLE 1 Compounds of active ingredients in Streptomyces albus HSB5 crude material
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (2)
1. A strain of Streptomyces albus, characterized in thatThe strain is named as streptomyces albus: (
Streptomyces albus) HSB5, wherein the strain is preserved in China general microbiological culture collection center in 2019, 10 and 28 months, and the preservation numbers are: CGMCC No. 18754.
2. The use of the Streptomyces albus strain as defined in claim 1 for the prevention and control of Aspergillus flavus and aflatoxin.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2355509A1 (en) * | 1976-06-21 | 1978-01-20 | Gist Brocades Nv | Streptomyces albus strain extract prepn. - by culturing strain, contacting with solvent, filtering, evaporating and sepg. extract into antifungal and antibacterial fractions |
| FR2355453A1 (en) * | 1976-06-21 | 1978-01-20 | Gist Brocades Nv | Use of Streptomyces albus as antibiotic and detoxicant - esp. for controlling aflatoxin on crops |
| US6183736B1 (en) * | 1998-04-07 | 2001-02-06 | Usda/Ars Southern Regional Research Center | Small peptides with antipathogenic activity, treated plants and methods for treating same |
| CN108300677A (en) * | 2018-02-22 | 2018-07-20 | 广东省微生物研究所（广东省微生物分析检测中心） | One plant of streptomyces albus and its application in preparing microbialpreservatives |
-
2019
- 2019-11-29 CN CN201911203900.5A patent/CN110777099B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2355509A1 (en) * | 1976-06-21 | 1978-01-20 | Gist Brocades Nv | Streptomyces albus strain extract prepn. - by culturing strain, contacting with solvent, filtering, evaporating and sepg. extract into antifungal and antibacterial fractions |
| FR2355453A1 (en) * | 1976-06-21 | 1978-01-20 | Gist Brocades Nv | Use of Streptomyces albus as antibiotic and detoxicant - esp. for controlling aflatoxin on crops |
| US6183736B1 (en) * | 1998-04-07 | 2001-02-06 | Usda/Ars Southern Regional Research Center | Small peptides with antipathogenic activity, treated plants and methods for treating same |
| CN108300677A (en) * | 2018-02-22 | 2018-07-20 | 广东省微生物研究所（广东省微生物分析检测中心） | One plant of streptomyces albus and its application in preparing microbialpreservatives |
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