关键词: 基质辅助激光解吸电离飞行时间质谱, 产毒霉菌, 建立数据库, 影响因素

Abstract: Objective To establish common toxigenic moulds in food identification database including 9 genus,15 species,and 15 strains in sum by a matrix-assisted laser desorption ionization time-of-flight mass spectrometry（MALDI-TOF MS）,aiming to provide supplementary means for the identification of toxigenic moulds in the standard GB 4789.16-2016 "Microbiological identification of common toxigenic molds in food " and improves the efficiency and accuracy. Simultaneously explore the effects of different culture conditions（media,time） and pretreatment methods on the MALDI-TOF MS identification results of 15 common toxigenic moulds. Methods From March 2022 to August 2023,15 strains of toxigenic mouldswere cultured in sabouraud liquid media at 28 ℃ for 24 hours were assigned for database construction,and their protein spectrum were used to establish their database. Compare the effects of different solid culture media,culture time,and pretreatment methods on the identification results through the established database. Results Taking penicillium purpureum as an example,the identification scores of different solid culture media,including sabouraud dextrose agar（SDA）,chalet agar media（CA）,potato dextrose agar（PDA） and Bengal red medium（RBC） were identification errors,1.555±0.007,1.875±0.022,and 2.138±0.048,respectively. There was a statistically significant difference in identification scores between different solid culture media（F=88.436,P<0.05）. The identification scores of three different pretreatment methods,namely direct application method,formic acid extraction method,and formic acid acetonitrile extraction method were 1.813±0.008,2.159±0.094,and 2.138±0.048 respectively. The differences in identification scores between different pretreatment methods were statistically significant（F=25.852,P<0.05）. The identification scores for different cultivation times from 3 to 7 days were 2.315±0.037,2.224±0.016,2.138±0.048,2.171±0.016 and 2.166±0.010,respectively. There was a statistically significant difference in identification scores for different cultivation times（F=22.893,P<0.05）. For the remaining 14 common toxigenic moulds,there were statistically significant differences in MALDI-TOF MS identification scores among different solid culture media,culture time,and pretreatment methods（all P<0.05）. Conclusion Due to the inability to use PDA as the solid culture medium for the identification of Streptomyces and Fusarium oxysporum,a self built database of common toxigenic moulds was ultimately used as the identification platform. Potato glucose agar was cultured at 28 ℃ for 4-5 days,and protein was extracted using formic acid acetonitrile method for mass spectrometry identification. A high confidence score of ≥1.80 was used as the evaluation criterion,which effectively identified 86.7% of the toxigenic moulds in 15 strains.

Key words: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry, Toxigenic moulds, Establishment identification database, Influence factors