CN109170509A - A kind of biological control method producing malicious aspergillus flavus - Google Patents
A kind of biological control method producing malicious aspergillus flavus Download PDFInfo
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- CN109170509A CN109170509A CN201811080492.4A CN201811080492A CN109170509A CN 109170509 A CN109170509 A CN 109170509A CN 201811080492 A CN201811080492 A CN 201811080492A CN 109170509 A CN109170509 A CN 109170509A
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- aspergillus flavus
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/30—Microbial fungi; Substances produced thereby or obtained therefrom
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Abstract
A kind of biological control method producing malicious aspergillus flavus, belongs to field of biological control, can inhibit the growth for producing malicious Aspergillus flavus using aspergillus oryzae and aspergillus flavus not producing aspergillus flavus toxin mutant TSJ-1 bacterial strain and produce poison.Virus mutants co-incubation is not produced and can obviously inhibit the synthesis of aflatoxin by the way that malicious aspergillus flavus and aspergillus oryzae or TSJ-1 will be produced.The nutrient growth of wild type aspergillus flavus and conidial generation, aflatoxin synthetic quantity are also remarkably decreased；Malicious aspergillus flavus further is produced with the processing of the culture solution of aspergillus oryzae and TSJ-1 bacterial strain, can equally inhibit the growth of aspergillus flavus, the synthesis of conidium development and aflatoxin；A kind of biological control method of production poison aspergillus flavus of the invention can reduce the malicious aspergillus flavus of production and infect and synthesize toxin to many agricultural product, have the function that prevention and control in advance and reduce economic loss.
Description
Technical field
The present invention relates to biological controls, and in particular to a kind of biological control method for producing malicious aspergillus flavus.
Background technique
Aspergillus flavus is a kind of aspergillus fungi being widely present, as the common pathogenetic bacterium of plant animal, not only to agricultural
Production, feed industry, farming industry cause great harm, also bring serious threat to human health.What aspergillus flavus generated
Aflatoxin is the native compound with virulent property and strong carcinogenicity, causes heavy economic losses and safety hidden to the mankind
Suffer from.Therefore, the prevention and treatment of research aflatoxin and inhibition aspergillus flavus produce poison and have a very important significance.
Compared with conventional physico chemical control, biological method, which prevents and treats aflatoxin, has treatment conditions relatively mild,
The advantages that quality and nutritive value to product do not generate destruction or relatively small extent of the destruction, be it is a kind of safely, effectively, it is economical
Detoxicating method.Carry out prevention and control to produce malicious aspergillus flavus flora and its toxin synthesis to be in agricultural production currently with atoxigenic aspergillus
The important means of utilization.
Summary of the invention
The purpose of the present invention is to provide a kind of biological control methods for producing malicious aspergillus flavus.
For achieving the above object, technical solution of the present invention is specific as follows:
A kind of biological control method producing malicious aspergillus flavus, the biological control method is one of following method:
Method 1: it is handled with the metabolite of the aspergillus oryzae of predetermined spore concentration and/or aspergillus oryzae easily by Aspergillus flavus infection
And/or by the host of Aspergillus flavus infection；
Method 2: with the metabolite processing of the aspergillus flavus of predetermined spore concentration not toxigenic bacterium and/or aspergillus flavus not toxigenic bacterium
Easily by Aspergillus flavus infection and/or by the host of Aspergillus flavus infection；
Method 3: with the aspergillus oryzae of the co-cultivation of predetermined spore concentration and aspergillus flavus, toxigenic bacterium processing is not invaded by aspergillus flavus easily
Dye and/or by the host of Aspergillus flavus infection；
Method 4: with the aspergillus oryzae of co-cultivation and aspergillus flavus not toxigenic bacterium metabolite handle easily by Aspergillus flavus infection
And/or by the host of Aspergillus flavus infection.
Preferably, the predetermined spore concentration is 106A/mL.
Preferably, the aspergillus oryzae Rib40.
Preferably, the aspergillus flavus not toxigenic bacterium TSJ-1.
Preferably, the host is peanut, (Groundnut products), corn or corn product.
