Studying On The Enzymatic Degradation Of Patulin By Lipase From Ralstonia Sp. SL312

Patulin is an unsaturated lactone mycotoxin produced by Penicillium expansum and other fungi under specific conditions,which is the most widely contaminated mycotoxins all around the world and found mainly in fruit especially apples and their by-products.Patulin can elicit a series of harmful acute chronic,and had neurotic,immunotoxic and genotoxic toxic effects,seriously threaten to food security and human health.The use of enzyme preparations in mycotoxins degradation is a promising strategy because of its advantages of safety and high efficiency.The structure of patulin reveals the presence of a tetrahydropyran ring and a lactone bond,ring-openingr has been reported as being an important activity and possible pathway for patulin detoxification.Lipases,which have been shown to have a wide variety of substrates and efficiently catalyze the ring-opening polymerization of lactones,may have the ability to degrade patulin.In this research,Ralstonia sp.SL312 was screened as a lipase producing strain,and an extracellular lipase secreted termed RL12,was successfully purified.The main research contents and results are as follows:（1）Screening the lipolytic strain capable of degrading patulin.Using triglyceride and olive oil as substrates,a lipase producing strain SL3-12 was isolated from soil by observing the hydrolysis circle and measuring lipase activity.The results of gene sequences based on16S r DNA indicated that the isolated strain SL312 displayed more than 99.7%sequence identity with Ralstonia sp.The cell-free culture supernatant of strain SL3-12 removed more than 80%of patulin within 54 hours,indicating that the extracellular lipase may play a key role in patulin degradation（2）Purification and characterization of patulin degradative enzyme.The activity of lipase of Ralstonia sp.SL312 accumulated primarily in the extracellular fraction,therefore the cell-free culture supernatant was used to enzyme purification.Lipase RL12 was successfully purified 6.02 fold through ammonium sulfate precipitation,dialysis and ion-exchange chromatography with DEAE-Sepharose Fast Flow.Purified enzyme corresponded to an apparent molecular mass of 38 k Da in SDS-PAGE and the specific activity of enzyme reached 398.62 U/mg proteins.The degradation ability of the purified enzyme was confirmed by HPLC.Partial properties of lipase RL12 were studied,the results showed that the optimum reaction temperature was 40℃,it has high stability at 50℃and p H 7.5～8.5;Mg²⁺could enhance its activity.（3）Degradation product analysis and cytotoxicity evaluation.The maximum UV absorption peak of patulin shifted after degradation by lipase RL12,indicating that patulin was most probably degraded to another substance whose chemical characteristics were different from patulin.The degradation product was analyzed by LC-MS.MS analysis showed that the molecular peak of m/z（[M+H]⁺）=158.99 was possible patulin degradation product.The characteristic fragments of MS/MS spectrometry inferred that the product was formed by the ring-opening of lactone bond and epoxy structure.After detoxification by purified lipase RL12,Caco-2,HEK293 and LO-2 cells were used for cytotoxicity evaluation,the result showed that the cell viability was significantly improved.Therefore,it is considered that lipase RL12 exhibited potential patulin detoxification ability.（4）Patulin degradation by RL12 and its application in apple juice.Degradation assays were systematically performed in aqueous solution preliminarily.The results of this analysis revealed that the optimum degradation conditions of RL12 for patulin were p H 7.5,temperature 37°C,enzyme amount 100μg/m L,initial patulin concentration 5μg/m L.Under the optimum conditions,the addition of RL12 permitted degradation of over 85%of patulin in apple juice within 24 h.After degradation treatment,the phenolic acids,citric acid and several volatile flavor components increased significantly.Similarly,several aroma components,such as acetic acid and acetone,were significantly increased or disappeared.Therefore,it is likely that the lipase-mediated degradation of patulin in apple juice may be responsible for slight changes in the nutritional and sensory qualities of apple juice.