高压对鲜切生菜品质与微生物的影响及机理研究
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摘要
本研究以国内外广泛消费的结球生菜（Iceberg lettuce）为材料，针对鲜切生菜存在的品质与微生物问题，通过应用生化分析并结合液质与微观结构分析及微生物检测等方法，较为系统地研究了高压对鲜切生菜品质的影响机理、高压对生菜保护酶系统及抗氧化能力的影响及高压、温度及货架期与鲜切生菜微生物的关系等，期望能为高压在鲜切生菜加工上的应用提供理论与技术支持。本研究取得的主要结果如下：
在高压对鲜切生菜品质的影响方面，本研究发现：1）适宜的高压（50MPa处理5min）可改善鲜切生菜的感官品质，除了保持新鲜生菜的脆与清香味外，比新鲜生菜口感更甜。研究证实，高压处理后生菜中葡萄糖、果糖含量有增加现象；随着压力的增加、保压时间的延长，生菜甜味的口感消失并产生异苦味，原因与糖的含量无关，而是可能与人体对异苦味物质的味感或增加有关。2）随着高压强度的增加，鲜切生菜的硬度呈下降趋势，在口感上失去脆度而变“韧”，UPLC/MS/MS鉴定表明，不论高压与否，生菜实验样本中均未发现与木质素合成有关的对香豆酸和阿魏酸的存在或增加或减少，口感变“韧”不是木质素合成的结果，扫描电镜图像进一步证实，随着高压强度的增加，细胞由饱满变得凹平，失去膨胀压，这是导致生菜在超高压下形成“韧”感的直接原因。3）100MPa、20min以上的高压处理导致鲜切生菜品质劣变，200MPa处理的鲜切生菜出现异苦味、高韧度、严重水浸与褐变。扫描及透射电镜图像证实，100MPa、20min以上处理时，细胞内容物被挤出，细胞膜受损伤，因而引起相关生化反应，造成上述品质劣变结果。4）实验范围内的高压处理对生菜主要单酚物质-绿原酸、咖啡酸及总酚含量无影响（p>0.05）；高压对鲜切生菜褐变无影响（p>0.05）；100MPa高压处理导致PAL活性增加（p<0.05），但总酚含量未见显著增加（p>0.05）；随着货架时间的延长，鲜切生菜的PAL活性增加(p<0.05)，但总酚含量呈下降趋势，而具有催化酚类聚合作用的POD活性增加(p<0.05)，说明酚的合成速率低于被利用速率；货架期间，PPO活性似乎与褐变无关，PPO活性最强时，褐变最轻，随后PPO活性下降，但褐变加重，而POD活性增强(p<0.05)，POD可能是导致货架期鲜切生菜褐变的原因。5）货架期间，在真空包装条件下，鲜切生菜的PAL活性显著增加（p<0.05），但总酚含量未见显著增加（p>0.05）；真空状态保持了鲜切生菜一定的PPO活性，但显著抑制了POD活性的增加（p<0.05），酚类物质的下降受到抑制，进一步说明鲜切生菜货架期褐变中，PPO活性不是关键因素。
在酶方面，本研究发现高压处理对鲜切生菜保护酶活性无影响（p>0.05），生菜中的酶对高压（室温，200MPa处理20min）的相对敏感性依次为PPO>ASO>GSH-Px>PAL>CAT>APX>POD。尽管本研究条件下，保护酶活性未受到高压破坏，但显微图像证实100MPa、20min以上的高压处理造成细胞膜受损，因而影响细胞代谢，引起品质劣变，因此，单一的高压处理（100MPa、20min以上）不适于鲜切生菜加工。
在高压对鲜切生菜抗氧化能力影响方面，高压对鲜切生菜DPPH清除能力无影响（p>0.05），但显著提高了FRAP总抗氧能力（p<0.05）。货架期间，鲜切生菜的DPPH清除能力提高（p<0.05），但总抗氧化能力下降（p<0.05）。
在高压对鲜切生菜微生物的影响方面，本研究证实：1）高压能够极显著降低鲜切生菜的菌落总数、大肠菌群和真菌的数量（p<0.01）；2）温度（5-25℃）对高压杀灭微生物无协同作用。3）大肠菌群对高压比真菌更敏感，与对照相比，高压处理的鲜切生菜在4℃货架期间，大肠菌群下降速度更快，这对于大肠杆菌如致病性的大肠杆菌O157:H7的控制可能具有启示意义。
在高压对鲜切生菜细胞水平的影响方面，本研究证实生菜细胞对压力敏感。200MPa下生菜细胞发生明显变形，细胞膜、细胞器膜破裂，细胞壁断裂。
总得来看，高压在同时保证鲜切生菜产品品质与安全上面临挑战。本研究认为，鲜切生菜适合采用低温、低压、短时间处理如5℃、50MPa处理5min，产品品质更佳，总抗氧化能力提高，大肠菌群得到显著抑制，其货架期（4℃）不宜超过3d。同其它加工技术一样，高压需要与产品品质及安全有关的因素的协同作用，如耐高压的鲜切生菜品种、减少原料初始微生物数量的技术及防止致病微生物污染的HACCP措施等，在此前提下，高压是一个能够改善鲜切产品品质及保证产品安全的新技术。
The control of quality and safety of fresh cut lettuce is directly related to the health of consumers,as one of new nonthermal technologies, high pressure has many advantages in the processing of food,whether high pressure can be used in the fresh cut lettuce industry or not depends on the effect of highpressure on the quality and microorganisms of fresh cut lettuce. This study aimed to investigate theeffect of high pressure on the sensory, nutrient, protective enzymes, antioxidant power, microorganismand cell ultrastructure of fresh cut iceberg lettuce by biochemical, UPLC/MS/MS, electron microscopyand microorganism analysis. The findings from this study are expected to direct the utilization of highpressure in the fresh cut lettuce industry as follows.
For the quality part, firstly, high pressure was found to improve the sensory quality of fresh cutlettuce, especially for50MPa with5min, which was tasted more sweet following with the originalcrisp and aroma of lettuce, and the data showed that the glucose and fructose content of lettuceincreased after high pressure processing. However, the sweetness can not be tasted with increasing ofpressure, and the disagreeable bitter was tasted obviously, which was attributed to the taste of tongue tobitter substance from lettuce or the possibly increasing of