Isolation, Identification and Liquid Culture of Antagonistic Actinomycetes to Penicillium Expansum
Patulin is a mycotoxin having ability in carcinogenesis, teratogenesis, mutagenesis and immunosupression. Penicillium expansum was proved to be the most important patulin-producing fungus in apple and apple products. Microorganisms that have ability in inhibiting Penicillium expansum have great potential in biocontrol of. P. expansium. The study aimed at searching antagonistic actinomycetes and evaluating their potential in biocontrol of P. expansium. Stability and conditions for production of the active components were also studied to get further information on practical application. Main contents and results of the study were listed as followings:(1)154 strains of actinomycetes were isolated from soil of apple orchard, kiwifruit orchard and vegetable orchard in Yangling Shaanxi province. Four strains of antagonistic actinomycetes named P50,C11,C16,C60 respectively were selected by prescreening for further screening, because they had high ability in inhibiting the growth of three strains of patulin-producing pecillium on the agar medium. Strain C16 was finally sreened out from four strains for further study on production and properties of active components because it showed the greatest inhibition ability to three patulin-producing P. expansium, and its average inhibitory diameter is 2.33cm in agar plate tests.(2)Actinomycetes C16 was classified and identified on the basis of morphologic and physi-biochemical characteristics and the analysis of 16 SrDNA sequence. It was identified as as a new species of the Genus of Micropolyspora, the Family of Micromonosporaceae, and the Order of Actinomycetale. It was named as Micropolyspora.C16 and patented as the code of CCTCC M207210.(3)The compositiona of media for cultivation seed of Actinomycetes CCTCC M207210 was optimized at 20.0 maltose g/L, 2.0 yeasty protein g/L, 1.0 K2HPO4 g/L, 1.0 MgSO4 g/L, and 1.0 NaCl g/L. Nutrients of the media for fermentation were optimized by using the central composite design. Results showed the highest value of cell growth was obtained at 2.70 maltose g/100mL,0.26 yeast protein g/100mL,0.17 K2HPO4 g/100mL,0.14 MgSO4 g/100mL,and 0.15 NaCL g/100mL. (4)Conditions for liquid cultivation of Actinomycetes CCTCC M207210 were optimized by using the Box-Behnken design. Response Surface Designs was used to analyze the data. The optimal conditions were obtained at inoculum’s amount of 13.5%(v/v), initial pH of 7.4, rotation speed of 180rpm, temperature of 27.6℃, and medium volume of 125mL in 250mL shake flask. Under the optimal conditions, the highest value of dry cell weight (DCW) was predicted as 6.27 g/L.( 5 ) In order to investigate the stability of active components for inhibiting patulin-producing Penicillium, the cell-free liquid culture of Actinomycetes CCTCC M207210 was tested on its ability in inhibiting the growth of P. expansium and patulin production in agar media after being treated for a period at different temperature, pH, and ultraviolet radiation.The cell-free liquid culture was also used in apple fruits and apple juice inoculated with P. expansium to test its potential in biological controller. The liquid culture was very stable at 28oC and pH 5.7 (the nature pH) but not stable at higher temperatures and when pH was too acidic or too alkaline. The inhibiting ability of the liquid culture declined by 39.5% when it was kept at 100oC for 30min and was completely destroyed when the treatment was prolonged to 60min. Comparatively, alkaline condition (pH8 to 10) showed higher inhibiting ability than acidic condition (pH2 to 4). The liquid culture lost the inhibiting ability when it was exposed to ultraviolet radiation for 90min. The liquid culture showed the highest inhibition of rotten ratio by 25% in apple fruit which was sprinked with the liquid culture 120h after inoculation of P. expansium. The patulin production was reduced by 90% when the liquid culture was added in apple juice at ratio of 40%(v/v). Therefore, the liquid culture had great potential in inhibiting the growth and patulin production of P. expansium.