猪肉解冻中损伤型气单胞菌的检测及修复机制

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3.0 陈辉 2024-11-19 7 4 2.02MB 50 页 15积分

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摘要

冷冻肉是国家调节肉制品市场的重要手段之一，是肉类产品在进出口贸易和

国内地区间流通的主要产品形态，也是各种肉类加工企业的原料。冷冻肉解冻后

的安全以微生物影响最为显著。肉类经过包括冷冻在内处理后，一部分微生物会

死亡，一部分可能存活，另一部分可能会亚损伤。肉类中的微生物经过冷冻冷藏

处理后，可遭受不同程度的亚致死性损伤而成为损伤菌，损伤菌在适宜的条件下

（如解冻）可进行修复，重新具有正常未损伤细菌的生理生化特性，恢复其对选

择性物质的耐受性，可重新具有产毒、致病性。传统检测微生物方法假阴性较高，

易于对失活和残存两类微生物计数，而亚损伤一类微生物很难在选择性培养基上

生长，常被归入失活类型而被忽略。因此，研究细菌的损伤种类来确定有效的修

复损伤细胞并将其检测出的方法是非常重要的。同时，确定微生物的冷冻损伤机

理可为更好的检测损伤型微生物提供理论依据。

本论文重点研究解冻猪肉中的优势腐败菌之一的气单胞菌，用固体和液体两

种修方法对损伤型气单胞菌进行优化检测；引入预测微生物学方法探讨猪肉解冻

过程损伤型气单胞菌失活模型的构建；在细胞膜和细胞质水平上对冷冻损伤的气

单胞菌进行损伤和修复机理开展初步分析，从而完善气单胞菌损伤模型的理论体

系，为冷冻导致的气单胞菌亚致死状态的检测提供理论支持，并指导肉类工业安

全生产。

本研究主要内容和结果如下：

（1）猪肉解冻过程中损伤型气单胞菌的检测

通过固体、液体修复方法的比较，得出以含 0.6%酵母浸膏的胰酪胨大豆琼脂

（TSAYE）为损伤修复培养基，气单胞菌的修复培养基中分别添加 1.0%、1.5%、

2.0%、2.5%和3.0%的NaCl，应用 SPSS17.0 软件分析不同的选择培养基计数的差

异显著性，经差异显著性分析得出 TSAYE+1.5%NaCl 为损伤培养基并用于后续研

究。损伤气单胞菌的数量以 TSAYE 的菌落数与损伤培养基 TSAYE+1.5%NaCl 的

菌落数之差来计数。

（2）猪肉解冻过程损伤菌失活模型的构建

将猪肉在 25℃解冻过程以修复与损伤培养基进行计数，测定此过程气单胞菌

的生长曲线，由 DPS9.50 软件分析不同曲线的显著性确定损伤气单胞菌的修复时

间大约为 75min。用 Origin8.0 软件模拟损伤菌的失活曲线，应用判定系数(R2)（通

过拟合软件得出）、预测标准误差（standard error of prediction, SEP）和均方误差

(mean square error, MSE)评定模型得出其失活符合 Polynomial 模型（R2=0.9973）及

Boltzmann 模型（R2=0.9884），本研究通过模型的检验选择 Polynomial 模型拟合损

伤型气单胞菌的失活过程。

（3）冷冻处理导致气单胞菌损伤的机理研究

为了进一步了解冷冻导致气单胞菌的损伤类型和修复的机理，对冷冻损伤的

气单胞菌进行细胞膜和细胞质水平上的分析。利用冷冻损伤菌体离心后上清液中

260nm 及280nm 吸光度值的改变及核酸、肽聚糖及 ATP 等物质合成代谢抑制作用

的实验来初步探讨冷冻损伤气单胞菌的结构及代谢的损伤。结果表明冷冻损伤气

单胞菌的细胞膜通透性发生改变，致使其对 1.5%NaCl 敏感。DNA 及肽聚糖等大

分子物质的合成并未遭到破坏，RNA 和ATP 的合成受到抑制。

本课题研究了猪肉解冻过程中损伤型气单胞菌的检测，失活模型的构建以及

气单胞菌冷冻损伤机理，探讨出损伤型气单胞菌的检测方法为 TSAYE 和

TSAYE+1.5%NaCl 的培养基计数之差，适宜修复温度为 25℃，修复时间大约为

75min，在猪肉解冻过程的失活模型符合 Polynomial 模型，冷冻造成其细胞膜通透

性改变以及 RNA 和ATP 的合成遭到破坏，以上结论可为食肉安全提供理论指导，

重溯消费者对食肉安全的信心。

关键词：气单胞菌 Polynomial 模型 ATP 合成代谢细胞膜通透性

Detection and Repair Mechanism of Injured Aeromonas

spp.from Thawing Pork

ABSTRACT

Frozen meat plays an important role in meat market of state regulation. It is not

only one of the main product forms in import and export of meat products in domestic

and international trade, but also raw materials of all kinds of meat processing enterprises.

The safety of thawing meat was significantly threatened by microorganism. After many

treatments (e.g thawing), some microorganisms may be killed, some may survive

(non-injured), the other may be sublethally injured. Microorganisms in meat can suffer

from some degrees of sub-lethal injury to become injured bacteria during freezing

processing. However injured bacteria can resuscitate and become functionally normal in

a favorable environment (e.g. thawing), and the repaired bacteria may restore its

tolerance of selective substances and has re-oxygenic, pathogenic. Traditional

microbiological detection methods were high false-negative and only fit for detecting

the inactivation and remaining microbial, while the injured was hard to grow on

selective medium, and they were often ignored as inactivation type. Therefore, it is

necessary to study the injured types and determine effective methods to detecte them.

