Duration: 10/01/2011 to 09/30/2016

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Consumption of fresh-cut produce increased at an annual rate of approximately 10% from 1995 to 2004 (UFPA, 2004) and the market for fresh-cut vegetables and fruits is estimated at $10-12 billion annually (UFPA, 2010). It is estimated that fresh-cut products currently make up more than 15% of all fresh produce marketed in the U.S (UFPA, 2010). Postharvest losses of fresh-cut produce are difficult to estimate but given the highly perishable nature of fresh-cuts compared to intact produce, the retail value of fresh-cut produce losses may exceed $1 billion annually.

The appearance, convenience, and generally high nutritive value of fresh-cut vegetables and fruits drive sales of fresh produce, but repeat sales of the fresh-cuts is dependent upon assurance of its safety and the products having pleasing texture and flavor. The industry primarily relies on established technologies derived mainly from practical experience to maintain visual quality and shelf-life with less consideration of the quality characteristics that drive repeat sales such as good flavor retention, maintenance of an appealing texture (crispness, crunchiness), and increased microbial quality leading to extended shelf stability and food safety. Through interaction with the industry we know that current technologies, especially for fresh-cut fruits, do not provide the shelf stability needed to supply long distance domestic markets with optimum flavor quality.

As a result of physiological and microbial deterioration occurring during storage and marketing of fresh produce, and especially fresh-cut produce, there is a need to develop effective, non-damaging treatments for maintaining the quality (appearance, flavor, texture, nutritional value) and food safety of fresh harvested produce (How, 1990). Most of the sales of fresh-cut produce have been in the vegetable (salad, carrot slice) area (Garrett, 2002) and commercial handling practices for fresh-cut vegetables have been described (Barth et al., 2004). Beginning about a decade ago, research and commercial interest has focused more on fresh-cut fruits and melons (Bai et al., 2004; Beaulieu et al., 2004; Beaulieu and Gorny, 2004; Beaulieu and Lancaster, 2007; Beaulieu and Lea, 2007; Bett-Garber et al., 2003; Dea et al., 2010; Kader, 2008; Plotto et al., 2010; Rojas-Grau and Martin-Belloso, 2008; Saftner et al., 2003; Soliva-Fortuny and Martin-Belloso, 2003). With over 200 different vegetable and fruit crops with potential for development as fresh-cut products, each with unique physiology and handling requirements, an integrated, scientific approach to research and development including microbiological interactions with these products is critically needed.

The conditions on the cut surface of fresh-cut products, with the presence of water and compounds that microbes can use for nutrition, provide ideal conditions for growth of microbes. Unfortunately, as produce consumption has increased in the U.S. in recent years, so has the number of produce-related outbreaks of foodborne illness. Produce-related outbreaks accounted for 12.3% of all reported foodborne outbreaks from 1990 to 2007, compared to only 0.7% in the 1970s (AFF, 2010, Sivapalasingam et al., 2004). About 23% of all foodborne illnesses from 1998 to 2007 were due to fresh produce (CSPI, 2009). Between 1996 and 2008, lettuce/leafy greens (32.9%), tomatoes (17.1%), and melons (15.9%) comprised two-thirds of produce-related outbreaks: (Gravani 2009). Pathogens of primary concern are E. coli O157:H7, Salmonella, L. monocytogenes, and Norwalk-like viruses. From 1996 to 2006, 72 foodborne illness outbreaks were associated with fresh produce consumption with 18 of these connected to fresh-cut produce (FDA 2008). The economic yearly losses due to acute foodborne illness are estimated to be $152 billion, with $39 billion of this loss associated with fresh, processed, and canned produce (Scharff 2010). The continuing nature of such produce-related outbreaks represents a threat to further increases in per capita consumption due to lowered confidence in the microbial safety of the product by the consuming public. Such outbreaks can also be very costly to growers, processors, shippers and restaurants.

It is very difficult to ascertain the efficacy of control measures for food safety as there are no direct measures of the effectiveness of intervention strategies on the rate of occurrence of foodborne illness in the general population. Instead, model systems are used to test the effectiveness of intervention strategies at selected stages of the processing chain. The hope is that by identifying and implementing numerous control strategies along the processing chain that were found to be effective in model systems, that the resulting net risk reduction will effectively reduce the real risk of foodborne illness. There are a number of opportunities to address food safety concerns as part of this project. Quality and safety concerns often overlap. For example, efforts to reduce spoilage organisms should also impact pathogenic organisms, and removing damaged produce prior to production will reduce the risks associated with pathogen colonization of wounds.

