摘要
PEF技术是一种非热技术，PEF处理是在室温下进行的，处理时间小于1s，并且PEF技术在很大程度上减少了食品感官和物理特性的有害变化。自20世纪90年代初期，国外开始对非热杀菌技术（冷杀菌）在食品工业中的应用研究进入高潮，PEF杀菌是研究的热点，它是通过高强度脉冲电场瞬时破坏微生物的细胞膜使细菌致死，由于利用高电位而非电流杀菌，因此杀菌过程中的温度低（最高温度小于50℃），从而可以避免热杀菌带来的副作用。
PEF技术主要是利用脉冲电场对微生物细胞的电刺激、电融合、以及可逆电穿孔，处理的强度均比较低。为这种新兴的生物物理技术，提供了一个更广阔的作用对象。近几十年来，PEF技术在食品工业的应用逐渐成熟和完善，主要应用于苹果汁、橘汁等处理，牛奶杀菌处理、鸡蛋制品处理等等，但最近在有效成分的提取方面有较多的应用，因此探讨其作用机理，为更好的促进PEF技术的推广和应用奠定基础。
PEF技术具有非热、作用时间短、效率高、操作简便、成本低等突出的优势，更重要的是其适用范围极广，在工业、农业、医学、临床以及许多基础学科研究的应用前景是十分可观。本研究的创新之处就在于首次把PEF浸提技术从工程提取、物理溶出和数学模型的角度研究，为PEF技术理论研究奠定了基础。因此，本研究不仅具有很大的应用价值，而且具有更重要的理论创新意义。
PEF技术的应用研究已广泛开展，但在食品工业方面中，还有一些问题需要解决。而针对不同系统、不同物料还没有建立正确的数学模型；科学解释和处理各影响因素的互作效应也不能在数学模型中科学体现。这都极大地限制了PEF技术在食品工业中的应用。本文即以大豆油脂、苹果渣胶、淋巴细胞和K562细胞等为研究对象，通过高电压脉冲电场作用，从中提取大豆油脂、苹果果胶及细胞的微观作用效果等研究PEF的作用机理，以使这种方法更好的投入到实际生产中，为高电压技术的发展奠定坚实的基础。
本文进行了PEF浸提机理研究，主要包括以下四个方面
（1）比较了几种大豆油脂的浸提溶剂，从油脂的得率、色泽、气味等因素考虑，选石油醚作为主要溶剂。通过单因素试验，得到高电压脉冲电场对大豆油的提取效果随脉冲数的增加显著增加。在脉冲数为5时提取率明显增加，之后随着脉冲数的增加大豆脂肪提取率增加且较平稳。随场强的增加显著增加，且在5kv/cm时达到最大值。之后随着场强的增加大豆脂肪提取率略有减少，且逐渐趋于恒定。通过正交试验发现，对大豆油脂提取影响最大的为脉冲数，且为显著性水平为0.05下的显著因素，其次为场强，为显著性水平0.25下的显著因素，料液比，为显著性水平0.25下的显著因素。确定的最佳参数附近安排二次通用旋转组合设计，建立了数学模型，回归方程：
Y=19.86310+0.05152x_2+0.10205x_3-0.14606x_1~2-0.08066x_2~2-0.50492x_3~2+0.11375x_1x_2+0.19875x_2x_3
得出了最佳的参数组合：脉冲数=47.58，场强=19.2kv/cm，石油醚：豆粉=11:1，达到了精确寻优。
（2）通过单因素分析，分别研究了电场强度、脉冲数、pH值、料液比、温度对PEF法提取苹果渣果胶提取效果的影响。通过对电场强度、pH值、脉冲数、料液比、温度五个因素取合理水平进行正交试验。利用PEF技术提取苹果渣果胶时，电场强度、pH值、脉冲数和料液比均是显著因素。其中，电场强度是最主要的影响因素，其显著性水平为0.01；其次为脉冲数，显著性水平为0.01；pH值的显著水平为0.05；料液比的显著性水平为0.05；通过正交试验确定电场强度、pH值、脉冲数、料液比及温度的最优水平分别为15kV/cm，3，10，1:19，62℃对该条件进行验证实验，苹果渣果胶得率为14.12%。
（3）针对脉冲电场非热效应造成微生物细胞膜电穿孔的特点，采用高电压脉冲电场设备，对血液中K562肿瘤细胞和淋巴细胞进行作用，试验结果表明，高电压脉冲电场能够诱导K562肿瘤细胞凋亡。