bitters substances induced by high pressure.Secondly, with the increasing of pressure, the firmness showed a decreasing trend, and the toughnesscan be tasted, while, it was not related to the synthesization of lignin, because UPLC-MS-MS findingsshowed that the lignin synthetic substances like p-coumaric acid and ferulic acid were not existed orincreasing or decreasing in experimental samples of lettuce whether high pressure processing or not, andthe ultrastructure image by Scanning Electron Microscop（ySEM）also showed the shapes of lettuce cellschanged from bulge to concave and flat, so the crispness was lost and the taste of toughness presented.Thirdly, the browning and blank taste of fresh cut lettuce were induced when pressure reached100MPawith20min, while200MPa made the lettuce disagreeable bitter taste, high toughness, serious wateryand browning. The ultrastructure image by SEM and Transmitting Electron Microscopy (TEM) showedthat the substances in the cells was squeezed out and the cell membranes was broken when the pressurewas over100MPa with20min, which can induced relative biochemical reactions and consequently thequality deteriorated. Fourthly, the content of chlorogenic acid and caffeic acid and total phenols were allnot affected by high pressure treatments used in this study (p>0.05). High pressure did not induce thebrowning of lettuce (p>0.05). The phenylalanine ammonialase (PAL) activity increased when lettucewas treated by high pressure at100MPa (p<0.05), while the total phenols content did not increasesignificantly (p>0.05). During shelf life of fresh cut lettuce processed by high pressure, PAL activityincreased significantly (p<0.05), but the total phenols content showed the decreasing trend, andperoxidase (POD) activity involved in phenols polymerization was observed to increase significantly(p<0.05), suggesting that maybe the synthetic rate of phenols was lower than the utilization rate ofphenols, which resulted in the increasing of browning. Polyphenol oxidase (PPO) activity appeared tohave no relationship with browning during the shelf life of fresh cut lettuce, maybe the browning was mainly induced by POD passway. Fifthly, during shelf life, vacuum packaging reduced the loss of totalphenols and inhibited the browning. Vacuum package increased PAL activity significantly (p<0.05),and kept the PPO activity, but inhibited the increasing of POD activity significantly（p<0.05），whichfurther supported that the PPO activity is not key factor during the browning of fresh cut lettuce。
For the enzymes of lettuce, the findings showed that the protective antioxidant enzymes were notaffected by high pressure processing (p>0.05), and the susceptibility of enzymes in lettuce to highpressure（200MPa with20min at ambient temperature）were PPO, ascorbate oxidase, glutathioneperoxidase, PAL, catalase, ascorbic peroxidase and POD in turn. Although the protective enzymesactivities were kept well under high pressure over100MPa with20min, the ultrastructure imagesshowed that the membrane of cells was broken and substances were out of cells, which will induce themetabolism disorder of cells and resulted in the deterioration of quality actually, suggesting the highpressure treatment over100MPa with20min can not be used in fresh cut lettuce industry.