Beneficial effects of the present invention: for the present inventor by extensive research, discovery for the first time is using aspergillus oryzae and does not produce yellow song
The aspergillus flavus mutant TSJ-1 bacterial strain of mould toxin can inhibit to produce the growth of malicious Aspergillus flavus and produce poison.It is malicious yellow bent by that will produce
It is mould not produce virus mutants co-incubation with aspergillus oryzae or TSJ-1 and obviously inhibit the synthesis of aflatoxin.Wild type is yellow bent
Mould nutrient growth and conidial generation, aflatoxin synthetic quantity are also remarkably decreased；Further with aspergillus oryzae and
The culture solution processing of TSJ-1 bacterial strain produces malicious aspergillus flavus, can equally inhibit the growth, conidium development and aspergillus flavus of aspergillus flavus
The synthesis of toxin；A kind of biological control method of production poison aspergillus flavus of the invention can reduce the malicious aspergillus flavus of production to many agricultural product
Infect and synthesize toxin, have the function that prevention and control in advance and reduce economic loss.
Detailed description of the invention
Fig. 1 is the influence of aspergillus oryzae and TSJ-1 mutant to aspergillus flavus strain growthform and dry mycelial weight, wherein (A)
The growing state that aspergillus flavus and aspergillus oryzae and TSJ-1 mutant co-culture；(B) aspergillus flavus and aspergillus oryzae and TSJ-1 mutant are total
The dry mycelial weight of culture；
Fig. 2 is the influence that aspergillus oryzae and TSJ-1 mutant synthesize aflatoxin, wherein (A) thin-layer chromatography detects rice
Aspergillus and TSJ-1 mutant and the production poison amount after aspergillus flavus co-cultivation 3 days；(B) thin-layer chromatography detection aspergillus oryzae and TSJ-1 mutation
Body and the production poison amount after aspergillus flavus co-cultivation 9 days；
Fig. 3 is that TLC detects different spore concentrations than aflatoxin synthesis situation under co-culture system；
Fig. 4 is the synthesis situation of aflatoxin after aspergillus oryzae and the processing of TSJ-1 mutant culture solution；
Fig. 5 is the influence that aspergillus oryzae and TSJ-1 mutant concentrate grow aspergillus flavus, wherein (A) wild type aspergillus flavus
Growth phenotype of the bacterial strain in the PDA culture medium that various concentration aspergillus oryzae and the processing of TSJ-1 mutant concentrate is added；(B) wild
Raw type aspergillus flavus strain is grown 4 days in the PDA culture medium that various concentration aspergillus oryzae and the processing of TSJ-1 mutant concentrate is added
Colony growth diameter afterwards；
Fig. 6 is the influence that aspergillus oryzae and TSJ-1 mutant culture concentrated liquor produce spore to aspergillus flavus.
Specific embodiment:
Following embodiment further illustrates the contents of the present invention, but should not be construed as limiting the invention.Without departing substantially from
In the case where spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, the present invention is belonged to
Range.Unless otherwise specified, the conventional means that technological means used in embodiment is well known to those skilled in the art, such as
The molecular cloning experiment handbooks such as Sambrook, or the condition according to product description suggestion.
Toxigenic bacterium TSJ-1 is not building early period to aspergillus flavus, is the kinase coding that aspergillus flavus CA14 has lacked MAPK approach
The mutant of gene, aflatoxin synthesize path blockade.
Embodiment
1. Fresh spores are collected
2 μ L wild type aspergillus flavus, aspergillus oryzae, TSJ-1 mutant spore liquid are drawn respectively is seeded in PDA solid medium
On, 7d is cultivated in dark at 37 DEG C；Spore is eluted with 0.001% 20 spore eluent of Tween, with four layers of lens wiping paper
Mycelia is filtered, conidium is collected into 10mL centrifuge tube.It is counted after spore is diluted, and spore liquid concentration is unified
It is diluted to 106A/mL.
