二氧化碳协同食品超高压杀菌研究
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摘要
超高压杀菌可以保持食品的营养成分、质构、色泽和新鲜的口感,避免了传统热杀菌对热敏性营养成分、色泽和风味的破坏。然而,要杀死食品中某些耐压的致病菌和腐败菌需要较高的压力(500MPa以上),导致设备造价及运行费用昂贵,阻碍了食品超高压杀菌技术的应用,为了降低杀菌压力,各种超高压协同措施的研究成为热点。其中包括添加天然抗菌化合物如nisin、Lactoperoxidase、CO2等,包括中等温度协同,循环脉冲压力的采用等,但由于抗菌化合物主要作用于革兰氏阳性菌的细胞壁,这类化合物对具有外膜保护的革兰氏阴性菌的作用效果较弱,且违背零添加的原则；而中温协同依然会造成热敏性果汁营养成分和口感的破坏。CO2作为天然的抑菌物质,廉价易得,使用安全,与超高压协同可以提高杀菌效果,以降低杀菌压力,但在超高压过程中应用CO2有协同浓度过低,且不稳定的问题,不能有效提高协同杀菌效果,因此限制了这种协同方法的研究和应用。另外,CO2协同超高压杀菌机理尚未明确,其中包括超高压对细菌细胞的作用、高压下CO2对细胞作用的机理以及二者协同的机理。
本论文以CO2为协同物质,设计和构建一种全新的CO2协同超高压试验方法和装置,实现在设定的CO2浓度协同超高压杀菌,以达到预期的协同效果,并且研究了CO2协同浓度、超高压压力、温度等因素对杀菌效果的影响。同时对CO2协同超高压杀菌的机理进行探索,涉及高压下CO2对细胞膜的渗透作用,对细胞内蛋白质的变性作用等。然后针对果蔬汁中常见的致病菌和腐败菌,采用协同处理对果汁进行巴氏杀菌,探索果汁pH值和基质对CO2协同超高压处理的影响,为超高压技术和工艺的开发和应用提供依据。主要研究成果如下：
1、在细胞悬浮液中添加.3～5.5NL/L CO2,能使金黄色葡萄球菌和大肠杆菌的超高压杀菌压力比无协同杀菌降低200～250MPa。
金黄色葡萄球菌的有效协同杀菌(>7对数级)条件为3.8NL/L CO2、350MPa、30℃、10分钟,大肠杆菌的有效协同杀菌条件为3.2NL/L10分钟,与单独超高压相比,降低了杀菌压力200-250MPa。灭活率随CO2增加而增加,当CO2浓度提高到5NL/L左右时,对杀菌效果的增强呈现饱和趋势。温度显著影响协同杀菌效果,在30℃或更高的温度时,有良好的杀菌效果,在低于20℃时,CO2协同效果降低,适当提高压力,或者采用循环压力也可以得到有效的杀菌效果。作为真正的冷杀菌方法,可以选择处理温度为25-35℃。CO2协同超高压造成细菌灭活的同时,也增加亚致死损伤。CO2协同对动态压力的杀菌效果也有一定的增强作用,但与高静压不同,高压均质处理没有造成细胞亚致死损伤。
2、CO2主要是通过破坏细胞膜完整性、降低细胞内pH、促进蛋白质聚集而发挥协同杀菌作用。
通过细胞扫描电镜(SEM)图像观察到协同处理的金黄色葡萄球菌和大肠杆菌细胞高度变形,表面凹陷,显示出其细胞壁遭到破坏,大肠杆菌的细胞壁的破坏程度要大于金黄色葡萄球菌。而单独高压处理的细胞仍具有光滑连续的表面,表明CO2协同处理增加了对细胞破坏的程度。
通过激光共聚焦显微镜和流式细胞仪观察细胞对PI的吸收,结果显示协同处理比单独高压处理大大提高细胞膜通透比例,将大肠杆菌细胞吸收PI的比例从12%提高到87%,将金黄色葡萄球菌细胞吸收PI的比例从5%提高到69%,证实了CO2增加了细胞膜的通透性,是细胞灭活的关键因素之一。
大肠杆菌细胞超薄切片的TEM图像显示,CO2协同高压作用使细胞内蛋白质聚集,由于CO2渗透导致细胞内pH下降,细胞内高浓度的蛋白质包括核糖体因此在压力作用下聚集,破坏了菌体内组织的活性,引起微生物失活。细胞质中蛋白质的聚集是CO2协同灭活的另一关键因素。
CO2渗透抑制试验表明,5mM PBS完全抑制0.2M CO2渗透,提出CO2对细胞膜的渗透模型如下：在细胞膜上吸附的HCO3-离子结合一个H+,生成CO2并渗透进入细胞,当H2PO4-取代HCO3-在细胞膜表面吸附,CO2渗透即被抑制,失去协同杀菌作用。
3、采用4.5NL/L CO2协同,对橙汁、番茄汁和胡萝卜汁均可在低于或等于350MPa有效杀灭其中的大肠杆菌、李斯特菌和植物乳酸杆菌,同时发现CO2协同增加了细菌的亚致死损伤和储存过程中的继续灭活。
在四种果汁中进行了超高压或CO2协同超高压杀菌：橙汁(pH分别为3.4和3.8)、番茄汁(pH4.2)和胡萝卜汁(pH6.3),结果显示,果汁的成分如可溶性固体和悬浮颗粒并没有对CO2的协同作用产生明显的影响,果汁pH的影响在pH<4和pH>4时有不同表现,在pH4.2的番茄汁和pH6.3的胡萝卜汁中,协同杀菌效果与在pH7.0的生理盐水中相近,pH没有明显的影响(p<0.05),在pH3.8的橙汁中,超高压本身的杀菌效果有一定提高,但pH3.8对CO2协同效应则有更大的增强作用。而在pH3.4的橙汁中,几种细菌都失去耐压性,仅超高压就可以在200MPa完全灭活大肠杆菌,在300MPa完全灭活李斯特无害菌和植物乳酸杆菌。