For the antioxidant capacity of fresh cut lettuce, high pressure had no effect on DPPH scavengingactivity (p>0.05), while it can improve the ferric reducing/antioxidant power (p<0.05). During shelflife, the DPPH scavenging activity was found to be increased (p<0.05) whether high pressuretreatments or not, while the ferric reducing/antioxidant power decreased with the decreasing of totalphenols (p<0.05).
For the microorganisms of lettuce, high pressure can reduced aerobic plate counts, coliform andfungi counts remarkably significantly (p<0.01). Temperature (5-25℃) had no synergism with highpressure on reduction of microorganisms of lettuce. Coliform was more sensitive to high pressurecompared to fungi, during shelf life at4℃, coliform counts of fresh cut lettuce treated by high pressuredecreased rapidly compared to the control, which suggested high pressure would help to controlfood-borne pathogen like E.Coli O157:H7.
At the cell level, lettuce cell was susceptible to the high pressure,200MPa can change the cellshapes obviously, and result in the collapse of cell membrane and cellular organelle membrane andbreak of cell wall by the observation of SEM and TEM.
For the application of high pressure in the fresh cut lettuce processing, high pressure faced thechallenges against assuring the quality and control of microorganisms of fresh cut lettucesimultaneously. The better high pressure treatment like50MPa with5min at5℃can produce moredelicious quality, keep high total antioxidant power and realize the significant reduction of coliformwithin3days’ shelf life at4℃. Like other processing technologies, high pressure processing also needother factors to realize the synergism on assuring the quality and safety of fresh cut lettuce, like pressureresistant varieties, and conducting HACCP plan to reduce the original microbes counts and prevent thepollution from food borne pathogen etc.. Under above premise, high pressure is a new technology withimproving the quality and assuring the safety of fresh cut products.