The Food & Drug Administration (FDA) in collaboration with the USDA and the Centers for Disease Control (CDC) issued a series of guidelines in 1998 (since updated) referred as Good Agricultural Practices (GAPs) to reduce the risk of foodborne diseases from fresh fruits and vegetables (U.S. FDA, 2008). Since that time, indicative of the importance of fresh fruit and vegetable food safety and security research, USDA has emphasized the enhancement of safety and security in its strategic planning and created the Food Safety and Quality National Education Initiative (FSQ), Special Research Grants for Food Safety, the Food Safety Institute of the Americas, and currently the National Food Integrated Safety Initiative. Members of S294 have been actively involved in all of these programs and many also are involved in extension food safety programs.

In order to ascertain food safety risks involving fresh and fresh-cut produce, it is critical to be able to determine the survival and persistence of viable or infectious human pathogens under environmental conditions occurring in produce handling and processing facilities, on harvested crops, and on intact or fresh-cut products. Therefore, methods for detection and enumeration of target microbes, including bacteria and viruses, are of core relevancy to this project. Approaches for detection and enumeration of microbes on fresh produce can play an important role in mitigation of fresh produce-associated spoilage or foodborne disease, as well, providing decision makers with timely and actionable data, especially on the presence of human pathogens in these products (Brehm-Stecher et al., 2009). These data could help guide interventions such as: refusal of contaminated product from the field, cessation of processing for line or equipment sanitation, destruction of contaminated product held in inventory pending testing results, or product recall. Due to the relatively short shelf lives of most types of fresh produce, rapid methods for detection and enumeration are of special relevance to the goals of this work.

Integration of physiological, pathological, food safety, and instrumental and sensory quality measurement concepts is essential for developing the most effective handling procedures and innovative, new technologies for maintaining quality and shelf stability of fresh-cut products. Much experimental work is needed to optimize and integrate new and emerging treatments in diverse fresh-cut products. This fact supports the proposed integrated approach of having parallel projects in different states and of focusing the research into specific areas of importance. Alternative and emerging technologies for maintaining the quality and shelf stability of fresh-cut produce are being introduced at a rate that often precludes thorough evaluation of instrumental and sensory quality attributes, and their impact on product nutritional value, microbial quality and food safety. To do so, a multidisciplinary approach as proposed herein also will be needed to optimize the new and emerging treatments.

Related, Current and Previous Work

A search of the CRIS database revealed no other multistate projects or coordinating committees dealing with fresh-cut vegetables and fruits, and also none with plant physiologists, food scientists, and microbiologists working together. This multistate project is needed in order to provide coordination and collaboration among the scientists working in this field if duplication of effort is to be avoided and the available time and resources are to be effectively applied. In this way, more effective approaches that are more widely applicable to different fresh-cut products may be more quickly developed.

A. Critical Review of Previous Project Accomplishments

This proposal is for a replacement project to S-294, Postharvest Quality and Safety in Fresh-cut Vegetables and Fruits, which has resulted in numerous collaborative activities including multiple federally funded grants with multistate collaboration. The project members developed information on postharvest treatment and storage effects on the nutritional value of fresh-cut products; standardized methods for subjectively evaluating postharvest quality-related changes; developed or evaluated new tools, treatments and cultivars to improve the quality and safety of fresh-cut vegetables and fruits; developed new information on the contamination and attachment of microbes to fresh-cut product as well as developing novel approaches to microbial control; developed standard practices for recovery, inoculation, detection, and enumeration of human microbial pathogens on produce; and elucidated the physiological processes underlying both positive and negative quality changes associated with fresh-cut processing.

The new project will emphasize standardization of microbiological procedures and instrumental and subjective methods for sensory quality analysis and flavor-based shelf life measurement and newly emerging treatments and techniques for assuring fresh-cut quality; similarly, we will consider physiological processes that control quality changes in fresh-cut products. We will develop standard protocols for evaluation of the efficacy of sanitizers and the appropriateness of experimental protocols for microbiological challenge studies with fresh-cut produce. New sanitizers, natural product antimicrobials, and physical treatments to control microbes will be tested. Because of the potential for treatment interactions between vegetable and fruit tissues and microbes, we plan on close coordination between microbiologists and plant/food scientists in all of the above activities.