协同处理造成的细胞亚致死损伤增加了细菌对环境pH的敏感性,使其在pH4.2番茄汁中储存时进一步灭活。而对于低酸性的果汁如pH6.3的胡萝卜,不能使亚致死细菌产生足够的继续灭活,应该采用较高的杀菌强度以确保安全。
High hydrostatic pressure (HHP) can be used to inactivate microorganisms while allows a better retention of product flavor, texture, color, and nutrient content than a comparable conventional heat pasteurization, avoiding heat destroying on these thermally sensitive ingredients. However, the expensive cost for the construction and running of HHP device is caused by very high pressure required for the inactivation of pressure resistant pathogenic and spoilage bacteria. This has limited the commercial breakthrough of HHP technology. Recently, to reduce the inactivation pressure, various effective synergistic treatments have attracted much more attention. These combined factors include antimicrobials, pH, moderate temperature and cycled pulse pressure. For the antimicrobials from natural sources, such as nisin, pediocin, lysozyme, lactoperoxidase and CO2, have been used to combine with HHP for microbial inactivation. It has been found that several natural biopreservatives, such as lysozyme and bacteriocins, are effective against some gram-positive bacteria by acting on the cell wall. Due to the protection from the outer membrane against the penetration of the above peptides and enzymes, gram-negative bacteria are normally insensitive to these antimicrobials. What is more, the addition of biopreservatives goes against "zero-added" principle. The combination of moderate heat also caused damage of nutrient and flavor to heat sensitive juices. Carbon dioxide is a natural inhibitory compound for microorganisms. It is cheap and safe for use. The combination of CO2with HHP can increase inactivation, thus decrease the treatment pressure. However, the low and unstable CO2concentration in the use with HHP has limited the inactivation effect. This limited the investigation and application of the combined method. Furthermore, the mechanisms of the synergistic bactericidal action on microorganisms remained unclear, which included the action of HHP, the action of CO2under pressure and the synergistic action of both on bacteria cells.