在高压对鲜切生菜品质的影响方面，本研究发现：1）适宜的高压（50MPa处理5min）可改善鲜切生菜的感官品质，除了保持新鲜生菜的脆与清香味外，比新鲜生菜口感更甜。研究证实，高压处理后生菜中葡萄糖、果糖含量有增加现象；随着压力的增加、保压时间的延长，生菜甜味的口感消失并产生异苦味，原因与糖的含量无关，而是可能与人体对异苦味物质的味感或增加有关。2）随着高压强度的增加，鲜切生菜的硬度呈下降趋势，在口感上失去脆度而变“韧”，UPLC/MS/MS鉴定表明，不论高压与否，生菜实验样本中均未发现与木质素合成有关的对香豆酸和阿魏酸的存在或增加或减少，口感变“韧”不是木质素合成的结果，扫描电镜图像进一步证实，随着高压强度的增加，细胞由饱满变得凹平，失去膨胀压，这是导致生菜在超高压下形成“韧”感的直接原因。3）100MPa、20min以上的高压处理导致鲜切生菜品质劣变，200MPa处理的鲜切生菜出现异苦味、高韧度、严重水浸与褐变。扫描及透射电镜图像证实，100MPa、20min以上处理时，细胞内容物被挤出，细胞膜受损伤，因而引起相关生化反应，造成上述品质劣变结果。4）实验范围内的高压处理对生菜主要单酚物质-绿原酸、咖啡酸及总酚含量无影响（p>0.05）；高压对鲜切生菜褐变无影响（p>0.05）；100MPa高压处理导致PAL活性增加（p<0.05），但总酚含量未见显著增加（p>0.05）；随着货架时间的延长，鲜切生菜的PAL活性增加(p<0.05)，但总酚含量呈下降趋势，而具有催化酚类聚合作用的POD活性增加(p<0.05)，说明酚的合成速率低于被利用速率；货架期间，PPO活性似乎与褐变无关，PPO活性最强时，褐变最轻，随后PPO活性下降，但褐变加重，而POD活性增强(p<0.05)，POD可能是导致货架期鲜切生菜褐变的原因。5）货架期间，在真空包装条件下，鲜切生菜的PAL活性显著增加（p<0.05），但总酚含量未见显著增加（p>0.05）；真空状态保持了鲜切生菜一定的PPO活性，但显著抑制了POD活性的增加（p<0.05），酚类物质的下降受到抑制，进一步说明鲜切生菜货架期褐变中，PPO活性不是关键因素。
在酶方面，本研究发现高压处理对鲜切生菜保护酶活性无影响（p>0.05），生菜中的酶对高压（室温，200MPa处理20min）的相对敏感性依次为PPO>ASO>GSH-Px>PAL>CAT>APX>POD。尽管本研究条件下，保护酶活性未受到高压破坏，但显微图像证实100MPa、20min以上的高压处理造成细胞膜受损，因而影响细胞代谢，引起品质劣变，因此，单一的高压处理（100MPa、20min以上）不适于鲜切生菜加工。
在高压对鲜切生菜抗氧化能力影响方面，高压对鲜切生菜DPPH清除能力无影响（p>0.05），但显著提高了FRAP总抗氧能力（p<0.05）。货架期间，鲜切生菜的DPPH清除能力提高（p<0.05），但总抗氧化能力下降（p<0.05）。
在高压对鲜切生菜微生物的影响方面，本研究证实：1）高压能够极显著降低鲜切生菜的菌落总数、大肠菌群和真菌的数量（p<0.01）；2）温度（5-25℃）对高压杀灭微生物无协同作用。3）大肠菌群对高压比真菌更敏感，与对照相比，高压处理的鲜切生菜在4℃货架期间，大肠菌群下降速度更快，这对于大肠杆菌如致病性的大肠杆菌O157:H7的控制可能具有启示意义。
在高压对鲜切生菜细胞水平的影响方面，本研究证实生菜细胞对压力敏感。200MPa下生菜细胞发生明显变形，细胞膜、细胞器膜破裂，细胞壁断裂。
总得来看，高压在同时保证鲜切生菜产品品质与安全上面临挑战。本研究认为，鲜切生菜适合采用低温、低压、短时间处理如5℃、50MPa处理5min，产品品质更佳，总抗氧化能力提高，大肠菌群得到显著抑制，其货架期（4℃）不宜超过3d。同其它加工技术一样，高压需要与产品品质及安全有关的因素的协同作用，如耐高压的鲜切生菜品种、减少原料初始微生物数量的技术及防止致病微生物污染的HACCP措施等，在此前提下，高压是一个能够改善鲜切产品品质及保证产品安全的新技术。
The control of quality and safety of fresh cut lettuce is directly related to the health of consumers,as one of new nonthermal technologies, high pressure has many advantages in the processing of food,whether high pressure can be used in the fresh cut lettuce industry or not depends on the effect of highpressure on the quality and microorganisms of fresh cut lettuce. This study aimed to investigate theeffect of high pressure on the sensory, nutrient, protective enzymes, antioxidant power, microorganismand cell ultrastructure of fresh cut iceberg lettuce by biochemical, UPLC/MS/MS, electron microscopyand microorganism analysis. The findings from this study are expected to direct the utilization of highpressure in the fresh cut lettuce industry as follows.