B. Fresh-cut Product Quality

Pre-harvest conditions that stress the plant will affect the quality and shelf-life of the postharvest crop (Monselise and Goren, 1987; Nigh, 1990). Knowledge of these conditions is important for assessing postharvest potential of fresh-cut products (Blacharski et al., 2001; Borve and Sekse, 2000; Gorny et al., 1998; Kim et al., 1993). The maturity of fruits and vegetables intended for fresh-cut processing is a critical factor determining potential quality and shelf life (Bai et al., 2009; Beaulieu, 2005, 2007; Beaulieu et al., 2004; Soliva-Fortuny et al., 2002, 2004). Integration of cultivar selection, pre-harvest and postharvest conditions and treatments is needed to obtain the best possible quality of the marketed fresh-cut product (Beaulieu and Lea, 2003).

Understanding the components of eating quality involves comparison of instrumental with sensory analysis (Beaulieu and Baldwin, 2002; Jordán et al., 2001a,b; Plotto et al., 2000; Schieberle and Hofmann, 1997). Much more detailed sensory work needs to be done on fresh-cut produce treated in ways that might affect flavor and textural attributes and impact overall eating quality. Many attempts at measuring texture have used sensory analysis coupled with instrumental measurements (Abbott et al., 1984; Beaulieu et al., 2004; DeBelie et al., 2002; Drake, 1962; Harker et al., 2002; Mohamed et al., 1982; Szczesniak, 1963; Vickers, 1981; Vickers and Bourne, 1976; Vincent, 1998). There are no generally accepted definitions of textural attributes applicable to fresh-cut products, sensory scale anchors, or methods for their measurement (Fillion and Kilcast, 2002; Harker et al., 1997). The proposed project will include study of practical methods for measuring flavor and texture of fresh-cut vegetables and fruits, and relating to sensory measurements using modern multivariate statistical techniques.

C. Technologies for Maintaining Quality and Shelf Stability

1. Modified atmosphere packaging. Altering the gas mixture surrounding fresh-cut produce to a composition different from that of normal air is an effective tool to prolong quality retention, and this can be achieved by using packaging technologies (Kader et al., 1989). Essentially all fresh-cut products are handled in packages designed to maintain a modified atmosphere surrounding the enclosed product. Importantly however, good temperature control is essential for effective use of this modified atmosphere packaging (MAP; Beaudry et al., 2006).

MAP can reduce or delay the physical, chemical and microbiological changes caused in the produce during preparation (Gorny, 1997; Sandhya, 2010) and is largely used to suppress global metabolic activity through respiratory inhibition (Kaji et al., 1993; Soliva-Fortuny et al., 2005), inhibition of ethylene synthesis and action (Gil et al., 1998; Gorny et al., 1999; Rosen et al., 1989; Soliva-Fortuny et al., 2005), inhibition of oxidative processes (Escalona et al., 2010; Smyth et al., 1998), retention of ascorbic acid (Agar et al., 1999), and suppression of microbial growth (Bai et al., 2001; Gonzalez-Fandos et al., 2006). MAP also provides an enclosed system that minimizes water loss and thereby reduces shriveling, wilting, and the loss of fresh and glossy appearance. Dangers associated with MAP include potential induction of fermentation and resultant off-flavors (Zhang and Watkins, 2005).

The potential exists for the inclusion of additional bioactive compounds in the package or applied to the product that target human and plant pathogens. Such compounds include a number of natural plant volatiles (Utama et al., 2002) such as hexanal and trans-2-hexenal (Kubo et al., 2004; Lanciotti et al.2003, Patrignani et al., 2008; Song et al., 1996; Utama et al, 2002). These volatiles can also be effectively encapsulated in cyclodextrins and their release manipulated (Almenar et al., 2007a; b).

2. 1-Methylcyclopropene (1-MCP). 1-MCP blocks ethylene receptors, preventing ethylene effects (Blankenship and Dole, 2003; Sisler and Blankenship, 1996; Sisler and Serek, 1997; Watkins, 2002). Thus, 1-MCP can potentially be used to control ripening and senescence of fresh-cut products (Jeong et al., 2004; Toivonen, 2008; Vilas-Boas et al., 2007), microbiological growth (Zhou, 2006), browning and hydrolysis of ascorbic acid (Buda et al., 2003). Further, the cyclodextrin can be incorporated into polymers to further modify release kinetics (Lee et al., 2006).