In this dissertation, a novel experimental method for the combined treatment of CO2and HHP was designed. A device was constructed to carry out the inactivation process at given CO2concentrations. The inactivation of Staphylococcus aureus and E. coli by the combined treatment, as well as the influence of CO2concentrations, treatment pressure and temperature on the bactericidal effect, were studied. The mechanisms of the synergy of CO2and HHP, including pressure induced CO2permeation of the cell membrane and denaturation of intracellular protein, were investigated. The combined treatment was applied in the pasteurization of the fruit juice to inactivate the common pathogenic and spoilage bacteria. The influence of fruit juice pH and matrix on the outcome of the combined treatment was studied to optimize the treatment parameters and establish the basis for the development and application of HHP technology. The main research results obtained as follows:
1. With the addition of3～5.5NL/L CO2in the bacteria suspension, the Staphylococcus aureus and Escherichia coli can be inactivated at pressure200-250MPa lower than that without CO2.
The effective treatment condition was at3.8NL/L CO2,350MPa and30℃for10min for S. aureus and3.2NL/L CO2,250MPa and30℃for10min for E.coli. The bactericidal pressures were reduced by200-250MPa compared to HHP alone. The inactivation rate increased rapidly with the increasing of the CO2concentration and leveled off, reaching the maximum inactivation at around5NL/L CO2. Temperature affected the inactivation significantly. The inactivation was effective at30℃or higher than30℃and declined clearly at below20℃. Higher or cycled pressure could be applied to acquire effective inactivation at lower temperature. Considering HHP as a true cold pasteurization method, the optional treatment temperature could be at25-35℃. While CO2in combination with HHP increased the inactivation, it increased the sublethal injury on bacteria cells. The combination of CO2also increased dynamic pressure inactivation to some extent. However, different from HHP treatment, hig pressure homogenization (HPH) did not caused sublethal injury on cells.
2. CO2destroyed the integrity of bacterial cell membrane, lowered the intracellular pH and facilitated protein aggregation under high pressure. These accounted for its bactericidal synergy with HHP.
The scanning electron microscope (SEM) images of the E. coli and S. aureus showed a highly deformed morphology of a rough surface with many concave defects and ruptured cell wall, whereas the cells treated with pressure alone only showed invaginations with the surface remaining smooth and continuous. The damage of E. coli cell wall was more serious than S. aureus. The results indicated that the combination of CO2increased the damage of bacteria cells.
Laser scanning cofocal microscope (LSCM) image and flow cytometry (FCM) analysis revealed that the combined treatment caused much more permeability of cell than HHP alone. The percentage of PI stained E. coli cells increased from12%to87%and S. aureus cells from5%to69%. CO2increased membrane permeability which was one of the key mechanisms involved in the inactivation of cells.
The transmission electron microscope (TEM) image of ultrathin section of E. coli cells showed that CO2in combination with HHP induced the intracellular protein aggregation. CO2permeation resulted in the decrease in intracellular pHi. The cytoplasmic protein with high concentration, including ribosome aggregated under high pressure, destroying the reactive compounds and inactivating cells. The intracellular protein aggregation was another key mechanism involved in the inactivation by the combined treatment.
The inhibition test of CO2permeation indicated that5mM PBS inhibited permeation of0.2M CO2entirely. A model of CO2permeation of the cell membrane was erected as follows:when a bicarbonate absorbed on the membrane phosphatide combined a proton, a CO2was created and permeated into the cell. When a dihydrogen phosphate ion displaced the bicarbonate on the membrane, CO2permeation was inhibited and its synergy on HHP inactivation was inhibited.
3. The selected vegetative bacteria E. coli, L. innocua and L. plantarum were effectively inactivated by the combination of dissolved4.5NL/L CO2and mild pressure350MPa or below in orange, tomato and carrot juices. Moreover, the synergy of CO2increased the sublethal damage and further inactivation during storage.
The present study used four fruit juices, orange (pH3.4and pH3.8), tomato (pH4.2) and carrot juice (pH6.3) in HHP treatment with or without dissolved CO2. The results indicated that the product matrix, such as soluble solids and suspended particles, did not exhibit an apparent influence on the action of the CO2. The juice pHs displayed different influences on the inactivation depending on pH<4or pH>4. The inactivation in tomato and carrot juices at pH4.2and6.3showed minor difference with that in physiological saline. The juice pH exerted no significant influence (p<0.05) on the inactivation at pH higher than4. By comparison, the combined effect was considerably promoted in orange juice at pH3.8, while the HHP inactivation was enhanced to a limited extent. In another orange juice of pH3.4, all the three strains lost their pressure resistance. HHP alone completely inactivated E. coli at relatively mild pressures of200MPa, and L. innocua and L. plantarum at300MPa.
