CN109030696B - 一种冻干双孢蘑菇中假单胞菌快速检测的方法 - Google Patents

一种冻干双孢蘑菇中假单胞菌快速检测的方法 Download PDF

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CN109030696B
CN109030696B CN201810794864.3A CN201810794864A CN109030696B CN 109030696 B CN109030696 B CN 109030696B CN 201810794864 A CN201810794864 A CN 201810794864A CN 109030696 B CN109030696 B CN 109030696B
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杨文建
王柳清
胡秋辉
方勇
裴斐
马宁
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Nanjing University of Finance and Economics
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Abstract

本发明涉及一种冻干双孢蘑菇中假单胞菌快速检测的方法，属于食品微生物检测技术领域。该方法基于顶空‑固相微萃取‑气相色谱‑质谱连用（HS‑SPME‑GC‑MS）技术来检测冻干双孢蘑菇中的苯甲醛含量的变化，通过最小二乘法分析估算其假单胞菌菌落总数，由此实现对冻干双孢蘑菇中假单胞菌的快速检测。本发明为微生物快速检测提供了新的理论支持和解决方式，开拓了对食用菌干制品中微生物快速检测的新思路。

Description

一种冻干双孢蘑菇中假单胞菌快速检测的方法
技术领域
本发明涉及一种冻干双孢蘑菇中假单胞菌快速检测的方法，属于食品安全检测领域。
背景技术
双孢蘑菇因其味道鲜美，营养价值高，药用和保健价值，而受到消费者的青睐。冻干双孢蘑菇由于其能够较好保持原有风味且耐储存，正在逐渐成为双孢蘑菇销售的主要形式。假单胞菌是冻干双孢蘑菇中的主要细菌，易引起细菌性褐斑，而目前冻干双孢蘑菇细菌生长的相关研究较少，需要新的检测技术来保证其商品性与安全性。在前期的研究中发现，冻干双孢蘑菇中的假单胞菌菌落总数与其苯甲醛含量呈显著正相关。
传统的微生物检测方法前处理复杂，试剂消耗量大，菌落培养周期长，无法对大量的样品进行实时检测。现代生物技术的微生物检测方法包括：核酸探针技术、PCR 技术、生物芯片技术、生物传感器检测技术等，操作步骤复杂，检测成本高。目前国内对假单胞菌的菌落总数测定通常使用选择性培养基培养样品得到粗略的菌落数量，或者应用PCR技术来准确测定。但现有的检测技术都难以实现对样品的快速检测，且操作步骤复杂，检测周期长。在安全检验中对菌落总数的检测并不需要准确的菌落数量，且允许一定的误差。
发明内容
针对上述问题，本发明提出了一种快速检测方法，基于顶空-固相微萃取-气相色谱-质谱连用（HS-SPME-GC-MS）技术来检测冻干双孢蘑菇中的苯甲醛含量，通过最小二乘法分析估算冻干双孢蘑菇中的假单胞菌菌落总数，由此实现冻干双孢蘑菇中假单胞菌的快速检测。
本发明通过预测卫生学的分析方法，建立苯甲醛含量与假单胞菌生长动态的数学模型，预测值与实测值的相对误差R² = 0.9225，所建模型可以较好地预测冻干双孢蘑菇中的假单胞菌生长状况。
具体操作为：将冻干双孢蘑菇置于20 mL的顶空进样瓶中，并添加正癸醇-甲醇溶液（80 μg/mL）作为内标，密封并置于55 ℃恒温中加热，平衡10min后将DVB/CAR/PDMS插入顶空进样瓶中萃取45min后进样。在 250℃进行解析5min后，化合物通过 DB-5MS 毛细管柱（30 m×0.25 mm，0.25 μm）进行分离。色谱条件：程序升温条件为初始温度 30 ℃，保持 2min，然后以 5 ℃/min 的速度升到60 ℃，无保留，最后以 10℃/min 的速度升至 200℃，保持时间 2 min；载气 He；流速 1.0 mL/min；分流比 1：1。质谱条件：EI 电离源；电子能量70 eV；离子源温度 250℃；质量扫描范围 35~550u。
冻干双孢蘑菇中假单胞菌菌落总数的预测模型为：y = 6.7483e5.4657x。
其中，y为冻干双孢蘑菇假单胞菌菌落总数（CFU/mL），x为样品中的苯甲醛含量（μg/g）。
本发明为冻干双孢蘑菇中的假单胞菌快速检测方向提供了绿色、经济、便捷的理论支持和解决途径，开拓了食用微生物高通量快速安全检测研究的新思路。
附图说明
图1为本发明的回归模型图；
图2为本发明观测值与预测值的线性回归图。
具体实施方式
以下结合具体实例对本发明作进一步说明。
将0.5 g冻干双孢蘑菇置于20mL的顶空进样瓶中，并添加1μL正癸醇-甲醇溶液（80μg/mL）作为内标，密封并置于55℃恒温中加热，平衡10min后将DVB/CAR/PDMS插入顶空进样瓶中萃取45min后进样。在 250℃进行解析5min后，化合物通过 DB-5MS 毛细管柱（30 m×0.25 mm，0.25 μm）进行分离。色谱条件：程序升温条件为初始温度 30 ℃，保持 2 min，然后以 5 ℃/min 的速度升到60 ℃，无保留，最后以 10℃/min 的速度升至 200℃，保持时间 2 min；载气 He；流速 1.0 mL/min；分流比 1：1。质谱条件：EI 电离源；电子能量 70eV；离子源温度 250℃；质量扫描范围 35~550u。
冻干双孢蘑菇中假单胞菌菌落总数的预测模型为：y = 6.7483e5.4657x。
其中，y为冻干双孢蘑菇假单胞菌菌落总数（CFU/mL），x为样品样品苯甲醛含量（μg/g）。
图1为冻干双孢蘑菇中的假单胞菌落总数实测值与苯甲醛含量的线性关系，用作模型比对。在观测值与预测值的线性回归图2中，R2=0.9225，表明该模型能够在一定程度上预测冻干双孢蘑菇中假单胞菌的生长情况。

