【作者】 方婷；

【导师】 陈锦权；

【作者基本信息】 福建农林大学 ， 农产品加工及贮藏工程， 2008， 博士

【摘要】 本文从高压脉冲电场对微生物的杀灭效果入手，通过建立高压脉冲电场强度和处理时间与微生物相对存活率之间的一级动力学方程，从而获得微生物的临界场强E_C和模型的回归系数k；将PEF杀菌技术应用于杀灭橙汁中大肠杆菌O157：H7和致病性沙门氏菌，并对比PEF杀菌与热力杀菌对橙汁品质的影响；在此基础上，通过Laplace方程研究处理器中电场分布，开展PEF处理室改进及其微生物杀灭效果研究。所开展的研究简介如下：1．本论文所使用的高压脉冲电场杀菌试验装置，主要由高压脉冲发生系统、同芯连续式处理室（co-field chamber）、蠕动泵、温度探头、温度记录仪、物料贮罐和冷却部分构成，其主要参数如下：电压范围0～15 kV；脉冲宽度5～20μs；连续处理室，流速范围5～100 mL／min；脉冲周期10～1KHz；单极脉冲；波型为方波。2．使用内径2mm电极处理室，流量u=10mL／min，脉冲电场频率为32Hz，脉冲宽度τ=17μs，脉冲电场处理的脉冲数为80个。高压脉冲电场处理啤酒酵母、青霉、大肠杆菌和金黄色葡萄球菌，脉冲电场强度分别为2．5 kV／cm、5kV／cm、10kV／cm、12．5kV／cm、15kV／cm、17．5kV／cm以及20 kV／cm，研究高压脉冲电场强度对啤酒酵母、金黄色葡萄球菌、青霉和大肠杆菌杀菌效果的影响，其结果如下：（1）随着电场强度和脉冲数增加，高压脉冲电场对微生物的杀灭效果提高。（2）不同对象菌对脉冲电场的承受力不同，革兰氏阴性菌的杀灭效果比阳性菌好，各菌种对高压脉冲电场的承受力从大到小如下：霉菌、金黄色葡萄球菌、大肠杆菌、啤酒酵母。（3）以大肠杆菌为指标菌，研究不同的介质对高压脉冲电场杀菌效果的影响，结果表明：酸性介质条件有利于杀灭细菌。（4）当样液温度分别是25℃、30℃、和35℃时，温度协同高压脉冲电场灭菌的处理效果不是很显著。（5）对象菌所处的生长周期也对杀菌效果也有一定的影响，处于延滞生长期的菌体比处于稳定期和对数生长期的菌体对电场更为敏感。3．通过建立高压脉冲电场强度和啤酒酵母、大肠杆菌和青霉存活率关系的数学模型，得到电场强度对微生物的致死作用符合一个分段函数N/N₀=1 E<E_CN/N₀=exp（-k（E-E_C）） E≥E_C,当电场强度小于临界场强时，不会对微生物产生致死作用，样液中的微生物数量不变，当电场强度大于等于临界场强时，高压脉冲电场对微生物产生致死作用，将试验数据经过一系列数学运算后，可以描绘出电场强度对微生物致死效果的拟合曲线，从而求得这三种菌的临界场强E_C和模型的回归系数k，青霉E_C=1．9893kV／cm>大肠杆菌E_C=1．8085kV／cm>啤酒酵母E_C=0．3339kV/cm，青霉k=0．1139<大肠杆菌k=0．1629<啤酒酵母k=0．4339，临界场强表示微生物对脉冲电场的耐受能力；k是微生物对高压脉冲电场敏感性参数，其值越大，表明微生物的对电场越敏感。k与微生物的种类和介质有关，在本试验中，介质均为无菌水，因此，k的大小与微生物种类有关，更进一步说，是和微生物的大小和细胞壁的组成有关。4．通过微生物存活率与处理时间关系的数学模型S=k₁×e^-k₂t，可知各种微生物对处理时间的耐受程度不一样，青霉k2=0．0036<大肠杆菌k2=0．0142<酵母k2=0．0153，k2是微生物对高压脉冲电场敏感性参数，其值越大，表明微生物的对电场越敏感。三种菌对高压脉冲电场处理时间的耐受能力为：青霉>大肠杆菌>啤酒酵母。5．在室温下，使用内径为3mm的处理室，电极距离为4mm，电场强度为12．5kV／cm，脉冲频率f=128Hz，脉冲宽度脉冲宽度τ=17μs，脉冲数为20个。研究此条件对大肠杆菌O157：H7和致病性沙门氏菌的杀菌效果，结果表明：高压脉冲电场可以有效地杀灭橙汁中的大肠杆菌O157：H7，其菌落数下降了2．12个对数值，而致病性沙门氏菌菌落数下降了4．00个对数值，但是该数值距离商业无菌还有差距，主要是由于在本试验中，高压脉冲电场强度有限，从另一方面来说，橙汁在常规预处理过程中不易产生10²以上的致病菌，这点在我们对橙汁原汁进行检测时得到了验证（未发现致病菌），因此高压脉冲电场是可以用于杀灭酸性介质中的致病菌的。6．在5．所述的高压脉冲电场条件下，可以有效地杀灭鲜橙汁中的微生物，但杀灭效果不及热力杀菌，这是由于在本试验中，受到设备的限制，所采用的电场强度还不够高，仅12．5kV／cm，贮藏120d后，细菌菌落总数和酵母、霉菌菌落总数符合国家标准，这可能是因为鲜橙汁pH值偏低4．0左右，结合低温贮藏可以有效抑制部分微生物的生长繁殖。对比高压脉冲电场杀菌技术和热力杀菌技术对鲜橙汁可溶性固形物、总酸和Vc，结果表明无论是PEF杀菌还是热杀菌对鲜橙汁中的可溶性固形物和总酸影响不大，并且在贮藏过程中这两个指标也没有明显的变化；鲜榨橙汁在经过热杀菌后Vc含量降低7．7％，而PEF杀菌处理鲜橙汁中Vc降低5％，在贮藏120d后，从鲜橙原汁的Vc含量下降最快，其次是热杀菌处理鲜橙汁，最好的是PEF杀菌处理鲜橙汁。可见，PEF杀菌处理与热杀菌处理相比，可有效抑制Vc在贮藏期间的损失。7．应用有限差分法解Laplace方程研究处理室中电场分布，改进连续式同芯处理室。当流量u=30mL／min，脉冲电场频率为128Hz，脉冲宽度τ=17μs，脉冲处理时间为3．92ms。高压脉冲电场处理啤酒酵母、青霉和大肠杆菌，当电场强度为2．5kV／cm、5．0kV／cm、7．5kV／cm、10．0kV／cm、12．5kV／cm和15．0kV／cm时，对比研究改进前处理室（内径18．00mm，电极间距4．00mm）和改进后处理室（内径18．00mm，内有4对厚度为1．00mm的不锈钢导体，电极间距4．00mm）在相同的高压脉冲电场条件下，改进后处理室的杀菌效果明显由于改进前的，当电场强度达到15．0kV／cm时，改进后处理室中啤酒酵母、大肠杆菌和青霉菌的相对存活率仅为0．00、0．01和0．05，而未改进的处理室则高达0．30、0．77和0．76。更多还原

【Abstract】 Based on the killing effect of Pulsed Electric Field（PEF） on microorganisms, the first order kinetics between PEF strength and treatment time and relative survival rate of microorganisms respectively were established with the critical PEF strength Ec and regression coefficients k . Then PEF was applied on killing Escherichia coli O157:H7 and pathogenic salmonella and the effects of PEF and thermal sterilization on orange juice quality were studied. At last, the Laplace equation was used to understand the distribution of electrical field strength in the treatment chamber of a pulse electric field process and improve the co-field chamber. Their effects on PEF