Sublethal damage induced by the combined treatment at very low pressure increased the sensitivity of the bacteria to the storage pH and caused further inactivation in tomato juice during storage, whereas, low acidic carrot juice did not induce sufficient further inactivation, indicating that intensive treatment is required to ensure safety.
本论文以CO2为协同物质,设计和构建一种全新的CO2协同超高压试验方法和装置,实现在设定的CO2浓度协同超高压杀菌,以达到预期的协同效果,并且研究了CO2协同浓度、超高压压力、温度等因素对杀菌效果的影响。同时对CO2协同超高压杀菌的机理进行探索,涉及高压下CO2对细胞膜的渗透作用,对细胞内蛋白质的变性作用等。然后针对果蔬汁中常见的致病菌和腐败菌,采用协同处理对果汁进行巴氏杀菌,探索果汁pH值和基质对CO2协同超高压处理的影响,为超高压技术和工艺的开发和应用提供依据。主要研究成果如下：
1、在细胞悬浮液中添加.3～5.5NL/L CO2,能使金黄色葡萄球菌和大肠杆菌的超高压杀菌压力比无协同杀菌降低200～250MPa。
金黄色葡萄球菌的有效协同杀菌(>7对数级)条件为3.8NL/L CO2、350MPa、30℃、10分钟,大肠杆菌的有效协同杀菌条件为3.2NL/L10分钟,与单独超高压相比,降低了杀菌压力200-250MPa。灭活率随CO2增加而增加,当CO2浓度提高到5NL/L左右时,对杀菌效果的增强呈现饱和趋势。温度显著影响协同杀菌效果,在30℃或更高的温度时,有良好的杀菌效果,在低于20℃时,CO2协同效果降低,适当提高压力,或者采用循环压力也可以得到有效的杀菌效果。作为真正的冷杀菌方法,可以选择处理温度为25-35℃。CO2协同超高压造成细菌灭活的同时,也增加亚致死损伤。CO2协同对动态压力的杀菌效果也有一定的增强作用,但与高静压不同,高压均质处理没有造成细胞亚致死损伤。
2、CO2主要是通过破坏细胞膜完整性、降低细胞内pH、促进蛋白质聚集而发挥协同杀菌作用。
通过细胞扫描电镜(SEM)图像观察到协同处理的金黄色葡萄球菌和大肠杆菌细胞高度变形,表面凹陷,显示出其细胞壁遭到破坏,大肠杆菌的细胞壁的破坏程度要大于金黄色葡萄球菌。而单独高压处理的细胞仍具有光滑连续的表面,表明CO2协同处理增加了对细胞破坏的程度。
通过激光共聚焦显微镜和流式细胞仪观察细胞对PI的吸收,结果显示协同处理比单独高压处理大大提高细胞膜通透比例,将大肠杆菌细胞吸收PI的比例从12%提高到87%,将金黄色葡萄球菌细胞吸收PI的比例从5%提高到69%,证实了CO2增加了细胞膜的通透性,是细胞灭活的关键因素之一。
大肠杆菌细胞超薄切片的TEM图像显示,CO2协同高压作用使细胞内蛋白质聚集,由于CO2渗透导致细胞内pH下降,细胞内高浓度的蛋白质包括核糖体因此在压力作用下聚集,破坏了菌体内组织的活性,引起微生物失活。细胞质中蛋白质的聚集是CO2协同灭活的另一关键因素。
CO2渗透抑制试验表明,5mM PBS完全抑制0.2M CO2渗透,提出CO2对细胞膜的渗透模型如下：在细胞膜上吸附的HCO3-离子结合一个H+,生成CO2并渗透进入细胞,当H2PO4-取代HCO3-在细胞膜表面吸附,CO2渗透即被抑制,失去协同杀菌作用。
3、采用4.5NL/L CO2协同,对橙汁、番茄汁和胡萝卜汁均可在低于或等于350MPa有效杀灭其中的大肠杆菌、李斯特菌和植物乳酸杆菌,同时发现CO2协同增加了细菌的亚致死损伤和储存过程中的继续灭活。
在四种果汁中进行了超高压或CO2协同超高压杀菌：橙汁(pH分别为3.4和3.8)、番茄汁(pH4.2)和胡萝卜汁(pH6.3),结果显示,果汁的成分如可溶性固体和悬浮颗粒并没有对CO2的协同作用产生明显的影响,果汁pH的影响在pH<4和pH>4时有不同表现,在pH4.2的番茄汁和pH6.3的胡萝卜汁中,协同杀菌效果与在pH7.0的生理盐水中相近,pH没有明显的影响(p<0.05),在pH3.8的橙汁中,超高压本身的杀菌效果有一定提高,但pH3.8对CO2协同效应则有更大的增强作用。而在pH3.4的橙汁中,几种细菌都失去耐压性,仅超高压就可以在200MPa完全灭活大肠杆菌,在300MPa完全灭活李斯特无害菌和植物乳酸杆菌。
协同处理造成的细胞亚致死损伤增加了细菌对环境pH的敏感性,使其在pH4.2番茄汁中储存时进一步灭活。而对于低酸性的果汁如pH6.3的胡萝卜,不能使亚致死细菌产生足够的继续灭活,应该采用较高的杀菌强度以确保安全。
High hydrostatic pressure (HHP) can be used to inactivate microorganisms while allows a better retention of product flavor, texture, color, and nutrient content than a comparable conventional heat pasteurization, avoiding heat destroying on these thermally sensitive ingredients. However, the expensive cost for the construction and running of HHP device is caused by very high pressure required for the inactivation of pressure resistant pathogenic and spoilage bacteria. This has limited the commercial breakthrough of HHP technology. Recently, to reduce