酱牛肉中的优势污染菌的微量热定量方法的实验研究

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摘要

微生物污染是影响我国食品安全的主要因素之一。目前，对于食品中微生物的

检测，仍以传统平板计数法为主，但该法所需时间过长且操作繁琐，并不适于食

品中微生物的快速检测以及产品的现场监控的需要。近年来新兴的快速检测方法

有酶联免疫吸附分析法、DNA 探针检测、聚合酶链式反应技术、ATP 生物发光技

术等，但这些新方法因为操作复杂，检测成本高，无法进行大面积推广使用。因

此寻找快速、简便、经济且适用面广的检测方法具有极其重要的意义。

微量热法是利用微生物生长代谢过程产热并且热量与微生物数量成比例关系

的原理，采用精密量热仪器测定微生物生长代谢热进而对微生物进行定量。利用

微量热法检测食品中细菌总数具备检测速度快、操作简单、应用范围广等优势，

可用于食品卫生检测测定菌落总数和不同的致病菌。鉴于这些优势，微量热法在

食品细菌总数快速检测上有着广阔的应用前景。

微量热法根据量热元件的不同有几种量热方式，本课题主要是以酱牛肉中主

要优势污染菌为研究对象，进行微生物生长热动力学分析并对其生长条件进行优

化，筛选出较好的测热方式，为开发量热式微生物快速检测仪并提高其检测效果

奠定基础。

本课题研究内容及结论如下：

1、以市售酱牛肉中主要优势污染菌葡萄球菌为实验菌种，通过热动力学分析

考察温度、初始接种量和培养基对细菌生长放热的影响，结果表明：该菌种的最

佳培养温度和检测温度为 37℃，初始接种量越高，细菌的生长繁殖速率越快；在

优化培养基中细菌的生长速率 k和细菌生长放热量得到明显的提高，有利于微量

热法快速测定细菌总数过程。

2、以酱牛肉中的优势污染菌作为实验菌，对混合池测热方式，循环池测热方

式和二氧化碳法测热方式这三种测热方式进行比较分析。得出三种测热方式均能

较好检测出细菌总数，且大大缩短了检测时间。同时得到二氧化碳测热法不仅放

大了热信号，也比其他两种方式操作更简便，是一种很有发展潜力的检测方法。

3、针对筛选出的二氧化碳法测热方式进行优化实验，对量热系统内温度、

NaOH 溶液浓度和气体流速三个主要影响因素进行了单因素和正交实验，得出放热

量随温度的升高而减小，随碱液浓度和气体流速的增加而增加，但温度和氢氧化

钠浓度对整个量热系统的影响均不显著，而通入量热系统的气体流速对整个量热

系统影响较显著，从环保和节能与将来实验的需要，其优化结果为细菌培养温度

37℃，通入细菌培养瓶的气体流速为 4 ml·min-1，量热系统内温度为 25℃，NaOH

溶液浓度 0.5 mol·L-1，通入量热系统的气体流速为 1.5 ml·min-1。在优化条件下该

方法具有较好的检测灵敏度、准确性和稳定性，优化后的条件有效提高该方法的

检测灵敏度、准确性和稳定性，为二氧化碳法量热装置的研究制作提供参考依据。

关键词：细菌热动力学微量热测热方式二氧化碳

ABSTRACT

Microbial contamination is one of the most primary reasons resulting in food

contamination. Presently, the conventional plate count method is still the standard

method for detecting microbe in our country though it is complicated and

time-consuming. This method is not suitable for rapid detection of microbes in food.

Although several faster detection methods have been developed, including PCR

technique，Enzyme linked immunosorbent assay and ATP bioluminescence technology,

the detection process is often complicated and the equipments are expensive. Hence, it

is necessary to establish a quick and inexpensive detection method for microbial

contamination in food.

It has been demonstrated that the microbial growth process involves an increase of

the number of cells and heat evolution, therefore, the microbial activity can be evaluated

by the thermal effect from all living cell metabolisms, and the microorganisms were

quantitatively evaluated to provide a correlation between thermal effects and the

microorganisms amount. Microcalorimetry, which is faster and operated easily method,

provides a potential new detection method that works with liquid media.

In this paper, the microbial growth conditions for multiplication were optimized

according to the thermokinetics analysis of microbial growth with heat flow rate-time

curve. Three different microcalorimetric methods were evaluated in the comparison

experiments to establish an effective microcalorimetric method for detection of

microbial contamination of food.

The main work and results of this research were as follows:

1. The main contamination bacteria in commercial sauce beef was isolated, and

identified as Staphylococcus by physiochemical experiments. the results suggested that

the best growth temperature of the bacteria was 37℃and the higher initial microbial

concentrations were, the faster microbial growth. Meanwhile, the microbial growth rate

k and the total thermal effect Q has been markedly improved in the optimized medium

culture.

2. Based on a number of testing and comparison experiments, the

microcalorimetric methods with mixed batch vessel, circulation vessel and carbon

dioxide were demonstrated to be good microbial detection methods. However, the

microcalorimetric measurement with the metabolic CO2was a potential advantageous

means to predict of microbial population, not only amplifying thermal but also simple

operation.

3. According to microcalorimetric method of the microbe metabolic CO2, the

calorimetric conditions of the CO2calorimetric detecting system were optimized. The

results suggested that the best growth temperature of the bacteria was 37 ℃and gas

flow velocity 4 ml·min-1, the temperature of detecting system 25 ℃, the NaOH

concentration 0.5 mol·L-1 and the flow velocity 1.5 ml·min-1 were chosen as the

calorimetric conditions in the experiment.

