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

摘要:

【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 rRNA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4.26×10¹⁰ CFU/mL、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。

Abstract:

[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 was α-ZEL. The other strain had a ZEN-adsorbing effect, with the adsorption rate reaching 62.9% within 20 min at a bacterial concentration of 4.26×10¹⁰ 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. tropicalis12-6 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.