the inactivation pressure, various effective synergistic treatments have attracted much more attention. These combined factors include antimicrobials, pH, moderate temperature and cycled pulse pressure. For the antimicrobials from natural sources, such as nisin, pediocin, lysozyme, lactoperoxidase and CO2, have been used to combine with HHP for microbial inactivation. It has been found that several natural biopreservatives, such as lysozyme and bacteriocins, are effective against some gram-positive bacteria by acting on the cell wall. Due to the protection from the outer membrane against the penetration of the above peptides and enzymes, gram-negative bacteria are normally insensitive to these antimicrobials. What is more, the addition of biopreservatives goes against "zero-added" principle. The combination of moderate heat also caused damage of nutrient and flavor to heat sensitive juices. Carbon dioxide is a natural inhibitory compound for microorganisms. It is cheap and safe for use. The combination of CO2with HHP can increase inactivation, thus decrease the treatment pressure. However, the low and unstable CO2concentration in the use with HHP has limited the inactivation effect. This limited the investigation and application of the combined method. Furthermore, the mechanisms of the synergistic bactericidal action on microorganisms remained unclear, which included the action of HHP, the action of CO2under pressure and the synergistic action of both on bacteria cells.
In this dissertation, a novel experimental method for the combined treatment of CO2and HHP was designed. A device was constructed to carry out the inactivation process at given CO2concentrations. The inactivation of Staphylococcus aureus and E. coli by the combined treatment, as well as the influence of CO2concentrations, treatment pressure and temperature on the bactericidal effect, were studied. The mechanisms of the synergy of CO2and HHP, including pressure induced CO2permeation of the cell membrane and denaturation of intracellular protein, were investigated. The combined treatment was applied in the pasteurization of the fruit juice to inactivate the common pathogenic and spoilage bacteria. The influence of fruit juice pH and matrix on the outcome of the combined treatment was studied to optimize the treatment parameters and establish the basis for the development and application of HHP technology. The main research results obtained as follows:
1. With the addition of3～5.5NL/L CO2in the bacteria suspension, the Staphylococcus aureus and Escherichia coli can be inactivated at pressure200-250MPa lower than that without CO2.