2. the influence that aspergillus oryzae, TSJ-1 mutant produce poison to aspergillus flavus is observed:
The aspergillus oryzae of fresh collection and TSJ-1 mutant spore concentration are diluted to 10 respectively3A/mL, 105A/mL, 106
A/mL；The each concentration spore liquid and 1mL concentration for taking 1mL to dilute respectively are 106The wild type aspergillus spore liquid of a/mL
Mixing, is added in 50mL YES fluid nutrient medium, and control group only adds 1mL wild type aspergillus spore liquid, and 30 DEG C, 150r/
Min after cultivating 4d, respectively takes the isometric chloroform toxin of 500 μ L culture solutions, draws organic phase, adds 20 μ L chlorine after dry
Imitative weight molten point sample, and carry out thin layer chromatography analysis (TLC).
3. aspergillus oryzae and TSJ-1 mutant culture solution, which influence aspergillus flavus, produces poison observation
Take 106The aspergillus oryzae and TSJ-1 mutant spore of a fresh collection, are added separately to 50mL YES fluid nutrient medium
In, 30 DEG C, 150r/min persistently cultivates 2d；With sterile four layers of gauze mycelia, culture solution is collected, and with 0.2 μm of diameter of filter membrane
(Millipore, Bedford, MA, USA) is filtered sterilizing；30mL filtered fluid is taken to be added to containing 20mL YES culture medium
In conical flask；Take 106The wild type aspergillus spore of a fresh collection is added in above-mentioned culture medium, and 30 DEG C, 150r/min is held
After continuous culture 4d, takes 500 μ L culture solutions to be added after isometric chloroform is stripped toxin, after dry organic phase, add 20 μ L chlorine
Imitative weight is molten, and contact plate detects toxin.
4. aspergillus oryzae and TSJ-1 mutant culture concentrated liquor are prepared and its are detected to aspergillus flavus development impact
(1) 10 are taken7A fresh collection aspergillus oryzae and TSJ-1 mutant spore are added in 500mL PDB culture medium, 30 DEG C,
150r/min collects culture medium clear liquid after cultivating 5d, culture solution is concentrated into 1/10th of original volume with the bag filter of 1KDa,
And it is spare with 0.2 μm of diameter of membrane filtration concentrate.
(2) 4% is separately added into 10mL PDA culture medium, 8% aspergillus oryzae/TSJ-1 mutant culture concentrated liquor；
After cultivating backbone, 2 μ L wild type aspergillus spore liquid are connect in good plate central point, control group is added without concentrate, and every group
Three parallel, and 30 DEG C, dark persistently cultivates 7d, measures colony diameter daily from 2d and takes pictures.After cultivating 7d, diameter is used
The punch of 1cm diametrically makes a call to three holes, is placed in 4mL centrifuge tube, and 2mL sterile water is added, and sufficient vortex concussion keeps spore complete
Full elution；200 μ L spore liquids are drawn, after 5 times of dilution, carry out spore count using blood counting chamber.
(3) 5%, 10% aspergillus oryzae, the concentration of TSJ-1 mutant culture solution is added in 50mL YES fluid nutrient medium respectively
Concentrate is not added in liquid, control group；1mL is added and contains 106A aspergillus spore liquid, cultivates 7d by 30 DEG C, 150r/min, from 3d,
500 μ L culture solutions are drawn every 2d, isometric chloroform is added, after organic phase is evaporated, add 20 μ L chloroforms weight molten, using thin layer
Analysis method is detected.
5. the influence that aspergillus oryzae/TSJ-1 mutant grows wild type aspergillus flavus under co-culture system
The spore suspension that wild type aspergillus flavus is produced virus mutants TSJ-1 bacterial strain with aspergillus oryzae, not respectively mixes in equal volume
Close, in YES fluid nutrient medium after co-incubation 9d, it has been observed that wild type aspergillus flavus individually cultivate the mycelium pellet of generation with
Aspergillus oryzae/TSJ-1 mutant co-cultures the mycelium pellet generated and produces after form has larger difference, aspergillus flavus and aspergillus oryzae to co-culture
Raw bigger mycelium pellet (Figure 1A)；And after being co-cultured with TSJ-1 mutant, aspergillus flavus then generates more tiny mycelium pellet, but
It is the dry mycelial weight no difference of science of statistics (Figure 1B) under three kinds for the treatment of conditions.
