摘要
高压脉冲电场(Pulsed electric field，简称PEF)是非热食品处理技术中最具有发展前景的技术，也是近年来国内外食品加工行业研究的热点问题之一。目前较多的研究集中在杀菌的机理和动力学方面，高压脉冲电场对酶影响的相关研究开展的较少，酶在脉冲电场作用下的活性变化机理也尚不清楚。研究者只是在对酶失活的比率进行测试时提出了几种失活可能性的假说，并没有得到相关的论证和深入的研究。
本研究使用大连理工大学设计的高压脉冲系统，选取热稳定性较高的辣根过氧化物酶(POD)为目标酶，从理论和实验两方面对高压脉冲电场处理条件下酶失活和酶构象的改变进行了系统的研究，并应用计算机分子模拟技术形象地模拟了酶三级构象的改变。从而对先前进行的有关酶失活动力学的研究进行了验证。
1、本实验使用的高压脉冲电场设备，电压范围0～22kV；脉冲宽度5～20μs；采用co-field连续处理室，流速范围5～100ml／min；脉冲周期10～1KHz；单极脉冲；波型为方波。
2、PEF处理可以钝化POD活力：方波条件下，脉冲宽度为17μs，PEF电场强度对POD酶失活有显著影响(P<0．01)，随脉冲电场强度(0 kV／cm～20kV／cm)的增强，POD酶活钝化效果增强。动力学方程组，(?)，能够描述高压脉冲电场作用与酶失活的关系，且酶失活过程存在有临界场强E_c(10．12±0．34 kV／cm)。
3、尺寸排阻色谱分析表明：高压脉冲电场条件下，POD的蛋白质肽链没有发生断裂，即POD的一级结构未发生明显改变。
4、红外光谱分析表明：高压脉冲电场条件下，POD的二级结构单元(α-螺旋、β-折叠、β-转角和无规卷曲)随着电场强度的增加，呈非单调型变化。并且高压脉冲电场的作用，使POD二级结构单元中的β-转角和无规卷曲向α-螺旋和β-折叠转化。
5、荧光光谱分析表明：高压脉冲电场处理POD可以使其荧光强度产生不同程度的下降，从而改变了POD的局部三级构象，并且电场强度越大，作用越明显。研究还表明，荧光淬灭过程存在与酶失活过程相同的临界场强E_c，说明酶三级构象改变与酶活改变关系密切。
6、计算机分子模拟分析形象地验证表明了：高压脉冲电场条件未改变POD分子片段的整体结构，但改变其部分基团的构象。
Pulsed electric field (PEF) is a very promising non-thermal food processing technology, and it has become the hot spot of food industry recently. Currently most researches focused on the mechanism and kinetics of microbial inactivation, however, few researches related to the effects of PEF on enzymes, and the mechanism of enzyme inactivation under PEF is still unclear. Several hypotheses about the inactivation of enzyme were proposed but these hypotheses still has not been proved yet.
PEF treatment was performed by using a laboratory scale pulse generator system (designed by Dalian University of Technology). The relative residual activity of commercial enzymes of horseradish peroxidase (POD) which is thermal resistance was investigated. The inactivation and conformational transitions of POD by PEF were studied systematically from theories and experiments. The change in tertiary structure of POD under PEF also was studied by computer molecular imitation. By doing so the enzyme inactivation kinetics studied previously was verified.
1、The PEF-treatment system consisted of a square wave generator, a co-field tubular treatment chamber and a cooling system. The voltage, pulse duration and pulse frequency were available from 0 to 22 kV, 5 to 20μs and 10 to 1000Hz respectively.
2、The activity of POD declined significantly after PEF treatment: the parameters in the experiment included square wave pulses of 17μs, electric field strength (0 kV/cm～20 kV/cm). The results showed that the inactivation of POD was significantly (P<0.01).And with the increasing of electric field strength, the inactivation effect of POD enhanced.
The kinetic equation (?) may describe therelationship between electric field strength and relative activities of POD. The kinetic equation showed that the critical electric strength (E_c) of POD existed, which was 10.12±0.34 kV/cm.
3、Measured by size exclusion chromatography, the result showed that polypeptide chain of POD was not fractured by PEF treatment which meant the primary structure of POD did not change after PEF treatment.
4、Measured by infrared spectrophometry the result showed that the secondary structure units of POD, including a-helix,β-strands, P-turn and random coil, presented nonmonotonic variation, with the increase of electric strength theβ-turn and random coil were transformed into a-helix andβ-strands.
5、Measured by spectrofluorimetry, the result showed that the fluorescence intensity of POD declined in different degree after being treated by pulsed electric fields. And then it led to local variations on tertiary structure of POD. The higher the electric field intensity, the more obvious the effect is. The result also showed that just as in the process of inactivation, there was a same critical electric strength (E_c) during the fluorescence quenching which meant the relationship between the tertiary structure variation and the inactivation of POD was close.
6、Computer molecular imitation vividly verified and showed that the whole molecular structure of POD did not change, but part of the groups of POD changed under PEF treatments.
引文
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