鲜榨苹果汁非热杀菌技术与设备的研究
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摘要
本论文研究内容是国家科技部“十五”重大科技专项一农产品深加工技术与设备研究开发课题《优质鲜榨苹果汁和浑浊型苹果汁加工关键技术研究》的一部分。
非热杀菌技术是制约我国鲜榨苹果汁生产和销售的重大关键技术难题，至今未能提出有效实用的解决方案。本论文主要针对几种在鲜榨苹果汁生产中具有应用前景的非热杀菌技术，为最大限度保留苹果汁的新鲜风味和热敏性营养成分，提高杀菌工艺效果，保证鲜榨苹果汁的卫生安全质量，延长产品货架期，从榨汁苹果原料中微生物的种类分析鉴定入手，将二氧化氯杀菌与清洗过程相结合，有针对性的研究其对原料中主要致病菌和腐败菌的杀菌规律；从实用和高效两方面考虑，重点开展脉冲强磁场杀菌和陶瓷膜过滤除菌技术研究，为优化杀菌／除菌工艺条件，控制鲜榨苹果汁中的微生物数量，探索了杀菌／除菌过程中主要影响因素对杀菌／除菌效果的影响规律，从细胞膜结构破坏和DNA损伤等方面推测脉冲强磁场杀菌技术的机理，并建立了陶瓷膜除菌过程膜污染机制的数学模型；同时根据已获得的杀菌规律和数据确定了脉冲强磁杀菌设备和陶瓷膜过滤除菌设备制造参数和样机设计方案，最终利用数学模拟方法描述了环境因素对鲜榨苹果汁中的常见致病菌和腐败菌生长状态的影响，建立了致病菌的生存/死亡概率模型，控制致病菌的存活，通过腐败菌最大生长速度的数学模型对鲜榨苹果汁的货架期进行预测。
本论文主要包括榨汁苹果原料表面微生物种类的分离和鉴定、二氧化氯对苹果原料的清洗与杀菌规律、脉冲强磁场杀菌规律与机理研究、陶瓷膜过滤除菌技术与膜污染机制、非热杀菌设备的设计、微生物在鲜榨果汁中的生存状态模拟等内容。主要结论如下：
1．榨汁苹果原料中微生物是鲜榨苹果汁中致病菌和腐败菌的主要来源。为有针对性地对苹果原料和鲜榨苹果汁中的主要污染菌采取有效的清洗与杀菌措施进行控制，利用API细菌鉴定系统，结合细菌的菌落形态特征，对榨汁苹果原料中的微生物种类和数量分布进行了研究。结果发现，烈性致病菌单增李斯特(Listeria monocytogenes)和大肠杆菌O157：H7在榨汁苹果原料中均有检出，微生物在榨汁苹果表面的数量分布在10~5～10~7cfu／g，引起果汁腐败变质的主要微生物是酵母菌、乳酸菌和芽孢杆菌，并对它们的种类进行了鉴定，共鉴定出酵母菌6种、乳酸菌3种、芽孢杆菌2种。
2．为降低鲜榨苹果汁中的微生物数量，减轻后续果汁非热杀菌过程的负担，原料中微生物数量必须尽可能降低到最低程度。本研究采用四因素中心组合试验设计(Central Composite Design)模型，对从榨汁苹果表面分离的单增李斯特氏菌(Listeria monocytogenes)、大肠杆菌O157：H7(E. coli. O157：H7)和几种典型腐败菌进行了二氧化氯杀菌实验，并将原料清洗与杀菌过程相结合，以杀菌效果(以杀菌效率的负对数表示，即log(N／N_0)为响应值，研究了二氧化氯溶液浓度、杀菌温度、处理时间和杀菌液pH值等因素对杀菌效果的影响，分析归纳了杀菌主要影响因素与杀菌效果的数学模型，为实现生产过程的在线自动优化控制提供了计算依据。
3．通过脉冲强磁场对主要致病菌和腐败菌杀菌效果的研究，发现影响杀菌效果的主要因素是磁场强度、脉冲次数、杀菌温度、介质pH值等。介质pH值对杀菌效果影响明显，较低pH值(＜4.5)均可提高对金黄色葡萄球菌、大肠杆菌和沙门氏菌的杀菌效果；介质温度
越高杀菌效果越好，提高介质温度也可以提高杀菌效果;随着磁场强度和脉冲数的增加，杀
菌效果提高;脉冲磁场对实验研究的几种微生物作用效果并不一致，对金黄色葡萄球菌的作
用最强，而对霉菌抱子和芽抱杆菌的作用效果相对较差。采用扫描电镜、流式细胞仪和单细
胞凝胶电泳技术揭示了脉冲磁场对大肠杆菌细胞膜和DNA的损伤情况，结果证实，脉冲磁
场对大肠杆菌的细胞膜结构和核内DNA有损伤作用。
4.为保证鲜榨苹果汁卫生指标达到要求，应用膜孔径为1 00nm的陶瓷膜对果胶酶酶解
后的鲜榨苹果原汁进行了过滤澄清与除菌效果研究。通过对经酶解除果胶的鲜榨苹果原汁进
行跨膜压力、膜面流速、过滤温度与膜通量关系的研究，初步确定了陶瓷膜过滤除菌的最佳
条件，经试验测定，本研究选用的loonm陶瓷膜过滤后可使细菌总数下降9个对数值，滤
后果汁澄清度得到明显提高，而果汁的各项质量指标如pH值、总酸、糖度等均未发生明显
变化;经过4℃，8℃，25℃和35℃28d贮存观察，果汁中均无微生物生长;4℃和8℃低温
贮藏的苹果汁其理化指标变化较小。通过研究压力、温度对膜通量的影响，分析了过滤过程
膜污染机制，确定了其适用的数学模型，证明选用1 00 nm陶瓷膜过滤果汁中的颗粒物质只
是对膜孔的覆盖，降低膜过滤面积，而不会对膜孔造成不可恢复性阻塞。
5.本研究对脉冲强磁杀菌设备和陶瓷膜过滤除菌设备进行了系统设计，并制作完成脉冲
强磁杀菌设备和陶瓷膜过滤除菌设备实验样机，根据选用的非热杀菌方法的特点，提出鲜榨
苹果汁非热杀菌技术方案，确定了鲜榨苹果汁生产过程的杀菌工艺和流程。
6.应用微生物预测技术(Predictive MicrobiologyT’e chnol
This paper was a part of the item - "The Study on Key Technologies of High-Quality Fresh Apple Juice and Concentrated Cloudy Apple Juice" in the Tenth-Five-Year Science and Technology Plan of China.