6. the influence that aspergillus oryzae/TSJ-1 mutant synthesizes wild type aflatoxin under co-culture system
The spore suspension that wild type aspergillus flavus is produced virus mutants TSJ-1 bacterial strain with aspergillus oryzae, not respectively mixes in equal volume
Conjunction is added in YES fluid nutrient medium, in 30 DEG C of co-incubation 9d.Detect 3d, 9d toxin synthesis situation discovery, with etc. it is biological
Amount aspergillus oryzae/TSJ-1 mutant co-incubation is compared with the control group individually cultivated, and the synthetic quantity of aflatoxin obviously subtracts
It is few；And AFB is nearly no detectable under the conditions of co-culturing with aspergillus oryzae1(Fig. 2).Since aspergillus oryzae and TSJ-1 mutant will not produce
Raw aflatoxin, therefore the co-cultivation of aspergillus oryzae/TSJ-1 mutant and wild type aspergillus flavus can obviously inhibit wild type yellow
Aspergillus synthesizes aflatoxin.
For further study aspergillus oryzae and TSJ-1 mutant under co-culture system inhibit wild type aspergillus flavus produce toxogen because,
Take 106A wild type aspergillus spore, respectively with containing be 103It is a, 105It is a, 106A aspergillus oryzae/TSJ-1 mutant spore
After liquid mixes in equal volume, it is added separately in 50mL YES fluid nutrient medium that (1mL wild type aspergillus spore is only added in control group
Liquid), 30 DEG C, cultivate 4d after detection toxin discovery, with contain 103When the aspergillus oryzae of a spore/TSJ-1 mutant co-cultures, AFB1It closes
There is no significant change compared with control group at amount；And with contain 105When the TSJ-1 mutant of a spore co-cultures, the AFB of aspergillus flavus1Synthesis
Amount significantly reduces；And when aspergillus flavus and containing 106When the aspergillus oryzae of a spore/TSJ-1 mutant co-cultures, wild type aspergillus flavus
It produces poison amount and significantly reduces (Fig. 3).These results suggest that aspergillus oryzae and TSJ-1 mutant are to wild type aspergillus flavus under co-culture system
The inhibiting effect for producing poison is positively correlated with its inoculum concentration.From result above speculate, due under co-culture system competitive growth or
The certain metabolites generated in aspergillus oryzae, TSJ-1 mutant growth course have inhibiting effect to aspergillus flavus Toxin producing C.
7. the influence that the metabolite of aspergillus oryzae and TSJ-1 mutant synthesizes wild type aflatoxin
For the influence that the metabolite of detection aspergillus oryzae and TSJ-1 mutant synthesizes wild type aflatoxin, originally grind
Study carefully fresh collection aspergillus oryzae and TSJ-1 mutant spore, is added separately in 50mL YES fluid nutrient medium, inoculum concentration is
106It is a, 30 DEG C, 150r/min, cultivate 2d；With four layers of filtered through gauze mycelia, culture medium clear liquid is collected, after filtration sterilization, take
30mL filtered fluid is added 10 in the conical flask of the sterile culture medium of YES containing 20mL6A wild type aspergillus spore, 30 DEG C,
150r/min after cultivating 4d, extracts toxin discovery, and the wild type handled respectively with aspergillus oryzae and TSJ-1 mutant culture solution is yellow
Aspergillus is less (Fig. 4) than untreated control group toxin synthetic quantity.Illustrate certain metabolites of aspergillus oryzae and TSJ-1 mutant
Producing poison to wild type aspergillus flavus has inhibiting effect.
