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Method for screening strain producing antifungal proteins and application

A technology for producing antifungal protein and strains, which is applied in the field of fermenting antifungal protein, to achieve the effects of reducing the addition of chemical reagent lime water, inhibiting pollution, and safe and environmentally friendly malting pollution

Inactive Publication Date: 2011-06-08
CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Currently, during the malting process, malting barley is easily contaminated with fungi such as Fusarium, Aspergillus fumigatus and Rhizopus due to the humid environment
These contaminated malts not only affect the brewing performance but also affect food safety. At present, lime water is generally used in the malting process to inhibit bacterial contamination, but this will also bring hidden dangers to food safety.
There are few reports on the use of antifungal proteins produced by microorganisms to inhibit contamination, especially about Aspergillus giganteus.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1 produces the screening of antifungal protein bacterial strain

[0016] 1. Add 10ml of sterile saline to 1.5g soil sample, and bathe in water for 30min at 60°C

[0017] 2. Remove the sterile water, add 65% ethanol for 10 minutes, and add 0.1% peptone solution for 10 minutes.

[0018] 3. Inoculate 0.1ml of the solution into sarcosine-sucrose medium, and culture for 7 days at 25°C.

[0019] 4. Select single colonies with different morphological characteristics and transfer to MEA (sucrose 25g / L, sodium nitrate 1.5g / L, dipotassium hydrogen phosphate 0.6g / L, potassium chloride 0.4g / L, magnesium sulfate 0.3g / L , ferrous sulfate 0.08g / L, agar 20g / L) on the slant solid medium.

[0020] 5. Collect and preserve the spores of a single colony with 10% glycerol.

[0021] 6. The isolated single colony was cultured for 4 days under the condition of MEA medium pH4.525°C.

[0022] 7. The puncher digs out the bacterial lawn with a diameter of 25mm, and moves it to a ster...

Embodiment 2A

[0025] Embodiment 2 AFP fermentation production

[0026] Inoculate AF-11 on the slant solid medium, culture at 28°C for 4 days, wash the slant with 10ml of bacteria-free water, and make spore suspension for seed cultivation. According to 10% inoculation amount, the spore suspension was inoculated into the seed culture solution (glucose 13g / L, peptone 3g / L, yeast extract 3g / L, NaCl 5g / L), and cultivated for 4 days at 28°C and 220rpm. According to the inoculum amount of 10%, the seed solution was inoculated into 2L fermentation culture, and cultivated at 30°C and 220rpm for 4 days. AFP was separated and purified by cation exchange chromatography, and the AFP obtained by coomassie brilliant blue colorimetric method was 10.2 mg.

Embodiment 3

[0027] Embodiment 3AFP malting microbial control

[0028] Weigh 1000g barley and soak it in 4000ml sterile water for 4 hours. The soaked barley is germinated at 15°C for 3-4 days. AFP was diluted with water to a concentration of 16 μg / g barley and added to the barley. After 4 days, the pollution of barley germination was detected, and the pollution situation was obviously improved. Compared with the control sample, the total number of polluted molds was reduced by 90%.

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PUM

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Abstract

The invention discloses a strain producing antifungal proteins, a method for screening the strain producing the antifungal proteins and a method for producing the antifungal proteins by fermenting the strain. The strain screened with the method is called as huge aspergillus Aspergillus giganteus AF-11 with the preservation number of CGMCC NO. 4342, and the strain has higher yield of the antifungal proteins. The antifungal proteins produced from the screened strain with the method disclosed by the invention can effectively inhibit the mildew during the malting barley production, in particular the fusarium, so as to improve the quality of malts; compared with the traditional processing method, the method disclosed by the invention is safer and more effective. In addition, the invention has a favorable application prospect.

Description

technical field [0001] The present invention relates to a strain producing antifungal protein, named Aspergillus giganteus AF-11, which was preserved in the General Microbiology Center of China Microbiological Culture Collection Management Committee on November 15, 2010. The address is: Beichen West, Chaoyang District, Beijing No. 3, Courtyard No. 1, the preservation number is CGMCC NO.4342. At the same time, it relates to a mutagenesis screening method for obtaining the strain and a method for using the strain to ferment antifungal protein and its application in malting barley, and belongs to the technical field of microbial malting. Background technique [0002] AFP (antifungal protein) is a small molecular weight basic protein secreted by Aspergillus giganteus, which can effectively inhibit the growth of filamentous fungi, especially animal and plant pathogenic fungi. Studies in recent years have found that AFP has the characteristics of safety, high efficiency, broad s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12P21/02C12C1/16
Inventor 宋绪磊陈洪元李建飞宋涛孙志伟江伟唐坤甜
Owner CHINA NAT RES INST OF FOOD & FERMENTATION IND CO LTD