老面中玉米赤霉烯酮消减乳酸菌的筛选、鉴定及机理解析

Screening,identification,and mechanism elucidation of zearalenone-reducing lactic acid bacteria from Chinese sourdough

【背景】玉米赤霉烯酮(zearalenone,ZEN)是一种由镰刀菌属真菌产生的具有雌激素效应的真菌毒素,是世界范围内严重危害人类健康和农业安全的污染物之一。乳酸菌作为一类被公认安全(generally recognized as safe,GRAS)的食品级非致病微生物,近年研究显示其具有良好的真菌毒素消减能力,为其在保障食品安全方面带来潜在应用前景。【目的】以来自山东、河南和甘肃等7份老面样品作为研究对象,从中筛选具有消减ZEN活性的乳酸菌,探究该菌对ZEN的吸附机理。【方法】利用稀释平板涂布法获得菌株;超高效液相色谱检测具有消减ZEN活性的菌株,质谱(mass spectrometry)确定代谢产物;16S r RNA基因序列分析比对确定菌株属种,透射电镜(transmission electron microscopy,SEM)观察菌株形态;分析不同初始毒素浓度及菌体浓度下的吸附效果,对其进行动力学模型拟合;定位吸附位点并通过傅里叶变换红外光谱(Fourier transform infrared spectroscopy,FTIR)分析确定菌株中参与吸附ZEN的官能团,阐明对ZEN的吸附机理。【结果】初步筛选获得63株乳酸菌,从中复筛获得3株具有消减ZEN活性的乳酸菌,经鉴定:菌株6-8和菌株18-2为短促生乳杆菌(Levilactobacillus brevis),菌株12-6为热带醋杆菌(Acetobacter tropicalis)。两株短促生乳杆菌具有降解效果,在ZEN浓度为10 mg/L的条件下,48 h内降解率分别达到85.5%和87.3%,质谱结果显示降解产物为α-ZEL;另一株具有吸附效果,在菌株浓度为4.26×10^(10)CFU/m L、ZEN浓度为10 mg/L条件下,20 min内吸附率达到62.9%,灭活后吸附率上升20%。吸附过程同时符合准一级(pseudo-first-order)动力学及准二级(pseudo-second-order)动力学模型。傅里叶变换红外光谱结果显示菌株12-6的主要吸附位点为细胞壁上的肽聚糖与磷壁酸分子,羟基、次甲基、羧基和酰胺基等作为主要官能团参与吸附。【结论】菌株6-8和菌株18-2对ZEN有较强的降解能力;菌株12-6对ZEN有较强的吸附能力。其吸附动力学符合准一级及准二级动力学模型,吸附位点为细胞壁上的磷壁酸及肽聚糖分子。本研究为乳酸菌清除食品和饲料中有害物质的应用提供了理论基础。

[Background]Zearalenone(ZEN),a mycotoxin with estrogenic effects produced by Fusarium,is one of the pollutants that seriously jeopardize human health and agricultural safety worldwide.As a class of food-grade non-pathogenic microorganisms generally recognized as safe(GRAS),lactic acid bacteria have been proved to have good mycotoxin-reducing ability in recent years,demonstrating the application potential in safeguarding food safety.[Objective]To screen the lactic acid bacteria capable of reducing ZEN from seven Chinese sourdough samples collected from Shandong,Henan,and Gansu,and investigate the reducing mechanisms of ZEN by the bacteria.[Methods]The strains were isolated by the dilution-plate coating method.Ultra-high performance liquid chromatography was employed to screen the strains with ZEN-reducing activity,mass spectrometry(Q exactive)to identify the metabolites,and transmission electron microscopy(SEM)to observe the morphology of the strains.The adsorption effects were examined at different initial toxin concentrations and bacterial concentrations,and a kinetic model was fitted.Fourier transform infrared spectroscopy(FTIR)was employed to identify the functional groups involved in the adsorption of ZEN and elucidate the adsorption mechanism of ZEN.[Results]A total of 63 strains of Lactobacillus were isolated from the preliminary screening,and then three strains with ZEN-reducing activity were obtained.Strains 6-8 and 18-2 were identified as Levilactobacillus brevis,and strain 12-6 as Acetobacter tropicalis.The two strains of L.brevis had ZEN-degrading effects,with the degradation rates of 85.5%and 87.3%,respectively,within 48 h at ZEN concentration of 10 mg/L.The mass spectrometry results showed that the degradation product wasα-ZEL.The other strain had a ZEN-adsorbing effect,with the adsorption rate reaching 62.9%within 20 min at a bacterial concentration of 4.26×10^(10)CFU/mL and a ZEN concentration of 10 mg/L.Moreover,the adsorption rate increased by 20%after strain inactivation.The adsorption process was fitted with both pseudo-first-order and pseudo-second-order models.Fourier transform infrared spectroscopy(FTIR)showed that the main adsorption sites of strain 12-6 were peptidoglycan and teichoic acid in the cell wall,involving hydroxyl,methenyl,carboxyl,and amide groups.[Conclusion]L.brevis 6-8 and 18-2 showed strong degradation capacity against ZEN,and A.tropicalis 12-6 had a strong adsorption capacity for ZEN.The adsorption kinetics conformed to pseudo-first-order and pseudo-second-order models,with the adsorption sites at teichoic acid and peptidoglycan in the cell wall.This study provides a theoretical basis for the application of lactic acid bacteria in the removal of harmful substances from food and feed.