8. the influence that aspergillus oryzae/TSJ-1 mutant metabolite develops aspergillus flavus
To primarily determine influence that aspergillus oryzae/TSJ-1 mutant metabolite grows wild type aspergillus flavus, this research
Wild type aspergillus spore is seeded in respectively containing in 4% and 8% aspergillus oryzae/TSJ-1 mutant concentrate PDA culture medium
(concentrate is not added in control group culture medium) is placed in 30 DEG C of incubators and cultivates 7d.As a result, it has been found that containing 4% aspergillus oryzae concentrate
Culture medium can inhibit the growth of wild type aspergillus flavus, and be ok containing 8% aspergillus oryzae and the concentrate of TSJ-1 mutant
Inhibit the growth (Fig. 5 A, 5B) of aspergillus flavus.Containing 4% and 8% TSJ-1 mutant concentrate and the concentration of 8% aspergillus oryzae
In the culture medium of liquid, the subiculum that wild type aspergillus flavus generates is thin compared with control group, and it generates less point compared to control group
Raw spore pigment (Fig. 5 A).The preliminary metabolite for inferring aspergillus oryzae and TSJ-1 mutant to the growth of wild type aspergillus flavus and
Conidium pigment synthesis has certain inhibiting effect.
Conidium is the important vegetative manner of aspergillus flavus and the important communication form of aspergillus flavus.Pass through observation
It was found that aspergillus oryzae/TSJ-1 mutant concentrate is able to suppress the formation of aspergillus flavus conidium pigment, further detection is yellow bent
Mould sporogenesis situation discovery is containing 4% and 8% TSJ-1 mutant concentrate in addition to 4% aspergillus oryzae concentrate is added
And 8% aspergillus oryzae concentrate culture medium in, wild type aspergillus flavus generate conidium amount it is significant compared to control group
It reduces (Fig. 6), illustrates that the metabolite of aspergillus oryzae and TSJ-1 mutant is able to suppress the formation of wild type aspergillus spore.
Claims (5)
1. a kind of biological control method for producing malicious aspergillus flavus, which is characterized in that the biological control method is one of following method:
Method 1: with the metabolite of the aspergillus oryzae of predetermined spore concentration and/or aspergillus oryzae handle easily by Aspergillus flavus infection and/
Or by the host of Aspergillus flavus infection；
Method 2: with the metabolite processing easily quilt of the aspergillus flavus of predetermined spore concentration not toxigenic bacterium and/or aspergillus flavus not toxigenic bacterium
Aspergillus flavus infection and/or by the host of Aspergillus flavus infection；
Method 3: with the aspergillus oryzae of the co-cultivation of predetermined spore concentration and aspergillus flavus, toxigenic bacterium is not handled easily by Aspergillus flavus infection
And/or by the host of Aspergillus flavus infection；
Method 4: with the aspergillus oryzae of co-cultivation and aspergillus flavus not toxigenic bacterium metabolite processing easily by Aspergillus flavus infection and/or
By the host of Aspergillus flavus infection.
2. a kind of biological control method for producing malicious aspergillus flavus according to claim 1, which is characterized in that the predetermined spore
Concentration is 106A/mL.
3. a kind of biological control method for producing malicious aspergillus flavus according to claim 1, which is characterized in that the aspergillus oryzae
Rib40。
4. a kind of biological control method for producing malicious aspergillus flavus according to claim 1, which is characterized in that the aspergillus flavus is not
Toxigenic bacterium TSJ-1.
5. a kind of biological control method for producing malicious aspergillus flavus according to claim 1, which is characterized in that the host is flower
Life, (Groundnut products), corn or corn product.
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| CN201811080492.4A CN109170509A (en) | 2018-09-17 | 2018-09-17 | A kind of biological control method producing malicious aspergillus flavus |
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| CN201811080492.4A CN109170509A (en) | 2018-09-17 | 2018-09-17 | A kind of biological control method producing malicious aspergillus flavus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334460A (en) * | 2020-03-30 | 2020-06-26 | 江苏师范大学 | Method for inhibiting temperature tolerance of aspergillus flavus |
| CN113354470A (en) * | 2021-06-03 | 2021-09-07 | 吉林大学 | Composite microbial slow-release bacterial fertilizer and preparation method thereof |
-
2018
- 2018-09-17 CN CN201811080492.4A patent/CN109170509A/en not_active Withdrawn
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111334460A (en) * | 2020-03-30 | 2020-06-26 | 江苏师范大学 | Method for inhibiting temperature tolerance of aspergillus flavus |
| CN113354470A (en) * | 2021-06-03 | 2021-09-07 | 吉林大学 | Composite microbial slow-release bacterial fertilizer and preparation method thereof |
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Application publication date: 20190111 |