Claims (2)

1.一种冻干双孢蘑菇中假单胞菌快速检测的方法，其特征在于：通过顶空-固相微萃取-气相色谱-质谱连用技术测定冻干双孢蘑菇中苯甲醛含量以检测其假单胞菌菌落总数；
冻干双孢蘑菇中假单胞菌菌落总数的预测模型为：y= 6.7483e5.4657x；
其中，y为样品假单胞菌菌落总数，CFU/mL；x为样品中的苯甲醛含量，μg/g。
2.根据权利要求1所述的冻干双孢蘑菇中假单胞菌快速检测的方法，其特征在于：采用顶空-固相微萃取法对样品挥发性物质进行萃取；将冻干双孢蘑菇样品置于20 mL的顶空进样瓶中，并添加80μg/mL正癸醇-甲醇溶液作为内标，密封并置于55℃恒温中加热，平衡10min后将DVB/CAR/PDMS萃取头插入顶空进样瓶中萃取45min后，将萃取头插入气相色谱-质谱联用仪进样口中进行苯甲醛物质检测；
色谱条件：在250℃进行解析5min，利用DB-5MS毛细管柱30m×0.25mm，0.25μm分离，色谱柱采用程序升温：初始温度30℃，保持2 min，然后以5℃/min的速度升到60℃，无保留，最后以 10℃/min的速度升至200℃，保持时间2min；载气He；流速1.0 mL/min；分流比1：1；
质谱条件：EI电离源；电子能量70 eV；离子源温度250℃；质量扫描范围35 ~550u。
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DE102013009610A1 (de) * 2013-06-07 2014-12-11 Kist Europe Forschungsgesellschaft Mbh Mittel und Verfahren zur Diagnose von Pseudomonas aeruginosa
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CN108318590A (zh) * 2017-12-25 2018-07-24 浙江大学 基于采用呼出气体标记物谱的常见病原菌感染所致的肺炎的诊断系统

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