3. Texture enhancers. Calcium salts have been used to decrease softening of fresh-cut fruits (Soliva-Fortuny et al., 2005). Calcium ions form cross-links between free carboxyl groups of pectin chains, resulting in strengthening of cell walls by formation of calcium pectates (Aguayo et al., 2008). Potassium has been reported to have both positive (Lester and Jifon, 2006) and negative (Pacheco et al., 2008) effects on fruit texture. Texture enhancers can be incorporated into the formulation of edible coatings (Oms-Oliu et al., 2008b; Rojas-Grau et al., 2009).

4. Antibrowning agents. Browning caused by polyphenoloxidase (PPO) can be controlled by reducing the O2 content of the surrounding atmosphere or by using antibrowning agents, which can be grouped into different categories: acidulants, reducing agents, chelating agents, complexing agents, enzyme inhibitors, and synergistic combinations (Fan et al., 2005). Son et al. (2001) reported that the antibrowning compounds oxalic acid, oxalacetic acid, ascorbic acid-2-phosphate, cysteine, glutathione, N-acetylcysteine, kojic acid and 4-hexyl resorcinol showed the highest browning inhibitory activity on fresh-cut apples among 36 known antibrowning compounds.

5. Naturally occurring compounds. Naturally occurring compounds have been also explored as flavor enhancers, and antibrowning agents. Some honeys and juices have shown antibrowning effects on fresh-cut apple slices (Jeon and Zhao, 2005; Song et al., 2007). Other natural antibrowning agents such as hexylresorcinol, N-acetylcysteine, and glutathione have been reported to reduce the browning of Fuji apple pieces with results significantly better than for ascorbic acid (Rojas-Grau et al., 2008a,b).

6. Edible coatings. Edible coatings act via their barrier properties to water and gases (Baldwin et al., 1995a). Benefits of edible coatings on fresh-cut produce are reduced browning (Beaulieu and Gorny, 2004, Baldwin et al., 1996), respiration (Banks, 1984), ethylene production (Wong et al., 1994), moisture loss (Avena-Bustillos et al., 1997), and surface discoloration (Banks, 1984), as well as enhanced flavor volatiles and texture retention (Baldwin et al., 1995b; Guilbert et al., 1996). Edible coatings can also extend fresh-cut shelf life by carrying active ingredients such as antibrowning agents, antimicrobial compounds, texture enhancers, and nutraceuticals (Han et al., 2004b; Hernandez-Munoz et al., 2008; Lee et al., 2003; Oms-Oliu et al., 2008a; Perez-Gago et al., 2006; Raybaudi-Massilia et al., 2008a; Rojas-Grau et al.; 2009; Waimaleongora-Ek et al., 2008; Zhao, 2010). New generation edible coatings focus on incorporating and controlling the release of active compounds using nanotechnology (Lopez-Rubio et al., 2006) and multi-layer nanolaminates (Weiss et al., 2006).

7. Mild heat stress. Wounding of plant tissues results in degradative oxidative processes including browning, lipid peroxidation, and flavor changes (Hodges et al., 2004; Toivonen, 2003). However, plants also respond to abiotic and biotic stresses by up-regulation of their antioxidant system (Baker and Orlandi, 1995; Bray et al., 2000; Wang et al., 2003), which serves to protect against those processes by acting as reducing agents, free radical terminators, metal chelators, and singlet oxygen quenchers and by mediating the activity of various oxidizing enzymes (Ho, 1992; Okuda, 1993; Toivonen, 2004). Nonlethal heat stress has been shown to stimulate production of bioactive compounds (Lemoine et al., 2009), delay senescence, and improve quality of fresh-cut products (Sgroppo et al., 2009).

8. Cyclodextrins. Cyclic oligosaccharides composed of ±-1,4-coupled D-glucose units have been tested as antibrowning agents on fresh-cut products because they have a chemical structure similar to that of the complexing agents used to trap or form complexes with the substrate of PPO or with reaction products. Their inhibitory effect on PPO has been reported to be more effective than some other chemicals on fresh-cut Red Delicious apples (Alvarez-Parrilla, 2007). Since cyclodextrins have a hydrophilic exterior and a hydrophobic cavity, they can form inclusion complexes with a variety of hydrophobic molecules. Ayala-Zavala et al. (2008a, b) described a cyclodextrin essential oil microcapsule that could be used to increase the shelf-life of fresh-cut products. Almenar et al. (2007a, 2007b) used cyclodextrins to control the release of the antimicrobial volatiles hexanal and acetaldehyde.