The non-thermal technology was the pivotal problem, which restricted the producing, and marketing of fresh apple juice in China. Up to now, there was no effective and practical method to resolve the processing problems. This study focused on the several prospective non-thermal inactivation technologies to keep the natural flavor and nutritional qualities of the apple juice. The presence of an inactivation processing unit operation aiming at microbial destruction was of primary importance to ascertain safety and stability of food. After the isolation and identification of the primary pathogenic and septic bacteria in the raw apple, the bactericidal effects of chlorine dioxide (ClO2) solution treatments on the microbia potted on pulp surface of apples were investigated to explore the inactivation rule. From the point of view of practicality and high efficiency, the effect of main parameters on the inactivation or sterilization efficiency of the selected pulsed magnetic field and membrane filtration technology
were explored to suppress microbial growth, and reduce or eliminate the microbial load. In this part, the damages inflicted on the membrane and DNA of Escherichia coli cells treated by pulsed magnetic field was investigated to reveal the mechanism of the inaction. The flux behavior and mechanism of ceramic membranes was examined during cross-flow ultrafiltration (UF) of depectinized fresh apple juice to evaluate the effects of transmembrane pressure (TMP) and temperature on membrane fouling. On the basis of data and rules induced from the experiments, the scheme to produce fresh apple juice was brought forward and the pilot scale equipment for non-thermal inactivation was designed. By the theory of predictive microbiology technology, predictive models for the lag phase and the maximum specific growth rate of a specific spoilage organism in fresh apple juice was developed and the logistic model to evaluate the growth/no growth boundary for the pathogenic bacteria in relation to the considered independent vari
ables was also established. From the studies above, the conclusions were:
1. The raw material apple was the main resource of the pathogenic and septic bacteria of the fresh apple juice. To sanitize the microbia on the apple surface effectively, the clones of bacteria and yeasts were isolated and identified respectively by their clone physiological characteristics and the API system. The microbial load in the raw apples was between 105 and 107 cfu/g. The pathogenic bacteria such as Listeria monocytogenes and E.coli O157:H7 were found in the raw apple. The primary kinds of septic microbia were yeast, lactic bacteria, and spores.