For the quality part, firstly, high pressure was found to improve the sensory quality of fresh cutlettuce, especially for50MPa with5min, which was tasted more sweet following with the originalcrisp and aroma of lettuce, and the data showed that the glucose and fructose content of lettuceincreased after high pressure processing. However, the sweetness can not be tasted with increasing ofpressure, and the disagreeable bitter was tasted obviously, which was attributed to the taste of tongue tobitter substance from lettuce or the possibly increasing of bitters substances induced by high pressure.Secondly, with the increasing of pressure, the firmness showed a decreasing trend, and the toughnesscan be tasted, while, it was not related to the synthesization of lignin, because UPLC-MS-MS findingsshowed that the lignin synthetic substances like p-coumaric acid and ferulic acid were not existed orincreasing or decreasing in experimental samples of lettuce whether high pressure processing or not, andthe ultrastructure image by Scanning Electron Microscop（ySEM）also showed the shapes of lettuce cellschanged from bulge to concave and flat, so the crispness was lost and the taste of toughness presented.Thirdly, the browning and blank taste of fresh cut lettuce were induced when pressure reached100MPawith20min, while200MPa made the lettuce disagreeable bitter taste, high toughness, serious wateryand browning. The ultrastructure image by SEM and Transmitting Electron Microscopy (TEM) showedthat the substances in the cells was squeezed out and the cell membranes was broken when the pressurewas over100MPa with20min, which can induced relative biochemical reactions and consequently thequality deteriorated. Fourthly, the content of chlorogenic acid and caffeic acid and total phenols were allnot affected by high pressure treatments used in this study (p>0.05). High pressure did not induce thebrowning of lettuce (p>0.05). The phenylalanine ammonialase (PAL) activity increased when lettucewas treated by high pressure at100MPa (p<0.05), while the total phenols content did not increasesignificantly (p>0.05). During shelf life of fresh cut lettuce processed by high pressure, PAL activityincreased significantly (p<0.05), but the total phenols content showed the decreasing trend, andperoxidase (POD) activity involved in phenols polymerization was observed to increase significantly(p<0.05), suggesting that maybe the synthetic rate of phenols was lower than the utilization rate ofphenols, which resulted in the increasing of browning. Polyphenol oxidase (PPO) activity appeared tohave no relationship with browning during the shelf life of fresh cut lettuce, maybe the browning was mainly induced by POD passway. Fifthly, during shelf life, vacuum packaging reduced the loss of totalphenols and inhibited the browning. Vacuum package increased PAL activity significantly (p<0.05),and kept the PPO activity, but inhibited the increasing of POD activity significantly（p<0.05），whichfurther supported that the PPO activity is not key factor during the browning of fresh cut lettuce。
For the enzymes of lettuce, the findings showed that the protective antioxidant enzymes were notaffected by high pressure processing (p>0.05), and the susceptibility of enzymes in lettuce to highpressure（200MPa with20min at ambient temperature）were PPO, ascorbate oxidase, glutathioneperoxidase, PAL, catalase, ascorbic peroxidase and POD in turn. Although the protective enzymesactivities were kept well under high pressure over100MPa with20min, the ultrastructure imagesshowed that the membrane of cells was broken and substances were out of cells, which will induce themetabolism disorder of cells and resulted in the deterioration of quality actually, suggesting the highpressure treatment over100MPa with20min can not be used in fresh cut lettuce industry.
For the antioxidant capacity of fresh cut lettuce, high pressure had no effect on DPPH scavengingactivity (p>0.05), while it can improve the ferric reducing/antioxidant power (p<0.05). During shelflife, the DPPH scavenging activity was found to be increased (p<0.05) whether high pressuretreatments or not, while the ferric reducing/antioxidant power decreased with the decreasing of totalphenols (p<0.05).
For the microorganisms of lettuce, high pressure can reduced aerobic plate counts, coliform andfungi counts remarkably significantly (p<0.01). Temperature (5-25℃) had no synergism with highpressure on reduction of microorganisms of lettuce. Coliform was more sensitive to high pressurecompared to fungi, during shelf life at4℃, coliform counts of fresh cut lettuce treated by high pressuredecreased rapidly compared to the control, which suggested high pressure would help to controlfood-borne pathogen like E.Coli O157:H7.
At the cell level, lettuce cell was susceptible to the high pressure,200MPa can change the cellshapes obviously, and result in the collapse of cell membrane and cellular organelle membrane andbreak of cell wall by the observation of SEM and TEM.
For the application of high pressure in the fresh cut lettuce processing, high pressure faced thechallenges against assuring the quality and control of microorganisms of fresh cut lettucesimultaneously. The better high pressure treatment like50MPa with5min at5℃can produce moredelicious quality, keep high total antioxidant power and realize the significant reduction of coliformwithin3days’ shelf life at4℃. Like other processing technologies, high pressure processing also needother factors to realize the synergism on assuring the quality and safety of fresh cut lettuce, like pressureresistant varieties, and conducting HACCP plan to reduce the original microbes counts and prevent thepollution from food borne pathogen etc.. Under above premise, high pressure is a new technology withimproving the quality and assuring the safety of fresh cut products.
引文
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