经过PEF处理后的K562细胞，细胞膜发生裂隙，细胞内出现气泡，线粒体膨大，胞浆内容物与大量细胞器外溢，核内染色质向核周边聚集逐渐形成凋亡小体，胞内的染色质趋边，细胞形成拉碎状，染色质呈凝固状，胞质空化。细胞膜结构破坏，厚度变薄，细胞无法保存正常的内环境，从而出现不可逆的损伤；利用AV/PI双标记流式细胞仪定量检测K562的四种亚群细胞：活细胞、早期凋亡细胞、晚期凋亡细胞与损伤细胞的分布结果，发现高电压脉冲电场诱发K562细胞凋亡的效果明显。利用FCM检测发现：K562肿瘤细胞的AnnexinV-FIT和PI双阳性细胞数由脉冲数4个时的2.24%（P＜0.05）提高到10个时的8.42%（P＜0.01），由此得到脉冲个数对K562肿瘤细胞的影响趋势，接近呈线性关系。在场强15kV/cm以下，对K562肿瘤细胞的影响，呈现平稳增长的趋势，斜率较小，在15kV/cm~20kV/cm区域，K562细胞凋亡发生剧烈变化，呈现陡增趋势，并在25kV/cm以后K562肿瘤细胞凋亡增长平缓，凋亡细胞数可以达到细胞总数的86.81%（P＜0.01）；淋巴细胞的凋亡率随脉冲数的增加而增加，并在4个脉冲数以后出现较为明显的变化，当脉冲个数为10个时，高电压脉冲电场对淋巴细胞的杀伤达到最大值；在0~20kV/cm区域，淋巴细胞的凋亡随场强的增加而增加明显，当场强达到20kV/cm时，淋巴细胞的凋亡进入平缓区，但在30kV/cm出现最大凋亡率。
（4）通过PEF对电介质的极化效应研究发现，细胞可以看成是偶极子式的极化效应，并同时伴有轻微的热效应，这与电场的频率（脉冲数）变化有关。PEF作用下K562细胞膜和胞腔受到巨大的影响，对膜造成的影响分析为穿孔效应。通过电导率的测定，验证了PEF对K562细胞溶液的作用。当电场强度小于15kV/cm，时电导率维持在较低值，平稳变化，此时，电场强度不足以引起细胞膜的变化，当场强提高到20kV/cm，电导率迅速增加，说明细胞膜开始被破坏的数量剧增，引起细胞内物的外溢，当继续增加场强至30kV/cm时，电导率变化不大，说明在20-30kV/cm之间，造成了细胞膜的不可逆破坏，继续增加场强电导率变化微小。根据固液两相浸提的原理，提出了PEF浸提技术的数学模型，并首次提出了多通道缩核浸提理论。PEF的作用恰恰极大提高了传质Biot数，从而打破了传统意义上的固液浸提过程，加速了传质过程。
Recently, non-thermal pasteurization of liquid stuff by using a high intensitypulsed electric fields (PEF) is a new technology. High intensity pulsed electricfields have the merits of uniformity in conducting heat, short processing time, lowenergy consumption and low quantity of heat. The technology of PEF is one of theprimary non-thermal pasteurizations. At present, the research in applying highintensity pulsed electric fields is mostly on food pasteurization, which has got somevery good effects in pasteurization.
This paper, combining the development of the PEF, manufactured a highintensity pulsed electric fields, and changed the technical parameters of the PEFsetting with the pasteurization mechanism by PEF, to selectively induce apoptosisof the K562leukemia cells and lymphocyte cells. Soybean of Northeast was usedas the main raw materials for lixiviating and extracting experiments of soybean oil,as a means to advanced high intensity pulsed electric fields, to test high intensitypulsed electric fields and to determine the related parameters. In order to providethe technology support of the pomace integrated utilization and pectin extraction,the research focused on the key problems of pectin extraction.
