ENTEROBACTER CLOACAE BIOCONTROL STRAIN CAPABLE OF EFFECTIVELY INHIBITING ASPERGILLUS FLAVUS FROM SYNTHESIZING AFLATOXINS AND APPLICATION THEREOF

Abstract

The present invention belongs to the field of microorganisms, and particularly relates to an Enterobacter cloacae biocontrol strain capable of effectively inhibiting Aspergillus flavus from producing aflatoxins and an application thereof. Enterobacter cloacae biocontrol strain 3J1EC was deposited in China Center for Type Culture Collection on Jun. 13, 2017, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC No. M 2017330. The strain can be used for inhibiting Aspergillus flavus from producing aflatoxins to prevent and control the contamination of food crops by aflatoxins.

Claims

1. An inhibitor for inhibiting production of aflatoxins by Aspergillus flavus, comprising a fermentation product of an Enterobacter cloacae biocontrol strain 3J1EC, and the Enterobacter cloacae biocontrol strain 3J1EC is deposited in China Center for Type Culture Collection (CCTCC) on Jun. 13, 2017, with an accession number being CCTCC No. M 2017330.

2. The inhibitor according to claim 1, wherein the inhibitor is a liquid, a spray powder, a dry wettable powder, or dry wettable granules.

3. The inhibitor according to claim 1, wherein the inhibitor is a liquid, and a final concentration of Enterobacter cloacae in the inhibitor is (1-9)10.sup.7 CFU/mL.

4. The inhibitor according to claim 3, wherein the inhibitor is a fermentation broth of Enterobacter cloacae 3J1EC, a final concentration of Enterobacter cloacae in the fermentation broth of Enterobacter cloacae 3J1EC is (1-9)10.sup.7 CFU/mL, and a method for preparing the fermentation broth of Enterobacter cloacae 3J1EC includes: activating Enterobacter cloacae 3J1EC on an LB plate, culturing the Enterobacter cloacae in an incubator at 25 C.-37 C. for 24 h, picking up a single colony of Enterobacter cloacae with a needle, transferring the single colony to a liquid medium, culturing with shaking for 12 h-24 h, pipetting and transferring 1%-3% of culture liquid to a fresh liquid medium, and culturing with shaking for 12 h-24 h to obtain an antagonistic fermentation broth of strain Enterobacter cloacae 3J1EC.

5. A method for inhibiting Aspergillus flavus from producing aflatoxins, comprising: coating a surface of a biological sample with an inhibitor of claim 1 or mixing an inhibitor of claim 1 with a biological sample to inhibit Aspergillus flavus from producing aflatoxins.

6. A method for preparing the fermentation broth of Enterobacter cloacae 3J1EC comprising: activating Enterobacter cloacae 3J1EC on an LB plate, culturing the Enterobacter cloacae in an incubator at 25 C.37 C. for 24 h, picking up a single colony of Enterobacter cloacae with a needle, transferring the single colony to a liquid medium, culturing with shaking for 12 h-24 h, pipetting and transferring 1%-3% of culture liquid to a fresh liquid medium, and culturing with shaking for 12 h-24 h to obtain an antagonistic fermentation broth of strain Enterobacter cloacae 3J1EC, wherein the Enterobacter cloacae biocontrol strain 3J1EC is deposited in China Center for Type Culture Collection (CCTCC) with an accession number being CCTCC No. M 2017330

Description

DETAILED DESCRIPTION

[0016] In the invention, a soil collected in a peanut field in Huangpi of Hubei was diluted with a gradient, and then 100 L of the dilution was taken and spread over a plate of an LB solid culture medium for culture, grown bacteria were picked up with an inoculating loop and streaked onto a fresh LB solid medium plate, a single colony was picked up after performing such plate streaking for several times and co-cultured in a culture medium with Aspergillus flavus, and was then tested for the toxin productivity, peanuts were ex vivo inoculated with Aspergillus flavus for a co-culture antimicrobial test, and a strain of 3J1EC, which has a significant effect on the toxin production of Aspergillus flavus, was screened out and deposited in China Center for Type Culture Collection (CCTCC), with an accession number being CCTCC No. M 2017330.

Example 1

[0017] 1) Enterobacter cloacae 3J1EC was activated on an LB plate, and cultured in an incubator at 37 C. for 24 hours, and a single colony of activated Enterobacter cloacae was picked up with a needle, and the single colony was transferred to a conical flask containing 15 mL of LB liquid medium, and cultured at 28 C. with shaking at 200 r.Math.min-1 for 12 hours. 1% of the culture liquid was pipetted and transferred to a conical flask containing 15 mL of LB liquid culture medium, and cultured at 28 C. with shaking at 200 r.Math.min-1 for 12 hours to obtain a fermentation broth of the antagonistic strain.

