CN104328161A

CN104328161A - Method of intuitively screening microorganism strains for inhibiting aflatoxin

Info

Publication number: CN104328161A
Application number: CN201410520947.5A
Authority: CN
Inventors: 闫培生; 王凯; 王琢; 史翠娟
Original assignee: Harbin Institute of Technology Weihai
Current assignee: Harbin Institute of Technology; Harbin Institute of Technology Weihai
Priority date: 2014-09-29
Filing date: 2014-09-29
Publication date: 2015-02-04

Abstract

The invention relates to a method of intuitively screening microorganism strains for inhibiting aflatoxin. The method includes following steps: (1) preparing a solid culture medium plate which are suitable for growth of a marine microorganisms, a land microorganisms, an endophyte and the like microorganisms; (2) inoculating four different microorganism strains on a periphery of the plate in a spot-inoculating manner; and (3) performing cultivation in a constant temperature incubation box at 25-35 DEG C for 0-2 days; (4) inoculating an aspergillus parasiticus DM strain on the center of the plate with cultivation continuously for 3-4 days and observing mycelial growth of the aspergillus parasiticus DM strain and generation of orange norsolorinic acid through eyes. A microorganism strain, which can inhibit the generation of the orange norsolorinic acid in the mycelia of the DM strain and/or the growth of the mycelia, is the microorganism strain capable of inhibiting generation of the aflatoxin. The method of intuitively screening the microorganism strains for high-efficiently inhibiting the aflatoxin is intuitive, quick, safe and low-cost.

Description

A kind of screening directly perceived suppresses the method for the microbial strains of aflatoxin

Technical field

The present invention relates to the technical fields such as using microbe, biological control and mycotoxins, a kind of screening directly perceived specifically suppresses the method for the microbial strains of aflatoxin.

Background technology

Aflatoxin is by the mycetogenetic poisonous secondary metabolite such as flavus, Aspergillus parasiticus, it is the mycotoxins that toxicity that occurring in nature has found is the strongest, its acute toxicity is more than 10 times of hypertoxic pharmaceutical chemicals potassium cyanide, and, also there is strong carinogenicity, inhibitive ability of immunity and cause abnormalities, is classified as a class carcinogenic substance by international cancer mechanism.Aflatoxin extensively can pollute cereal crop, oil crops, legume crop with duration of storage before crop harvesting, and processed goods, very big to human and animal's Health hazard, and it is serious to cause rural economy to be lost.Therefore, how effectively to prevent aflatoxin contamination agricultural-food from being all key subjects urgently to be resolved hurrily, the method of control aflatoxin contamination food in the past is mainly removed by physics and chemistry method the toxin that polluted in food or is made it lose toxicity, not only cost is large, and effect is also undesirable, chemical pesticide control Aspergillus flavus is utilized to infect crop and food was also once utilized, but in recent years, the problem of environmental pollution caused due to chemical pesticide is more and more paid attention to by various countries scientist and government, environment amenable biological control is utilized to become study hotspot.

The key of biological control aflatoxin contamination filters out aflatoxin producing strains efficiently to be suppressed to grow and produce the Biocontrol microorganism bacterial classification of poison, due to the fungus metabolite that aflatoxin is extremely poison, after generation, naked eyes can not directly observe again, the production of toxin just must can be detected by pertinent instruments and analysis means, not only formality is loaded down with trivial details, and easily cause environmental pollution, the health of harm operator, therefore, set up a kind of directly perceived, fast, safety, low cost screening can efficiently suppress the method for the microbial strains of aflatoxin extremely important.

Summary of the invention

Technical problem to be solved by this invention overcomes above-mentioned the deficiencies in the prior art, provide a kind of be suitable for directly perceived from the microorganism in many different niches such as marine microorganism, land microorganism, endophyte of plant source, fast, safety, low cost filter out the method that efficiently can suppress the microbial strains of aflatoxin.

The technical scheme that the present invention solves the problems of the technologies described above employing is: a kind of screening directly perceived suppresses the method for the microbial strains of aflatoxin ,its feature comprises the steps:

(1) the aflatoxin indicator strain used is Aspergillus parasiticus DM bacterial strain;

(2) screening culture medium used is for providing the various solid mediums of all kinds of microorganism growth such as marine microorganism, land microorganism, endophyte of plant;

(3) according to the difference of microbe species to be screened, the solid medium flat board that preparation is suitable;

(4) the microbial strains that dull and stereotyped surrounding point inoculation 4 is different, cultivate 0-2 days in 25-35 degree constant incubator after, then at dull and stereotyped central authorities inoculation DM bacterial strain, continue to cultivate after 3-4 days, observe cultivation results;

(5) eyes directly observe the generation that the acid of loose dish clothing falls in the mycelial growth of DM bacterial strain in flat board and orange, everyly orange in DM bacterial strain mycelia can be suppressed to fall the generation of loose dish clothing acid and/or the microbial strains of mycelial growth, be namely the microbial strains that aflatoxin can be suppressed to produce.