D. Physiology of Fresh-cut Products

The physiology of fresh-cut vegetables and fruits is typical of that observed in plant tissues that have been wounded or exposed to stress conditions (Brecht et al., 2004). This includes increased respiration and ethylene production. Other consequences of wounding include oxidative browning, lipid oxidation, and enhanced water loss. Minimizing the negative consequences of wounding in fresh-cut products can result in increased shelf-life and greater maintenance of nutritional, appearance, and taste quality.

1. Appearance versus flavor-based shelf life. Shelf life is generally based on appearance, but internal quality characteristics may deteriorate faster than external characteristics. There are three main reasons for the general decline in flavor of fresh produce: genetics, harvest maturity, and postharvest handling (Baldwin and Plotto, 2007). Both harvest maturity and postharvest handling techniques are often geared toward extending the shelf life of fresh produce after harvest, sometimes with negative impacts on flavor quality. This is especially problematic for fresh-cut products, for which flavor loss may be due to metabolic changes related to wounding, off-gassing of volatile compounds due to removal of diffusion barriers, and altering of atmosphere through necessary packaging (Forney, 2008).

Quality and quantity of flavor components have been shown to vary among varieties of fruits and vegetables. Preharvest factors, including environmental influences, during the production season can affect flavor quality (Baldwin et al. 1995c; Beaulieu, 2005; Romani et al. 1983; Thybo et al., 2006; Wright and Harris 1985). Harvest maturity, especially for climacteric fruits that ripen after harvest, strongly affects flavor life (Bai et al., 2009; Beaulieu, 2005, 2007; Beaulieu et al., 2004; Soliva-Fortuny et al., 2002, 2004). Even nonclimacteric fruits like citrus have an optimal harvest maturity window for flavor (Obenland et al., 2009).

Quality of fresh-cut products is highly dependent on minimizing injury to the product. Wounding of plant tissues may cause elevated ethylene production and membrane lipid degradation (Deschene et al., 1991; Picchioni et al., 1994; Rolle and Chism, 1987; Zhuang et al., 1997). Extensive enzymatic degradation occurs in damaged membrane systems (Marangoni et al., 1996). Discoloration occurs when the products of phenylpropanoid metabolism and possibly other substrates (e.g., anthocyanins) are oxidized by phenolases such as polyphenoloxidase (PPO) or peroxidases. Wounding also induces synthesis of enzymes involved in browning reactions (Martinez and Whitaker, 1995) or substrate biosynthesis (Ke and Saltveit, 1989). It has been clearly shown that the degree of injury incurred during the cutting process has a tremendous influence on slice quality and shelf-life (Portela and Cantwell, 2001; Toivonen et al., 2005).

2. Nutritional components in fresh-cut produce. Intact and fresh-cut vegetables and fruits are important dietary sources of vitamin A, C and E, minerals, carotenoids, polyphenols (flavonoids), and other antioxidant phytochemicals. However, little is known of the effects of fresh-cut processing technologies on the nutritional quality of fresh-cut products. Cutting increased the phenolic content and antioxidant capacity of fresh-cut lettuce and other vegetables (Heredia and Cisneros-Zevallos, 2009). This suggests that stressful treatments such as fresh cutting can increase nutrient levels in some commodities under certain circumstances. More information is needed related to phytonutrient levels in fresh-cut produce and how emerging fresh cutting technologies are impacting phytonutrients. Measurement of antioxidant capacity will also be important in order to better understand the potential health implications of these changes. This can most expediently be done by measuring changes in the two most important antioxidant functionalities: 1) reducing capacity, and 2) peroxyl radical scavenging capacity (Toivonen and Hampson, 2010).

E. Microbiology and Food Safety of Fresh-cut Products

1. Methods for recovery, inoculation, detection, and enumeration/quantification of fecal contamination, biofilms, and human pathogens. Pre-analytical sample preparation: Although numerous rapid detection methods are available commercially or are being developed, detection methods alone are inadequate. As detection is a final downstream event, the value of the information that it provides is dependent on critical upstream inputs such as effective separation of target cells from the food matrix and their subsequent concentration to analytically suitable volumes (Brehm-Stecher et al., 2009; DSouza et al., 2009). Therefore, development of methods for effective pre-analytical sample preparation will play an integral role in the work proposed here.

Enumeration: Although presence/absence testing for pathogens serves a vital role in food safety surveillance, quantitative information on pathogen loads is required to understand pathogen behavior in foods or in processing environments, to explore kinetic phenomena such as pathogen growth or inactivation and to provide critical input for microbial risk analyses (Brehm-Stecher et al., 2009). Therefore, methods capable of providing enumerative data on target organisms, especially human pathogens, are expected to be of special importance to the proposed work.

Biofilms and inoculation studies: The dominant microbial phenotype in nature is the biofilm, not individual, free-living cells (Bisha and Brehm-Stecher, 2009a; Fett et al., 2006). Understanding the complex microbial interactions on produce surfaces will be an important focus of the proposed project. Approaches for artificial inoculation of microbes onto produce surfaces, with the goal of more accurately representing processes occurring in the field or in processing environments, will also be a focus of this work.

Microbial interactions on produce or processing surfaces: Tape-based sampling methods have recently been combined with whole-cell molecular identification via fluorescence in situ hybridization (FISH), to enable rapid, visual identification of specific pathogens on produce surfaces (Bisha and Brehm-Stecher, 2009b). Because tape-based sampling approaches preserve the spatial relationships of microflora present on plant surfaces, they could enable characterization of competitive or metabiotic interactions between plant saprophytes or spoilage organisms on these surfaces or on inanimate processing surfaces (Bisha and Brehm-Stecher, 2009b).

Viruses: Human noroviruses remain the leading cause of viral gastroenteritis outbreaks worldwide (Siebenga et al., 2009). Newly emergent strains are highly virulent and can be life threatening, especially to the elderly and immuno-compromised (Siebenga et al., 2009). Other viruses, such as hepatitis A, are significant because of the severity of disease they cause. Outbreaks of foodborne viruses have been linked to fresh produce such as green onions, lettuce, raspberries or frozen strawberries and raspberries. Methods for rapid and sensitive detection of viruses are needed. Promising approaches include real-time reverse-transcriptase-PCR, nucleic acid sequence based amplification (NASBA), and loop-mediated isothermal amplification (LAMP) assays.

2. Sanitizers; alternatives/natural products; antimicrobial efficacy and sensory effects. Washing fresh-cut produce after cutting and prior to packaging is an important step in reducing microbial populations. Different washing chemical agents have been studied to determine their efficacy in the inactivation of pathogenic bacteria (Fan et al., 2009; Narciso and Plotto, 2005; OConner-Shaw et al., 1996; Ukuku and Fett, 2004; Ukuku et al., 2004; Yuk et al., 2005). Combining levulinic acid with sodium dodecyl sulfate (SDS) dramatically increased the bactericidal activity against both Salmonella and E. coli O157:H7 populations (Zhao et al., 2009). However, lettuce treated with levulinic acid and SDS developed sogginess and loss of texture (Guan et al., 2010). Organic acids have shown to be effective in reducing bacteria populations on some fresh-cut fruits and vegetables.

Gaseous antimicrobials such as chlorine dioxide (ClO2) and ozone (O3) can penetrate to sites that liquid sanitizer cant. This makes gaseous antimicrobials attractive for fresh-cut applications. Chlorine dioxide has been successfully applied to a number of crops (Du et al. 2003, Han et al. 2001; 2004a; Lee et al., 2004; Sapers et al. 2003), but ClO2 gas can have a negative impact on visual quality of leafy vegetables (Mahmoud and Linton 2008). Therefore, processing conditions would likely need to be altered for consumer acceptance and commercial applications. Gaseous O3 has been found to be effective in reducing S. enteritidis on tomatoes (Da_ et al., 2006) and Salmonella on cantaloupe melons (Selma et al., 2008).

Ionizing irradiation is effective in inactivating pathogens either on the surface or inside of fresh produce (Fan et al., 2008). Other new food processing technologies such as cold plasma, in-packaging sanitization, ultraviolet irradiation, and high hydrostatic pressure need to be investigated for their feasibility on fresh and fresh-cut produce.