[0018] 2) The fermentation broth of Enterobacter cloacae (with a final concentration of 110.sup.7 CFU/mL) was co-cultured in a Sabouraud's liquid culture medium together with a suspension of vigorously grown Aspergillus flavus (with a final spore concentration of 5.010.sup.5 spores.Math.mL.sup.1), which was cultured for 7 days. The co-culture was carried out at 28 C. and 200 rpm for 5 days. 3 replicates were set for each process.

[0019] 3) The content of aflatoxin B1 in the culture liquid was measured (Table 2).

TABLE-US-00002 TABLE 2 Prevention and control effect of the biocontrol bacteria against Aspergillus flavus A. flavus + CCTCC M Treatment A. flavus 2017330 AFB1 toxin content (ng/ml) 215 36.67 11.2 1.79

[0020] It can be seen from the above experimental results that the inhibition rate of Enterobacter cloacae CCTCC No. M 2017330 against the aflatoxin is about 94.8%, indicating that it has the ability to inhibit the production of aflatoxin.

Example 2

[0021] 1) Peanuts kernels of Zhonghua No. 6 were taken from a peanut field in Hubei and ground into powder, 1 g of the peanut powder was weighed into a culture dish, and 1 ml of an Aspergillus flavus spore solution (510.sup.5 spores/mL) and 1 ml of an Enterobacter cloacae 3J1EC (CCTCC M 2017330) solution (110.sup.7 CFU/mL) were simultaneously added thereto; in addition, a control was provided by replacing the CCTCC M 2017330 solution with a Sabourand culture medium;

[0022] 2) the inoculated peanut powder was cultured in an incubator at 28 C. for 9 days, 15 mL of 70% aqueous methanol was added thereto, and the mixture was vortexed and then placed on a shaker for 30 min. 3 mL of a supernatant was taken, 8 mL of ultrapure water was added thereto, and vortex centrifugation was carried out; and

[0023] 3) 8 mL of a supernatant was taken and tested by using an immunoaffinity column-HPLC method for the content of aflatoxin B1 (Table 3), with three replicates being set in the test.

TABLE-US-00003 TABLE 3 Prevention and control effect of the biocontrol bacteria against Aspergillus flavus A. flavus + Treatment A. flavus CCTCC M 2017330 AFB1 toxin content (ng/ml) 516.61 51.91 78.27 2.98

[0024] It can be seen from the above experimental results that the inhibition rate of the strain of CCTCC M 2017330 on Aspergillus flavus from producing toxins on the peanuts of Zhonghua No. 6 is about 85%, indicating that the strain has a good prevention and control effect against aflatoxins during the storage of peanuts.

Example 3

[0025] 1) 10 peanut kernels of Luhua No. 8 were taken from a peanut field in Anhui, the surface of the peanuts was coated with a fermentation broth of Enterobacter cloacae 3J1EC, while 1 ml of Aspergillus flavus spore solution (510.sup.5 spores/mL) was added thereto; in addition, a control was provided by replacing the fermentation broth of CCTCC M 2017330 with a Sabourand culture medium;

[0026] 2) the inoculated peanut kernels were cultured in an incubator at 28 C. for 9 days. Thereafter, the peanut kernels were ground into a peanut powder, and added with 15 mL of 70% aqueous methanol to obtain a mixture. The mixture was vortexed and then placed on a shaker for 30 min.

[0027] 3 mL of a supernatant was taken, 8 mL of ultrapure water was added thereto, and vortex centrifugation was carried out; and

[0028] 3) 8 mL of a supernatant was taken and tested by using an immunoaffinity column-HPLC method for the content of aflatoxin B1 (Table 4), with three replicates being set in the test.

TABLE-US-00004 TABLE 4 Prevention and control effect of the biocontrol bacteria against Aspergillus flavus in peanuts Treatment A. flavus A. flavus + CCTCC M 2017330 Toxin content (ng/ml) 449.95 42.51 58.23 2.23

[0029] It can be seen from the above experimental results that the inhibition rate of the strain of CCTCC M 2017330 on Aspergillus flavus from producing toxins on the peanuts of Luhua No. 8 is about 87%, indicating that the strain also has a good prevention and control effect for peanuts of different varieties.