The present invention is significantly different from other screening methods is that first is use Aspergillus parasiticus DM bacterial strain to be indicator strain, the ability of loose dish clothing acid falls in the DM bacterial strain intermediate product had in the aflatoxin biosynthetic pathway of accumulation eye visible orange in mycelium, therefore, directly can be observed by eyes and whether show orange in the mycelium of Aspergillus parasiticus DM bacterial strain and judge whether that the generation of loose dish clothing acid falls in intermediate product, and then judge whether the generation of aflatoxin further, every microbial strains that orange can be suppressed to fall loose dish clothing acid generation, namely the ability suppressing aflatoxin to produce is possessed, therefore, result is observed directly perceived, simply, convenient, fast, second is that DM bacterial strain does not produce aflatoxin, therefore, can not bring the harm of aflatoxin to environment and operator, therefore, and this invention operational safety, 3rd is the eye observation result only needing people, do not need complicated plant and instrument to detect the inhibition that can judge aflatoxin, and incubation time only has 3-6 days, has feature that is quick, low cost, can screen a large amount of sample.

Embodiment

Below in conjunction with embodiment, the present invention will be further described, but therefore do not limit the present invention among described scope of embodiments.

Embodiment 1: the marine microorganism bacteria selection suppressing aflatoxin

Solid medium flat board (yeast extract 3 g is prepared by the formula in bracket, malt extract 3 g, peptone 5 g, glucose 10 g, agar 15g, natural sea-water 1000mL), four marine microorganism bacterial classifications are inoculated respectively in dull and stereotyped surrounding, 25 degree of constant temperature culture after 2 days, at dull and stereotyped central authorities inoculation Aspergillus parasiticus DM bacterial strain, continue cultivation after 4 days, observe cultivation results, the sour bacterial classification produced in mycelia of loose dish clothing falls in every orange that can suppress, and is namely the marine microorganism bacterial classification that aflatoxin can be suppressed to synthesize, as shown in Figure 1.

Fig. 1 is that plate screening can suppress orange to fall the figure of the marine microorganism bacterial classification that the acid of loose dish clothing produces.In Fig. 1, the bacterial strain of forward side is have the marine microorganism bacterial classification suppressing orange to fall loose dish clothing acid generation, also can suppress the marine microorganism bacterial classification that aflatoxin produces.

Embodiment 2: the screening suppressing the land microbial strains of aflatoxin

Solid medium flat board (yeast powder 5g, casein 2 g, Zulkovsky starch 10 g is prepared by the formula in bracket, sucrose 10g, agar 20g, tap water 1000mL), after dull and stereotyped surrounding inoculates four land microbial strainss respectively, at dull and stereotyped central authorities inoculation Aspergillus parasiticus DM bacterial strain, at 32 degree after constant temperature culture 3-5 days, observe cultivation results, every bacterial classification that orange can be suppressed to fall the acid of loose dish clothing produce in mycelia, namely be the land microbial strains that aflatoxin can be suppressed to synthesize, as shown in Figure 2.

Fig. 2 is that plate screening can suppress orange to fall the figure of the land microbial strains that the acid of loose dish clothing produces.In Fig. 2, left hand view is contrast, and display produces a large amount of orange and falls dish clothing acid of faling apart, and right part of flg display can suppress orange to fall loose 4 land microbial strainss of coiling clothing acid and producing, and also can suppress 4 land microbial strainss that aflatoxin produces.

Embodiment 3: the endophyte of plant bacteria selection suppressing aflatoxin

Solid medium flat board (yeast powder 5g is prepared by the formula in bracket, glucose 20g, agar 18g, peanut seed extracting solution 1000mL), after dull and stereotyped surrounding inoculates four endophyte of plant bacterial classifications respectively, at dull and stereotyped central authorities inoculation Aspergillus parasiticus DM bacterial strain, at 30 degree after constant temperature culture 3-5 days, observe cultivation results, every bacterial classification that orange can be suppressed to fall the acid of loose dish clothing produce in mycelia, namely be the endophyte of plant bacterial classification that aflatoxin can be suppressed to synthesize, as shown in Figure 3.

Fig. 3 is that plate screening can suppress orange to fall the figure of the endophyte of plant bacterial classification that the acid of loose dish clothing produces.In Fig. 3, left hand view is contrast, and display produces a large amount of orange and falls dish clothing acid of faling apart, and right part of flg display can suppress orange to fall loose 4 the endophyte of plant bacterial classifications coiling clothing acid and produce, and also can suppress 4 endophyte of plant bacterial classifications that aflatoxin produces.

Claims

1. one kind is intuitively screened the method for the microbial strains suppressing aflatoxin ,its feature comprises the steps: