Quality and Safety of Fresh-Cut Vegetables and Fruits

Non-Technical Summary:<br/>
This research addresses the critical issues facing the fresh fruit and vegetable processing industries for enhancing the product safety and quality. Over the last decade, there have been an increasing frequency of multistate outbreaks of Listeria monocytogenes and Salmonella poisoning related to cantaloupes, tomatoes, or other fresh produce according to the Centers for Disease Control and Prevention. Diverse groups of L. monocytogenes and Salmonella are widely distributed in the fresh fruit and vegetable processing environments while some strains are more endemic and persistent for years. Current knowledge on L. monocytogenes and Salmonella attachment of the fruits and vegetable surfaces is limited. The attachment of these bacteria on the surface of the fruit and vegetables is governed by number of factors including environmental temperature, produce pH and water activity, and presence and distribution of surface structures. Also, these foodborne bacterial pathogens are capable of surviving and growing for long periods (especially on cut surfaces or damaged tissues) on fruits and vegetables. L. monocytogenes frequently appears on the cleaned surfaces and nearly 1/3 of the processing plants commonly isolate this pathogen. Presence of this pathogen in the processing environment is indicative of increased possibility of occurrence in finished ready-to-eat products. The natural persistence of L. monocytogenes is due to its wide-spread distribution and its ability to withstand adverse environmental conditions. This includes the ability of this pathogen to survive and grow at low temperatures, resistance to high osmolarity, acidic and alkaline environments. The critical factors contributing to the ability of L. monocytogenes to produce highly stable stress-resistant phenotypes that are capable of surviving in sublethal and lethal concentrations of acid, alkali, heat, oxidative stress for their potential cross resistance to disinfectants and other antimicrobials is unknown. Our work will give insights into the role of stress-resistant phenotypes of L. monocytogenes and Salmonella that may be surviving in different produce processing environments and in fresh fruits and vegetables. New ways to prevent or decontaminate stress-adapted cells of these pathogens on ready-to-eat fresh fruits and vegetables are required to protect fresh produce at all these stages. Also, since current market dynamics of fresh fruits and vegetable industries have shifted towards large scale operation and bagging of fresh-cut minimally processed fruits and vegetables, even if small contamination occur, target foodborne bacterial pathogens can easily spread to a large volume of the final product.
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Approach:<br/>
Our goal is to identify the potential role of mild stress conditions, contact time and exposure concentration, strain diversity, surrounding temperature, growth phase and the number of generation cycles on the selection of highly stable stress-resistant phenotypes of L. monocytogenes. We will do a step-wise process of triggering mild stress and sever stress environments to induce genetic instability and natural selection in certain serotypes in L. monocytogenes (Lm) in broth and agar models containing esculin and ferric ammonium citrate that will distinguish Lm growth by black color. Highly stable acid-stress resistant sub-populations of Lm will be isolated by preexposing Lm cells to sublethal pH concentrations of 5.5 or 5.25 for 2 days and then post exposure to lethal pH concentration of 4.0 for 2 days. Highly stable salt-stress resistant sub-populations of Lm will be isolated by preexposing Lm cells to sublethal salt concentrations of 4% for 2 days and then post exposure to lethal salt concentration of 12% for 2 days. Highly stable alkaline-stress resistant sub-populations of L. monocytogenes will be isolated by preexposing Lm cells to sublethal pH concentrations of 9.5 for 2 days and then post exposure to lethal pH concentration of 11.0 for 2 days. Highly stable oxidative-stress resistant sub-populations of Lm will be isolated by preexposing Lm cells to sublethal H2O2 concentrations of 0.01% for 2 days and then post exposure to lethal H2O2 concentration of 0.05% for 2 days. Highly stable heat-stress resistant sub-populations of Lm will be isolated by preexposing L. monocytogenes cells to sublethal heat of 48C for 1 h and then post exposure to lethal heat of 65C for 3 min and then surviving sub-populations will be recovered. The non-stressed Lm cells will be prepared using the same generation cycles except that the growth will be at a normal pH of 7.0 in the absence of target stress. Quantitative viability assays will be conducted for each stress-resistant phenotype of Lm to determine their growth limits in sublethal stress and their survival in lethal stress when exposed to a target environment and in the presence of common disinfectants and antimicrobials used in fresh produce processing industries. These stress-resistant phenotypes of L. monocytogenes will be tested for their ability to form biofilms in fresh produce processing environments and for their ability to attach/survive in intact/fresh-cut products. Based on these findings, best-practices and standardized models will be developed for use in food safety risk assessment of fresh-cut product quality and safety.