2. Production of safe fresh apple juice includes scrutinizing raw materials and reducing or eliminating the microbial load by chlorine dioxide (ClO2) solution. The inactivation rule of chlorine dioxide (C1O2) treatments on the microbiology was investigated in the process of
apple rinsing. The Central Composite Design model was used to describe the influence of main, quadratic, and cross-product effects of the environmental factors such as concentration of chlorine dioxide (C1O2), temperature, treating time and pH on the response of inactivation efficiency (log (NO/N)).
3. The major factors on the inactivation efficiency of the pulsed magnetic field were strength, the number of pulses, temperature and pH of the medium. The pH of the medium effected the inactivation markedly. In low pH (<4.5), the efficiency of inactivation was higher for the treatment of Escherichia coli, Salmonella enterica and Sarcina aurea Henrici. The increase in the strength and the number of the pulses increased the efficiency of inactivation. The inactivation efficiency of pulsed magnetic field was not consistent to all kinds of microbiology. In the investigated microbiologies t
非热杀菌技术是制约我国鲜榨苹果汁生产和销售的重大关键技术难题，至今未能提出有效实用的解决方案。本论文主要针对几种在鲜榨苹果汁生产中具有应用前景的非热杀菌技术，为最大限度保留苹果汁的新鲜风味和热敏性营养成分，提高杀菌工艺效果，保证鲜榨苹果汁的卫生安全质量，延长产品货架期，从榨汁苹果原料中微生物的种类分析鉴定入手，将二氧化氯杀菌与清洗过程相结合，有针对性的研究其对原料中主要致病菌和腐败菌的杀菌规律；从实用和高效两方面考虑，重点开展脉冲强磁场杀菌和陶瓷膜过滤除菌技术研究，为优化杀菌／除菌工艺条件，控制鲜榨苹果汁中的微生物数量，探索了杀菌／除菌过程中主要影响因素对杀菌／除菌效果的影响规律，从细胞膜结构破坏和DNA损伤等方面推测脉冲强磁场杀菌技术的机理，并建立了陶瓷膜除菌过程膜污染机制的数学模型；同时根据已获得的杀菌规律和数据确定了脉冲强磁杀菌设备和陶瓷膜过滤除菌设备制造参数和样机设计方案，最终利用数学模拟方法描述了环境因素对鲜榨苹果汁中的常见致病菌和腐败菌生长状态的影响，建立了致病菌的生存/死亡概率模型，控制致病菌的存活，通过腐败菌最大生长速度的数学模型对鲜榨苹果汁的货架期进行预测。
