草酸青霉中新型线性五肽的发现及对柑橘采后致腐菌拮抗活性研究

A novel linear pentapeptide from Penicillium oxalicum and its antagonism against postharvest decay mold of citrus

【背景】柑橘是我国南方农村地区的一种重要经济作物,每年因采后真菌感染导致大量柑橘腐烂和劣变,造成了巨大的经济损失,严重影响柑农的积极性。【目的】丝状真菌SG-4对柑橘采后致腐菌橘青霉(Penicillium citrinum)和指状青霉(Penicillium digitatum)具有很强的拮抗作用,为明确其抑菌的物质基础,对该菌进行了生物学鉴定和活性成分分析。【方法】将SG-4菌株的核糖体DNA的内转录间隔区进行测序,结合菌株形态进行鉴定;对其菌体相和培养液进行抑菌活性追踪,利用醇沉淀和反相高效液相色谱分离得到主效物质,通过高分辨质谱和核磁共振等波谱分析方法确定结构;采用离体的牛津杯抑菌圈法和柑橘的体内接种试验来评价其抑菌活性;通过液相分析测定了活性物质在发酵液中的含量。【结果】分子和形态鉴定表明SG-4是一株草酸青霉(Penicillium oxalicum),其抑制柑橘采后致腐菌的活性物质位于培养液的水相提取物中,且具有强极性。进一步研究表明其活性成分为一种新型线性醇溶五肽,含3个具羟基的氨基酸,并且富含甲基化修饰,构成寡肽的第一个氨基酸为β-氨基丁酸(beta-aminobutyric acid,BABA),其结构为O-Me-BABA-N-Me-Thr-Thr-NMe-Val-Ser。该五肽对柑橘致腐菌的抑制效果明显优于正对照,且在培养液中的初始含量为90 mg/L,达到了工业化生产的水平。【结论】来自于草酸青霉的新型线性五肽具有较强的拮抗柑橘致腐菌的作用,其结构和活性在国内外均属首次报道,未来可作为抑制真菌的抗生素在农业、食品领域进行开发。

[Background]Citrus is an important economic crop in rural areas of South China.Due to the influence of the postharvest fungi,a large number of fruits are rotted and deterioratedannually resulting in huge economic losses and seriously affecting the enthusiasm of citrus farmers.[Objective]The filamentous fungus SG-4 exhibited strong antagonism to the postharvest decay mold Penicillium citrinum and Penicillium digitatum.In order to figure out the fungicidal substance,here we performed biological identification and active ingredient analysis.[Methods]The internal transcribed spacer(ITS)of strain SG-4 nuclear ribosomal DNA was sequenced to identify the strain together with the morphology.The antifungal activities of the mycelium phase and culture liquid were traced and the main active substance was separated by alcohol precipitation and reverse high performance liquid chromatography(RP-HPLC).The structure was determined by high resolution mass spectrometry and nuclear magnetic resonance spectroscopy.The antimicrobial activity was evaluated both by Oxford cup method as in vitro and by inoculation test of citrus as in vivo.The content of the fungicidal substance in fermentation broth was determined by RP-HPLC.[Results]Molecular and morphological identification showed that the strain SG-4 was a Penicillium oxalicum.The active substance inhibiting postharvest decay mold of citrus in the aqueous extract was highly polar.Further studies showed that the substance was a novel linear alcohol-soluble pentapeptide,which contained three hydroxyl amino acids and was rich in methylation modification.The first amino acid was beta-aminobutyric acid(BABA),and the linear structure was O-Me-BABA-N-Me-Thr-N-Me-Val-Ser.The inhibitory effect to postharvest decay mold is significantly better than the positive control,and the initial content in the culture liquid got to 90 mg/L,reached the level of industrial production.[Conclusion]The new linear pentapeptide from P.oxalicum showed strong antagonism to citrus decay fungi.Since it is the first time to report both the structure and the activity at home and abroad,the pentapeptide can be developed as an antibiotic to inhibit fungi in agriculture and food industry in the future.