扩展青霉拮抗放线菌的分离、鉴定及其液体培养研究
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摘要
展青霉素有是一种具有致癌、致畸、致突变及免疫抑制效应的真菌毒素,广泛存在于苹果汁中,其根本原因是毒素产生菌对苹果原料的污染与产毒。寻找对展青霉素主要产生菌扩展青霉生长及其产毒特性有抑制作用的菌株,对于进一步开发展青霉素的生物防治用菌种和制剂具有重要的理论和实际意义。为此,本文的主要研究内容与研究结果如下:
(1)从陕西杨凌地区,不同的生境土壤(苹果园、猕猴桃园、菜园)中,共分离到154株放线菌。经过初筛选,得到4株对3株试验用展青霉素产生菌均有显著抑制作用的拮抗放线菌。其中,1株来自苹果园,3株来自辣椒园,编号分别为P50,C11,C16,C60;采用液体发酵滤纸片法进行复筛,得到1株拮抗效果最好的优势拮抗放线菌株C16,对3株扩展青霉的平均抑菌直径达2.33 cm。
(2)对拮抗放线菌C16进行了分类与鉴定。综合其形态学、生理生化特征及16SrDNA序列分析的结果,最终将其分类鉴定为放线菌目(Actinomycetales)小单胞菌科(Micromonosporaceae)小多胞菌属(Micropolyspora)的一个新种,命名为Micropolyspora.C16,并申请专利保藏,专利保藏号为放线菌CCTCC M207210。
(3)对C16液体培养基进行筛选,并得到适宜生长和抑菌产物形成的基础培养基为:麦芽糖20g,酵母膏2.0g,K2HPO4 1.0g,MgSO4 1.0g,NaCl 1.0g,自来水1000mL。应用中心旋转组合(CCC)试验设计,对以上基础培养基中五种主要的营养组分进行了响应面分析,得出各因素及其交互作用与发酵菌体生长量之间的多元回归方程,通过方程求解,获得最佳营养条件为:麦芽糖2.70 g/100mL,酵母膏0.26 g/100mL,MgSO4 0.17 g/100mL,K2HPO4 0.14 g/100mL,NaCl 0.15 g/100mL。
(4)对C16的液体培养条件进行了优化,采用Box-Behnken试验设计对主要影响因子,即温度、接种量、装液量等三个因素与菌体生长量之间的关系进行了探讨,获得最佳培养条件为接种量13.5%(v/v)、培养基初始pH7.4、摇床转速180 r/min、发酵温度27.6℃、250 mL摇瓶装液量125mL。
(5)为了明确发酵液中抑菌活性物质的稳定性及其实际应用潜力,在琼脂平板上考察了发酵抑菌活性粗提物对热、酸、碱、紫外线等处理方法的稳定性,并考察了该物质在新鲜苹果及果汁发酵中对两株展青霉素产生菌的生长及毒素产量的影响。结果发现,高温破坏发酵液抑菌活性粗提物,100℃处理30 min,抑菌活性降低39.5%,处理60 min,其活性完全丧失;酸、碱处理都会使活性物的稳定性降低,在发酵液原始pH(5.7)下最稳定,相对而言,碱性条件下稳定性高于酸性条件;活性物质对紫外线敏感,经紫外线照射90 min,其抑菌活性完全丧失。接种青霉120 h后,在苹果表面喷涂无菌体发酵液可使苹果的腐烂程度降低25%;在果汁中加入40%(v/v)的无菌体发酵液,可使产毒菌的展青霉素产量降低90%。
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.
(1)从陕西杨凌地区,不同的生境土壤(苹果园、猕猴桃园、菜园)中,共分离到154株放线菌。经过初筛选,得到4株对3株试验用展青霉素产生菌均有显著抑制作用的拮抗放线菌。其中,1株来自苹果园,3株来自辣椒园,编号分别为P50,C11,C16,C60;采用液体发酵滤纸片法进行复筛,得到1株拮抗效果最好的优势拮抗放线菌株C16,对3株扩展青霉的平均抑菌直径达2.33 cm。
(2)对拮抗放线菌C16进行了分类与鉴定。综合其形态学、生理生化特征及16SrDNA序列分析的结果,最终将其分类鉴定为放线菌目(Actinomycetales)小单胞菌科(Micromonosporaceae)小多胞菌属(Micropolyspora)的一个新种,命名为Micropolyspora.C16,并申请专利保藏,专利保藏号为放线菌CCTCC M207210。
(3)对C16液体培养基进行筛选,并得到适宜生长和抑菌产物形成的基础培养基为:麦芽糖20g,酵母膏2.0g,K2HPO4 1.0g,MgSO4 1.0g,NaCl 1.0g,自来水1000mL。应用中心旋转组合(CCC)试验设计,对以上基础培养基中五种主要的营养组分进行了响应面分析,得出各因素及其交互作用与发酵菌体生长量之间的多元回归方程,通过方程求解,获得最佳营养条件为:麦芽糖2.70 g/100mL,酵母膏0.26 g/100mL,MgSO4 0.17 g/100mL,K2HPO4 0.14 g/100mL,NaCl 0.15 g/100mL。
(4)对C16的液体培养条件进行了优化,采用Box-Behnken试验设计对主要影响因子,即温度、接种量、装液量等三个因素与菌体生长量之间的关系进行了探讨,获得最佳培养条件为接种量13.5%(v/v)、培养基初始pH7.4、摇床转速180 r/min、发酵温度27.6℃、250 mL摇瓶装液量125mL。
(5)为了明确发酵液中抑菌活性物质的稳定性及其实际应用潜力,在琼脂平板上考察了发酵抑菌活性粗提物对热、酸、碱、紫外线等处理方法的稳定性,并考察了该物质在新鲜苹果及果汁发酵中对两株展青霉素产生菌的生长及毒素产量的影响。结果发现,高温破坏发酵液抑菌活性粗提物,100℃处理30 min,抑菌活性降低39.5%,处理60 min,其活性完全丧失;酸、碱处理都会使活性物的稳定性降低,在发酵液原始pH(5.7)下最稳定,相对而言,碱性条件下稳定性高于酸性条件;活性物质对紫外线敏感,经紫外线照射90 min,其抑菌活性完全丧失。接种青霉120 h后,在苹果表面喷涂无菌体发酵液可使苹果的腐烂程度降低25%;在果汁中加入40%(v/v)的无菌体发酵液,可使产毒菌的展青霉素产量降低90%。
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.
引文
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