本论文主要包括榨汁苹果原料表面微生物种类的分离和鉴定、二氧化氯对苹果原料的清洗与杀菌规律、脉冲强磁场杀菌规律与机理研究、陶瓷膜过滤除菌技术与膜污染机制、非热杀菌设备的设计、微生物在鲜榨果汁中的生存状态模拟等内容。主要结论如下：
1．榨汁苹果原料中微生物是鲜榨苹果汁中致病菌和腐败菌的主要来源。为有针对性地对苹果原料和鲜榨苹果汁中的主要污染菌采取有效的清洗与杀菌措施进行控制，利用API细菌鉴定系统，结合细菌的菌落形态特征，对榨汁苹果原料中的微生物种类和数量分布进行了研究。结果发现，烈性致病菌单增李斯特(Listeria monocytogenes)和大肠杆菌O157：H7在榨汁苹果原料中均有检出，微生物在榨汁苹果表面的数量分布在10~5～10~7cfu／g，引起果汁腐败变质的主要微生物是酵母菌、乳酸菌和芽孢杆菌，并对它们的种类进行了鉴定，共鉴定出酵母菌6种、乳酸菌3种、芽孢杆菌2种。
2．为降低鲜榨苹果汁中的微生物数量，减轻后续果汁非热杀菌过程的负担，原料中微生物数量必须尽可能降低到最低程度。本研究采用四因素中心组合试验设计(Central Composite Design)模型，对从榨汁苹果表面分离的单增李斯特氏菌(Listeria monocytogenes)、大肠杆菌O157：H7(E. coli. O157：H7)和几种典型腐败菌进行了二氧化氯杀菌实验，并将原料清洗与杀菌过程相结合，以杀菌效果(以杀菌效率的负对数表示，即log(N／N_0)为响应值，研究了二氧化氯溶液浓度、杀菌温度、处理时间和杀菌液pH值等因素对杀菌效果的影响，分析归纳了杀菌主要影响因素与杀菌效果的数学模型，为实现生产过程的在线自动优化控制提供了计算依据。
3．通过脉冲强磁场对主要致病菌和腐败菌杀菌效果的研究，发现影响杀菌效果的主要因素是磁场强度、脉冲次数、杀菌温度、介质pH值等。介质pH值对杀菌效果影响明显，较低pH值(＜4.5)均可提高对金黄色葡萄球菌、大肠杆菌和沙门氏菌的杀菌效果；介质温度
越高杀菌效果越好，提高介质温度也可以提高杀菌效果;随着磁场强度和脉冲数的增加，杀
菌效果提高;脉冲磁场对实验研究的几种微生物作用效果并不一致，对金黄色葡萄球菌的作
用最强，而对霉菌抱子和芽抱杆菌的作用效果相对较差。采用扫描电镜、流式细胞仪和单细
胞凝胶电泳技术揭示了脉冲磁场对大肠杆菌细胞膜和DNA的损伤情况，结果证实，脉冲磁
场对大肠杆菌的细胞膜结构和核内DNA有损伤作用。
4.为保证鲜榨苹果汁卫生指标达到要求，应用膜孔径为1 00nm的陶瓷膜对果胶酶酶解
后的鲜榨苹果原汁进行了过滤澄清与除菌效果研究。通过对经酶解除果胶的鲜榨苹果原汁进
行跨膜压力、膜面流速、过滤温度与膜通量关系的研究，初步确定了陶瓷膜过滤除菌的最佳
条件，经试验测定，本研究选用的loonm陶瓷膜过滤后可使细菌总数下降9个对数值，滤
后果汁澄清度得到明显提高，而果汁的各项质量指标如pH值、总酸、糖度等均未发生明显
变化;经过4℃，8℃，25℃和35℃28d贮存观察，果汁中均无微生物生长;4℃和8℃低温
贮藏的苹果汁其理化指标变化较小。通过研究压力、温度对膜通量的影响，分析了过滤过程
膜污染机制，确定了其适用的数学模型，证明选用1 00 nm陶瓷膜过滤果汁中的颗粒物质只
是对膜孔的覆盖，降低膜过滤面积，而不会对膜孔造成不可恢复性阻塞。
5.本研究对脉冲强磁杀菌设备和陶瓷膜过滤除菌设备进行了系统设计，并制作完成脉冲
强磁杀菌设备和陶瓷膜过滤除菌设备实验样机，根据选用的非热杀菌方法的特点，提出鲜榨
苹果汁非热杀菌技术方案，确定了鲜榨苹果汁生产过程的杀菌工艺和流程。
6.应用微生物预测技术(Predictive MicrobiologyT’e chnol
This paper was a part of the item - "The Study on Key Technologies of High-Quality Fresh Apple Juice and Concentrated Cloudy Apple Juice" in the Tenth-Five-Year Science and Technology Plan of China.