Through extraction by high intensity pulsed electric fields, we finally obtainedthe main findings summarized as follows:
1. PEF is found a highly efficient, non-thermal, continuous operationtechnology. It was found that the extraction rate of soybean oil increasedsignificantly with the increase of pulse by orthogonal test. The extraction rate wasincreased in the pulse at5. After that along with the increase in the pulse, soybean oil extraction rate increased and became more stable. Increased significance withthe increase in field reached the maximum in term of5kv/cm. Along with theincrease in field soybean oil extraction rate decreased slightly, and graduallybecame invariableness. It was found that the major factor affecting soybean oilextraction was pulse number by orthogonal test. It was the notable factor whoselevel of significance was0.05. Next one is field strength, and it was also thenotable factor whose level of significance was0.25and soybean: isopropanol wasalso the notable factor whose level of significance was0.25.
2. The electric field intensity, pH, pulse number, stuff and liquid ratio andtemperature were the remarkable factors by PEF extraction pectin from pomace.And the electric field intensity was the most important factor. Then it was followedby pulse number; and pH was0.05, stuff and liquid ratio was0.05. The optimumparameter of electric field intensity, pH, pulse numbers, stuff and liquid ratio aredetermined by orthogonal test, i.e.
3. When observing the erythroleukemia K562cells, which was induced by thehigh intensity pulsed electric fields detected with transmission electron microscope,we may find many vacuoles and crannies in cells, mitochondria swelling, vacant,shelvse of the inner membrane break, the hetero-chromatins are on the edge,nuclear pycnosis, nuclear fragmentation. The ultra-structure approves that the highintensity pulsed electric fields can make huge damage in K562cells and induceK562cells apoptosis. From the analysis of orthogonal array, we know when weinduce the K562cells apoptosis by the high intensity pulsed electric fields, the PEFstrength and pulse number are significant factors, therefore, the PEF strength isdominant and pulse extent is not significant. The best parameter combination isA1B3C3in the apoptosis of K562cells by PEF, which is the pulse extent, is1s,the PEF strength is25kV/cm, the pulse number is10and the PEF pulse is30Hz.The apoptosis rate is59.65%in the best condition. From the observation in FCM,we can find that the apoptosis rate of K562cells increases gradually along with thehoist of the PEF pulse number, when the PEF strength and pattern’s velocity offlow hold the line, and the biggest apoptosis rate of the K562cells is59.13%, P＜ 0.01, when the PEF pulse number is10. Meanwhile The apoptosis rate oflymphocyte cells achieve8.42%in10PEF pulse number from2.24%in4PEFpulse number, and when the pulse number is10, there is significant difference, P＜0.01. From the graphs of the K562cells and lymphocyte cells induced by the PEF,we can be conscious that the apoptosis rate of the K562cells and lymphocyte cellsis close in the change of the PEF pulse number, when the PEF pulse number is10,the difference in apoptosis rate of the K562cells and lymphocyte cells is thebiggest. When the PEF pulse number and pattern velocity of flow hold the line, theapoptosis rate of the K562cells increases along with the PEF strength, from the2.73%in0kV/cm PEF strength to86.81%in30kV/cm, and after20kV/cm PEFstrength, the apoptosis rate of the K562cells tends to immovability, which accountsfor the apoptosis action in the K562cells induced by the PEF is very notable.Besides in the same conditions, the apoptosis rate of lymphocyte cells alsogradually increases along with the PEF strength, when the PEF strength is20kV/cm or bigger, the apoptosis curve of lymphocyte cells tends to be smoothedline, and there is the biggest apoptosis rate47.08%in30kV/cm, P＜0.01.
4. A theoretical analysis of PEF mechanism modeling was studied.Shrinking-core model for multi-passage was advanced and Dynamic models wereestablished and it was validated for the solid theoretical foundation for the future ofthe widely used of PEF. It is the least20kV/cm electric field intensity; more2pulsenumbers to damage cell membrane non-reversibly. Electric field intensity isdecisive factor to damage K562cell membrane, and the pulse numbers decides theexistent duration of cell membrane structural changes. But it is improper toincrease the electric field intensity and pulse numbers overly.
In summary, PEF technology has the advantages of non-thermal, reactionspeed quick, efficient, easy to operate and low cost. It has significant social benefit and economic benefit.
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