Moreover, determining the injury mechanism of microorganisms can provide a

theoretical guide for better detecting microbial counts.

This paper focuses on Aeromonas spp. on the thawing meat, because it is one of

the specific spoilage bacteria. Predictive microbiology theory was used to build

inactivation model of injured Aeromonas spp. during thawing process. Besides, in order

to better understanding injury model and substrate for detection of sub-lethal

Aeromonas spp., researches about freezing injury and repair mechanisms were carried

out, and the injury was explained on both cell membrane and cytoplasm levels, All

these results would contribute for the safety of meat industry.

Content and results of this study are as follows:

(1) Comparing solid with liquid repair method showed that medium (TSAYE) was

suit for repair research. The medium was added 1.0%, 1.5%, 2.0%, 2.5% and 3.0% of

NaCl, respectively. Results showed that TSAYE +1.5% NaCl medium could be used for

counting injured Aeromonas spp. by analysis of SPSS 17.0 software. The number of

injured Aeromonas spp. colonies was counted by difference between TSAYE and

TSAYE +1.5% NaCl medium

(2) This study indicated that repair time of injured Aeromonas spp.was about 75

min during meat thawing process at 25℃, also this result was obtained on both TSAYE

and TSAYE +1.5% NaCl medium. The Origin8.0 software was used to fit the

inactivation curve, and results showed that the inactivation curve could be fitted with

Polynomial model (R2=0.9937) and Boltzmann model (R2=0.9884), respectively.

Moreover, Polynomial model might be better fitting injury inactivation process by the

model evaluation.

(3) To explore freezing injury mechanism preliminarily, experiments on measuring

260nm and 280nm absorbance variations of cell supernatant centrifugation and

inhibition of nucleic acids, peptidoglycan and ATP were carried out. The results

indicated permeability of cell membrane was damaged, thus leading it sensitive for

1.5% NaCl. Moreover, DNA and peptidoglycan synthesis were not destroyed, but RNA

and ATP synthesis were inhibited.

This study contains detection of injuried Aeromonas spp. on thawing meat,

establishing its inactivation model and exploring freezing injury mechanism. Results

showed that difference between TSAYE and TSAYE+1.5%NaCl medium is the number

of injured Aeromonas spp., suitable temperature and repair time are 25ºC and 1h,

respectively; Polynomial model could be used for fitting the inactivation model;

membrane permeability, synthesis of RNA and ATP were damaged.These conclusions

can provide a guidance for meat safety, making consumer regain their confidence for the

safety of meat.

Key words: Aeromonas app.,Polynomial model, ATP synthesis and

metabolism, cell membrane permeability

中文摘要

ABSTRACT

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

§1.1 冷冻食品的研究进展....................................................................................................... 1

§1.1.1 冷冻食品的种类和特点........................................................................................ 1

§1.1.2 冷冻肉品在储藏期间的品质变化........................................................................ 1

§1.1.3 冷冻食品的保鲜机理............................................................................................ 3

§1.2 肉类食品的微生物安全................................................................................................... 3

§1.2.1 肉类食品的安全现状............................................................................................ 4

§1.2.2 肉制品的腐败菌.................................................................................................... 5

§1.3 微生物损伤及修复研究现状........................................................................................... 6

§1.3.1 微生物损伤............................................................................................................ 6

§1.3.2 微生物冷冻损伤类型............................................................................................ 7

§1.3.3 损伤修复培养基.................................................................................................... 7

§1.3.4 损伤修复方法的研究............................................................................................ 8

§1.4 肉类预测微生物学应用进展........................................................................................... 8

§1.4.1 预测食品微生物学的发展.................................................................................... 8

§1.4.2 预测微生物的生长与失活模型............................................................................ 9

§1.4.3 微生物预测模型在肉类工业中的应用.............................................................. 10

§1.5 本研究主要内容和意义................................................................................................. 10

§1.5.1 课题的主要内容.................................................................................................. 11

§1.5.2 课题的意义.......................................................................................................... 11

第二章猪肉解冻过程中损伤型气单胞菌的检测............................................................... 13

§2.1 引言................................................................................................................................. 13

§2.2 实验材料......................................................................................................................... 13

§2.2.1 冷冻猪肉原料及菌种接种.................................................................................. 13

§2.2.2 培养基与试剂...................................................................................................... 13

§2.2.3 仪器与设备.......................................................................................................... 14

§2.3 实验方法......................................................................................................................... 14

§2.3.1 气单胞菌固体修复方法的优化.......................................................................... 14

§2.3.2 选择培养基的优化.............................................................................................. 14

§2.3.3 液体修复方法...................................................................................................... 14

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摘要：

猪肉解冻中损伤型气单胞菌的检测及修复机制摘要冷冻肉是国家调节肉制品市场的重要手段之一，是肉类产品在进出口贸易和国内地区间流通的主要产品形态，也是各种肉类加工企业的原料。冷冻肉解冻后的安全以微生物影响最为显著。肉类经过包括冷冻在内处理后，一部分微生物会死亡，一部分可能存活，另一部分可能会亚损伤。肉类中的微生物经过冷冻冷藏处理后，可遭受不同程度的亚致死性损伤而成为损伤菌，损伤菌在适宜的条件下（如解冻）可进行修复，重新具有正常未损伤细菌的生理生化特性，恢复其对选择性物质的耐受性，可重新具有产毒、致病性。传统检测微生物方法假阴性较高，易于对失活和残存两类微生物计数，而亚损伤一类微生物很难在选择性培养基...

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