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Progress:<br/>
2012/01 TO 2012/12<br/>
OUTPUTS: Fresh-cut vegetables and fruits due to minimal processing are vulnerable to contamination by Listeria monocytogenes and other foodborne bacterial pathogens. New ways to prevent or decontaminate these pathogens on ready-to-eat fresh fruits and vegetables is required to protect fresh produce at all stages. Many sanitizers such as chlorine or derivatives of chlorine such as chlorine gas, sodium hypochlorite or chlorine dioxide are extensively used in the fruits and vegetable industries with mixed results against foodborne bacterial pathogens. L. monocytogenes can survive in almost all environments and can adapt to changing conditions. Designing any decontamination technologies require defeating the potential of L. monocytogenes to survive under these stress conditions. The critical factors contributing to the ability of L. monocytogenes to produce highly stable stress-resistant phenotypes that are capable of surviving in common disinfectants is not known. The conditions that induced acid-stress and alkali-stress adaptation and their stability were investigated for L. monocytogenes Scott A (serotype 4b) and Bug 600 (serotype 1/2a). We have also determined the effect of temperature on stress adaptation in L. monocytogenes and their cross resistance to other physiological stresses and disinfectants. Our results showed that 1 h pre-exposure in pH 5.0 was required to induce strong acid adaptation in L. monocytogenes. Also, just 15 min of pre-exposure in pH 9.0 was required to induce strong alkali-stress adaptation in L. monocytogenes. The inactivation effect of pH 3.5 or pH 12.5 was influenced by temperature which was as follows: 37C > 22C > 4C. Furthermore, acid adapted cells exhibited enhanced survival under lethal alkaline pH 11.5 or in 20 ppm lauric arginate. Compared to the non-adapted cells, acid-stress adapted cells were more sensitive to 1000 ppm hydrogen peroxide challenge. In another study, a group of 37 strains of L. monocytogenes representing all 13 serotypes were analyzed for their heat stress resistance. These strains were categorized into three group of low (strains with <2 log survival), medium (2 to 4 log survival) and high (4-6 log survival) heat tolerance. No specific serotype-based correlation was observed but extensive variation was observed for heat tolerance within L. monocytogenes strains. For example, of the three 1/2b serotypes tested, one was non-detectable and the other survived as high as 5.4 log CFU/ml. The heat-stress adapted cells of L. monocytogenes were found to be stable even if the cells are cooled to 4C for 24 h. These data suggests the potential for occurrence of acid-stress, alkali-stress and heat-stress adapted cells of L. monocytogenes serotypes that should be taken into account for enhancing the safety of fresh-cut vegetables and fruit industries.
<br/>PARTICIPANTS: R. Nannapaneni, Q. Shen, P. Pandare, P. Jangam, and K. Soni This research provides practical information to help fresh produce processing industries in Mississippi for enhancing the microbial quality and safety of minimally processed fresh fresh-cut vegetable and fruit products. This project also provides extensive research training to our graduate students so they are able to perform independent research.
<br/>TARGET AUDIENCES: Fresh produce processors in Mississippi, Fresh-cut vegetables and fruit processors in Mississippi, FDA, and National Integrated Food Safety Initiative.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: Current market dynamics have shifted towards large scale operation and bagging of fresh-cut minimally processed fruits and vegetables, and so if any contaminations occur, target bacterial pathogens can easily spread to the final product. Safety of fresh-cut vegetables and fruits against L. monocytogenes contamination is controlled by carefully selected hurdles. Ability of L. monocytogenes cells to survive in adverse physiological conditions is a serious food safety and public health concern. Any such stable physiological changes in response of environmental stress stimuli reflect the key changes instituted by the microbial cells at the gene or protein expression levels. Only by improved understanding of the fundamental changes occurring at the phenotypic and genotypic level in L. monocytogenes sub-populations in response to such adverse environmental conditions, we will get new insights that can be harnessed in developing more effective practical decontamination approaches for fresh-cut vegetables and fruits. Fruit and vegetable processing industries are continuously finding it a formidable challenge to develop ways to keep this organism out of the processing environments. The critical physiological and genetic properties of L. monocytogenes that make it persistent or nonpersistent in a produce processing environment are not well understood. Until now, all previous research has focused on the importance of general stress response and biofilm formation in L. monocytogenes. We have recently demonstrated the ability of some serotypes within L. monocytogenes that are strong biofilm producers than the others. We are currently indentifying the factors that contribute to the innate ability of L. monocytogenes to produce highly stable stress-resistant phenotypes that persist under various environmental conditions. This project is uncovering the critical factors responsible for triggering the occurrence/selection of highly stable stress-resistant phenotypes of L. monocytogenes in target stress environments. Further studies will determine the effect of such adaptation on the increased survival of L. monocytogenes cells in mildly processed fresh-cut vegetables and fruits during low temperature storage.