Isolation, Identification And Degradation Characteristics Of An Efficient Patulin-degrading Marine Yeast

The contamination of patulin in food industry is very serious, which is harmful to human health. It is a powerful and prospective method to degrade patulin by microorganisms. This study aims to screen and identify marine yeasts able to degrade patulin efficiently, study the yeast growing characteristic, safety, patulin degrading properties, and patulin degradation mechanism.1. The method for patulin determination in YPD liquid medium is established and verified by some indexes. The method for sample pretreatment is to extact with ethyl acetate, purified with Na2CO3 (1.5 g/100 mL) and dried with Na2SO4, then the organic phase is dried under a stream of nitrogen and redissolved in 1 mL of pH 4.0 water acidified by glacial acetic acid. The high performance liquid chromatography method uses UV detector at 276 nm, and an Inertsil ODS column is used for the separation. The mean recovery values are 78.01%-83.64% and the relative standard deviations are 3.09%-9.70%. LOD and LLOQ are 0.08 ?g/mL and 0.24 ?g/mL respectively. The method for patulin determination has high stability and precision, which could meet requirements for determining patulin fastly and economically by simple sample pretreatment and few reagent consumption.2. A strain of marine yeast, No.34, was screened with high patulin degradation ability and patulin tolerance. Compared with terrestrial yeast, the marine yeast has great advantage in patulin degradation. The yeast is identified as Kodameae ohmeri by Biolog identification system, partial 26S rRNA gene sequencing and phylogenetic tree construction, and then it is named Kodamare ohmeri HYJM34.3. Growing characteristics of K. ohmeri HYJM34 are studied. It is indicated that temperature plays an important role in the growth of K. ohmeri HYJM34 and the optimum temperature is 25-28 ?. K. ohmeri HYJM34 could survive under pH 2-12 and the biomass is highest at pH 6.0. Inoculum size has no obvious effect on yeast growth, and yeast biomass decreases gradually with the increase of loading volume. By the analysis of single factor experiment, fractional factorial experiment, central composite design and response surface, the optimum culture medium for K. ohmeri HYJM34 is glucose 2%, sucrose 8.19%, yeast extract 3%, MgSO4 0.25%, K2SO4 0.18%. The yeast biomass could reached to 19.54±0.28 g/L after 24 h of incubation. Compared with the YPD medium, the yeast biomass increased by 174.1%, which shows that the optimum culture medium has significantly promoted the growth of yeast. The results of acute toxicity test show that the maximum tolerable dose of mouse is more than 15 g/kg·bw, meaning that K. ohmeri HYJM34 is nontoxic grade.4. Patulin degrading properties by K. ohmeri HYJM34 is studied. The presence of patulin(10 ?g/mL) in YPD medium slightly restrained the yeast growth, for the yeast biomass treated with patulin is less than control. There is no patulin after 24 h by patulin degradation kinetics curve, revealing that the patulin degradation efficiency of K. ohmeri HYJM34 is better than other yeasts reported. Great degradation rate is observed at 35 ?, pH between 3 and 6 and more than 5×108 cells/mL of cell concentration. The patulin degradation performance by K. ohmeri HYJM34 is reduced with the increase of patulin concentration. The patulin degradation is significantly inhibited in the presence of Cu2+, Zn2+ and Fe3+, and promoted in the presence of Fe2+ and Mg2+. The patulin degradation rate is highest when the concentration of Mg2+ was 5 mmol/L.5. A preliminary study on the mechanism of patulin degradation by K. ohmeri HYJM34 is done. Inactivated yeast cells have no ability to degrade patulin. No patulin is found in the cell and cell wall, indicating the patulin reduction is a biodegradation process. Patulin is degraded by the reaction of enzymes within the cell and the cell membrane, and patulin-degrading enzyme was mainly distributing on cell membrane. HPLC and LC-TOF/MS analysis is used to search the major breakdown products of patulin. The results show that the major breakdown products by the reductase of K. ohmeri HYJM34 are (E)-ascladiol and (Z)-ascladiol, which are less toxic than patulin.