The non-thermal technology was the pivotal problem, which restricted the producing, and marketing of fresh apple juice in China. Up to now, there was no effective and practical method to resolve the processing problems. This study focused on the several prospective non-thermal inactivation technologies to keep the natural flavor and nutritional qualities of the apple juice. The presence of an inactivation processing unit operation aiming at microbial destruction was of primary importance to ascertain safety and stability of food. After the isolation and identification of the primary pathogenic and septic bacteria in the raw apple, the bactericidal effects of chlorine dioxide (ClO2) solution treatments on the microbia potted on pulp surface of apples were investigated to explore the inactivation rule. From the point of view of practicality and high efficiency, the effect of main parameters on the inactivation or sterilization efficiency of the selected pulsed magnetic field and membrane filtration technology
were explored to suppress microbial growth, and reduce or eliminate the microbial load. In this part, the damages inflicted on the membrane and DNA of Escherichia coli cells treated by pulsed magnetic field was investigated to reveal the mechanism of the inaction. The flux behavior and mechanism of ceramic membranes was examined during cross-flow ultrafiltration (UF) of depectinized fresh apple juice to evaluate the effects of transmembrane pressure (TMP) and temperature on membrane fouling. On the basis of data and rules induced from the experiments, the scheme to produce fresh apple juice was brought forward and the pilot scale equipment for non-thermal inactivation was designed. By the theory of predictive microbiology technology, predictive models for the lag phase and the maximum specific growth rate of a specific spoilage organism in fresh apple juice was developed and the logistic model to evaluate the growth/no growth boundary for the pathogenic bacteria in relation to the considered independent vari
ables was also established. From the studies above, the conclusions were:
1. The raw material apple was the main resource of the pathogenic and septic bacteria of the fresh apple juice. To sanitize the microbia on the apple surface effectively, the clones of bacteria and yeasts were isolated and identified respectively by their clone physiological characteristics and the API system. The microbial load in the raw apples was between 105 and 107 cfu/g. The pathogenic bacteria such as Listeria monocytogenes and E.coli O157:H7 were found in the raw apple. The primary kinds of septic microbia were yeast, lactic bacteria, and spores.
2. Production of safe fresh apple juice includes scrutinizing raw materials and reducing or eliminating the microbial load by chlorine dioxide (ClO2) solution. The inactivation rule of chlorine dioxide (C1O2) treatments on the microbiology was investigated in the process of
apple rinsing. The Central Composite Design model was used to describe the influence of main, quadratic, and cross-product effects of the environmental factors such as concentration of chlorine dioxide (C1O2), temperature, treating time and pH on the response of inactivation efficiency (log (NO/N)).
3. The major factors on the inactivation efficiency of the pulsed magnetic field were strength, the number of pulses, temperature and pH of the medium. The pH of the medium effected the inactivation markedly. In low pH (<4.5), the efficiency of inactivation was higher for the treatment of Escherichia coli, Salmonella enterica and Sarcina aurea Henrici. The increase in the strength and the number of the pulses increased the efficiency of inactivation. The inactivation efficiency of pulsed magnetic field was not consistent to all kinds of microbiology. In the investigated microbiologies t
引文
[1] 边藏丽，杨远荣，熊贻该，等．激光杀菌的实验研究．中国消毒学杂志，1995，12(3)：168
[2] 陈长娥，刘跃进，熊双喜，等．二氧化氯的杀菌研究．湘潭大学自然科学学报，1994，16(2)：45～47
[3] 陈春田，李东力，刘希真，等．二氧化氯作用后细菌中DNA漏出的实验观察．中国公共卫生，2002，18(1)：57
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