Key Words: Microorganism ，thermodynamics ，Microcalorimetry

Calorimetric method，Carbon dioxid

中文摘要

ABSTRACT

第一章绪论 .................................................................................................................1

§1.1 我国食品安全现状概述 ....................................................................................... 1

§1.2 现有食品微生物快速检测方法概述 ................................................................... 3

§1.2.1 基于代谢学的快速检测技术 ........................................................................3

§1.2.2 基于分子生物学的快速检测技术 .................................................................4

§1.2.3 基于免疫学的快速检测技术 .........................................................................5

§1.2.4 基于生物传感器的检测技术 .........................................................................5

§1.3 微量热法在微生物领域的应用研究 .................................................................. 6

§1.3.1 细菌分类鉴定研究 .........................................................................................6

§1.3.2 细菌代谢过程的热动力学的研究 .................................................................7

§1.3.3 不同药物对细菌生长的抑制作用及其规律的研究 .....................................8

§1.3.4 食品中微生物的快速检测研究 .....................................................................8

§1.4 本课题的背景和意义 .......................................................................................... 9

§1.5 主要研究内容 ..................................................................................................... 10

第二章酱牛肉中优势菌的生长热动力学分析 .........................................................11

§2.1 实验菌株、试剂及设备 .....................................................................................11

§2.1.1 实验菌株 .......................................................................................................11

§2.1.2 实验试剂 .......................................................................................................11

§2.1.3 实验设备 ......................................................................................................12

§2.2 实验方法 ............................................................................................................ 13

§2.2.1 温度对细菌生长代谢的影响 .......................................................................14

§2.2.1.1 细菌最适生长温度 ................................................................................ 14

§2.2.1.2 循环池法中温度对细菌生长放热的影响 ............................................ 14

§2.2.2 初始接种量对细菌生长放热的影响 ...........................................................15

§2.2.3 培养基对细菌生长放热的影响 ..................................................................15

§2.3 实验结果及分析 ................................................................................................ 15

§2.3.1 细菌最适生长温度的确定 ...........................................................................15

§2.3.2 循环池法中温度对细菌生长放热的影响 ...................................................16

§2.3.3 初始接种量对细菌生长放热的影响 ...........................................................17

§2.3.4 培养基对细菌生长放热的影响 ...................................................................18

§2.4 本章小结 ............................................................................................................ 19

第三章不同细菌测热方式的比较选择 .....................................................................21

§3.1 实验菌株、试剂及设备 .................................................................................... 22

§3.1.1 实验菌株 .......................................................................................................22

§3.1.2 实验试剂 .......................................................................................................22

§3.1.3 实验设备 .......................................................................................................22

§3.2 实验方法 ............................................................................................................ 23

§3.2.1 混合池测热方式 ...........................................................................................23

§3.2.2 循环池测热方式 ...........................................................................................24

§3.2.2.1 停流法清洗循环池参数的确定 ........................................................... 24

§3.2.2.2 循环池测热实验 ................................................................................... 25

§3.2.3 二氧化碳法测热方式 ...................................................................................25

§3.2.3.1 二氧化碳法测热方式中气体流速对细菌生长的影响 ..................... 25

§3.2.3.2 二氧化碳法测热实验 ......................................................................... 26

§3.3 实验结果及分析 ................................................................................................ 27

§3.3.1 停流法清洗循环池参数的确定 ...................................................................27

§3.3.2 二氧化碳测热方式中气体流速对细菌生长的影响 ...................................28

§3.3.3 三种测热方式实验结果及比较分析 ...........................................................28

§3.3.4 酱牛肉中优势菌测热方式的选择 ..............................................................33

§3.4 本章小结 ............................................................................................................ 34

第四章二氧化碳测热方式的条件优化及验证实验 ...............................................35

§4.1 实验菌株、试剂及设备 ..................................................................................... 35

§4.1.1 实验菌株 .......................................................................................................35

§4.1.2 实验试剂 .......................................................................................................36

§4.1.3 实验设备 .......................................................................................................36

§4.2 实验方法 ............................................................................................................. 37

§4.2.1 量热系统内温度对放热的影响 ...................................................................37

§4.2.2 量热系统内气体流速对放热的影响 ...........................................................37

§4.2.3 量热系统内 NaOH 溶液浓度对放热的影响 .............................................. 37

§4.2.4 正交优化实验 ..............................................................................................37

§4.2.5 验证实验 ......................................................................................................38

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摘要：

摘要微生物污染是影响我国食品安全的主要因素之一。目前，对于食品中微生物的检测，仍以传统平板计数法为主，但该法所需时间过长且操作繁琐，并不适于食品中微生物的快速检测以及产品的现场监控的需要。近年来新兴的快速检测方法有酶联免疫吸附分析法、DNA探针检测、聚合酶链式反应技术、ATP生物发光技术等，但这些新方法因为操作复杂，检测成本高，无法进行大面积推广使用。因此寻找快速、简便、经济且适用面广的检测方法具有极其重要的意义。微量热法是利用微生物生长代谢过程产热并且热量与微生物数量成比例关系的原理，采用精密量热仪器测定微生物生长代谢热进而对微生物进行定量。利用微量热法检测食品中细菌总数具备检测速度快、操作...

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