sterilization were also investigated. The brief introduction of whole research was as follow:1. The pilot device of PEF was made up of high voltage pulse generator, co-field chamber, peristaltic pump, temperature sensors, temperature recorder, material tank and cooling system. The major parameters of PEF device were as follow: voltage from 0 to 15 kV; pulse duration from 5 to 20μs; flow rate from 5 to 100 mL/min; pulse frequency from 10 to 1000Hz; monopolar pulse and square waveform.2. The co-field chamber with inner diameter 2.00mm, and the PEF system with pulse frequency 32Hz, pulse duration 17μs was used in the research. Saccharomyces cerevisiae CICC 1001, Penicillium citrinum CICC4010 and Escherichia coli CGMCC1.90 were treated by PEF strength 2.5kV/cm, 5.0kV/cm, 7.5kV/cm, 10.0kV/cm, 12.5kV/cm and 15.0kV/cm respectively. The results were as follow:（1） With the strength and pulse number increasing, the killing effect of PEF was improved.（2） Different target microorganisms had different tolerance to PEF. The killing effect of Gram-negative Bacteria was better than Gram-positive Bacteria. The tolerance to PEF ranged from big to small as follow: Penicillium citrinum, Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae.（3） The influence of different mediums on PEF kill effect on Escherichia coli CGMCC1.90 was investigated. The result showed that acid medium could benefit for killing bacteria.（4） When the medium temperatures were 25℃, 30℃and 35℃respectively, the effect of temperature combined with PEF on sterilization was not significant. （5） The growth period of target microorganism would also influence the killing effect. The microorganism staying in arrearage growth period was more sensitive to PEF than that in stationary or logarithmic growth period.3. By establishing the mathematical model of PEF strength and the survival rate of Saccharomyces cerevisiae, Penicillium citrinum and Escherichia coli, the effect of PEF strength on sterilization could be described as a piecewise functionIf the strength （E） was below a critical value （E_c）,no lethal effect can be exerted to the microorganisms, while E was above E_c, there would be lethal effect. The fitting curve of lethal effect could be gained after series of mathematical operation. The critical field strength E_c was 1.9893kV/cm, 1.8085kV/cm and 0.3339kV/cm for P. citrinum CICC 4010, E. coli CGMCC1.90, and S. cerevisiae CICC1001, respectively. And the sensitivity coefficient k was 0.1139, 0.1629 and 0.4339 for P. citrinum CICC 4010, E. coli CGMCC1.90, and S. cerevisiae CICC 1001, respectively. E_c represented the tolerance to PEE and a large k value would suggest that the microorganism be more sensitive to the change in the field strength. In this research, with the same medium, k was relative to the kind of microorganism, furthermore to the size and the construction of microorganism.4. As the mathematical model of treatment time and survival rate could be describedas s = k₁×e^-k₂t , the results showed that the tolerance of microorganisms to thetreatment time was different. The sensitive parameter k2 was 0.0036, 0.0142 and 0.0153 for P. citrinum CICC 4010, E. coli CGMCC1.90, and S. cerevisiae CICC 1001, respectively. A large k₂ value would suggest that the microorganism be more sensitive to