The effective treatment condition was at3.8NL/L CO2,350MPa and30℃for10min for S. aureus and3.2NL/L CO2,250MPa and30℃for10min for E.coli. The bactericidal pressures were reduced by200-250MPa compared to HHP alone. The inactivation rate increased rapidly with the increasing of the CO2concentration and leveled off, reaching the maximum inactivation at around5NL/L CO2. Temperature affected the inactivation significantly. The inactivation was effective at30℃or higher than30℃and declined clearly at below20℃. Higher or cycled pressure could be applied to acquire effective inactivation at lower temperature. Considering HHP as a true cold pasteurization method, the optional treatment temperature could be at25-35℃. While CO2in combination with HHP increased the inactivation, it increased the sublethal injury on bacteria cells. The combination of CO2also increased dynamic pressure inactivation to some extent. However, different from HHP treatment, hig pressure homogenization (HPH) did not caused sublethal injury on cells.
2. CO2destroyed the integrity of bacterial cell membrane, lowered the intracellular pH and facilitated protein aggregation under high pressure. These accounted for its bactericidal synergy with HHP.
The scanning electron microscope (SEM) images of the E. coli and S. aureus showed a highly deformed morphology of a rough surface with many concave defects and ruptured cell wall, whereas the cells treated with pressure alone only showed invaginations with the surface remaining smooth and continuous. The damage of E. coli cell wall was more serious than S. aureus. The results indicated that the combination of CO2increased the damage of bacteria cells.
Laser scanning cofocal microscope (LSCM) image and flow cytometry (FCM) analysis revealed that the combined treatment caused much more permeability of cell than HHP alone. The percentage of PI stained E. coli cells increased from12%to87%and S. aureus cells from5%to69%. CO2increased membrane permeability which was one of the key mechanisms involved in the inactivation of cells.
The transmission electron microscope (TEM) image of ultrathin section of E. coli cells showed that CO2in combination with HHP induced the intracellular protein aggregation. CO2permeation resulted in the decrease in intracellular pHi. The cytoplasmic protein with high concentration, including ribosome aggregated under high pressure, destroying the reactive compounds and inactivating cells. The intracellular protein aggregation was another key mechanism involved in the inactivation by the combined treatment.
The inhibition test of CO2permeation indicated that5mM PBS inhibited permeation of0.2M CO2entirely. A model of CO2permeation of the cell membrane was erected as follows:when a bicarbonate absorbed on the membrane phosphatide combined a proton, a CO2was created and permeated into the cell. When a dihydrogen phosphate ion displaced the bicarbonate on the membrane, CO2permeation was inhibited and its synergy on HHP inactivation was inhibited.
3. The selected vegetative bacteria E. coli, L. innocua and L. plantarum were effectively inactivated by the combination of dissolved4.5NL/L CO2and mild pressure350MPa or below in orange, tomato and carrot juices. Moreover, the synergy of CO2increased the sublethal damage and further inactivation during storage.
The present study used four fruit juices, orange (pH3.4and pH3.8), tomato (pH4.2) and carrot juice (pH6.3) in HHP treatment with or without dissolved CO2. The results indicated that the product matrix, such as soluble solids and suspended particles, did not exhibit an apparent influence on the action of the CO2. The juice pHs displayed different influences on the inactivation depending on pH<4or pH>4. The inactivation in tomato and carrot juices at pH4.2and6.3showed minor difference with that in physiological saline. The juice pH exerted no significant influence (p<0.05) on the inactivation at pH higher than4. By comparison, the combined effect was considerably promoted in orange juice at pH3.8, while the HHP inactivation was enhanced to a limited extent. In another orange juice of pH3.4, all the three strains lost their pressure resistance. HHP alone completely inactivated E. coli at relatively mild pressures of200MPa, and L. innocua and L. plantarum at300MPa.
Sublethal damage induced by the combined treatment at very low pressure increased the sensitivity of the bacteria to the storage pH and caused further inactivation in tomato juice during storage, whereas, low acidic carrot juice did not induce sufficient further inactivation, indicating that intensive treatment is required to ensure safety.
引文
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