PEF.5. Under the room temperature, the co-field chamber with inner diameter 3mm and the distance between two electrodes 4.00mm and the PEF system with strength 12.5kV/cm, pulse frequency 128Hz, pulse duration 17μs, pulse number 20 was adopted for experiment. The killing effect of PEF on Escherichia coli O157:H7 and pathogenic salmonella was studied. The result showed that Escherichia coli O157:H7 decreased for 2.12 log-value while pathogenic salmonella went down for 4.00 log-value. However it’s a long way for commercial sterilization because in our experiment the strength and treatment was limited. In another point, it’s hard to get more than 10² pathogenic bacteria in orange juice which was verified in test. In the matter of fact, PEF could be used to kill the pathogenic bacteria in acid medium.6. PEF can effectively kill microorganisms in the fresh orange juice under conditions of 5, whose effect was inferior to the thermal sterilization. Restricted by the equipment, the electric filed strength was only 12.5kV/cm in this experiment, which was not intense enough. After 120 days storage, the total bacterial, yeast and mold colonies conformed to the national standards. That probably because comparatively low pH value about 4.0 combined with low temperature preservation can effectively inhibited the growth of some microorganisms. Compared the effect on soluble solid content, total acid and Vc content in fresh orange juice by PEF treatment with by thermal sterilization, the result showed that either PEF or thermal sterilization had little effect on the changes the soluble solid content and total acid, and the two indexes didn’t change much during the storage; but Vc content decreased to 7.7% after thermal sterilization compared to 5% after PEF treatment, Vc content decreased fastest in untreated group followed by thermal sterilization, and PEF group was the slowest after 120 days storage. It is concluded that PEF treatment could effectively reduce Vc content losses compared to thermal sterilization during storage.7. Laplace equation solved by finite difference method was adopted to investigate the electric fields distribution in co-field chamber. The flow rate was 30mL/min, the pulse electric frequency was 128Hz, the square pulse duration time was 17μs and the treatment time was 3.92 ms. Saccharomyces cerevisiae CICC1001, Penicillium citrinum CICC4010 and Escherichia coli CGMCC1.90 were treated by PEF strength 2.5kV/cm, 5.0kV/cm, 7.5kV/cm, 10.0kV/cm, 12.5kV/cmand 15.0kV/cm respectively. And effects of unimproved （the inner diameter was 18.0mm and the distance between two electrodes was 4mm） and improved （the inner diameter was 18.0mm, the distance between two electrodes was 4mm, and 4 pairs of stainless steel conductors with thickness of 1 mm were included） chamber on relative survival rate were studied. Under the same PEF conditions the inactivation effect of improved chamber was much better than the conventional chamber design. When the electric filed strength was up to15.0kV/cm, relative survival rates in the improved chamber were 0.00, 0.0 and 0.05, respectively, for Saccharomyces cerevisiae CICC 1001, Escherichia coli CGMCC1.90 and Penicillium citrinum CICC4010, compared to the rates of 0.30, 0.77 and 0.76 in the unimproved chamber.更多还原