Unlike chemical sanitizers that only affect the surface of produce, hot water can inactivate bacteria below the produce surface (Breidt et al. 2000), and thus, is potentially more effective than chemical washes (Annous et al. 2004, Breidt et al. 2000, Lichter et al. 2000). Treatment of whole cantaloupes with 85°C water for 60 sec reduced Salmonella spp. by ca. 4.6 log CFU per cm2 of rind (Solomon et al., 2006). Hot water pasteurization of cantaloupes reduced native bacteria, yeast and mold populations, which also frequently resulted in lower microbial loads on fresh-cut fruit (Fan et al., 2008). No negative effect by the hot water treatment on sensory quality or vitamin C content of fresh-cut cantaloupes was observed.

Natural products and generally regarded as safe (GRAS) substances that often have antimicrobial/antioxidative activities or otherwise maintain the freshness and quality of the fresh-cut produce are of interest (Tiwari et al., 2009). In many cases, the concentration of the antimicrobial compound in its natural form is too low to be successfully used without damaging the sensory characteristics of the final product (Bagamboula et al., 2004).

Most chemical sanitizers are oxidants that can cause bleaching and other discoloration of fresh and fresh-cut produce. Because of their oxidative nature, these chemicals may not be able to be applied directly with antioxidants. However, treatment with oxidative sanitizers followed by treatment with an antioxidant may effectively maintain microbial quality of fresh-cut fruits. Several classes of surface active agents approved for food use can aid penetration of substances within apples (Saftner et al., 1997) and other produce, but their compatibility with fresh-cuts and their efficacy when combined with sanitizers have not been well studied.

3. Antimicrobial edible coatings. Some coating materials have antimicrobial functions (Chien et al., 2007; Iverson and Ager, 2003; Park et al., 2005; Vargas et al., 2006; Zhang and Quantick, 1998) and antimicrobial agents may be incorporated into edible coatings (Du Plooya et al., 2009; Franssen and Krochta, 2003; Garcia et al., 2001; Lee et al., 2003; Park et al., 2004; 2005; Raybaudi-Massilia et al., 2008a; b; Rojas-Grau et al., 2007) to extend shelf-life and enhance microbial safety of fresh-cut produce (Lin and Zhao, 2007; Vargas et al., 2006). A new patented edible film comprising organic acids, protein, and glycerol can inhibit human pathogen growth, including L. monocytogenes, S. gaminara, and E. coli 0157:H7 (Hettiarachchy and Satchithanandam, 2007). The function and application of some antimicrobial coatings in fresh and fresh-cut produce have been discussed extensively in several review articles including Lin and Zhao (2007), McHugh et al., (2009), and Rojas-Grau et al. (2009).

Note that while we will not be conducting research on HACCP, the results from our project may help strength HACCP programs, such as development of rapid and sensitive detection methods for the identification of biological hazards (foodborne pathogens), and also development of new sanitizers, treatments and technologies for controlling pathogens.

Objectives

1. Evaluate methods of sampling and measuring flavor and nutrition of fresh-cut products to facilitate comparison to traditional shelf life factors.
   
2. Develop new strategies to improve and better maintain inherent fresh-cut product quality and nutrition.
   
3. Improve understanding of physiological mechanisms that affect fresh-cut product quality.
   
4. Determine critical factors in controlled inoculation studies with human pathogens and surrogates that influence the outcome of quantitative microbial risk assessments.
   
5. Evaluate and control unintentional and intentional microbial contamination of intact and fresh-cut produce.
   

Methods

All produce will be appropriately prepared and cut under highly sanitary conditions at refrigerated temperatures where the processing area, tools, and gloved hands are appropriately sanitized and personnel wear proper clothing to protect the cut produce from contamination. Standard sanitation procedures to be used in conducting experiments will be determined by consensus of the participants. Any post-cutting treatments and packaging will also be performed using good manufacturing practices. After treatment, the fresh-cut products, along with intact control samples, will be stored using appropriate refrigerated temperatures and durations depending on the commodity, its stage of maturity at harvest or upon treatment, and the storage temperature of the intact produce prior to processing. Standard sensory and instrumental measures of flavor quality to be used in work encompassing the first three objectives will be developed by project members in CA, FL, ARS-FL, and ARS-LA. Visual quality of fresh-cut products will be evaluated by applying standard hedonic scoring systems, reflectance color measurements, and spectrophotometric analysis of chlorophyll, anthocyanin, carotenoid, and phenolic pigments. Textural alterations will be analyzed using mechanical measurements of tissue firmness. Since apparent responses to temperature, ethylene, etc. can be strongly affected by different fresh-cut preparation procedures, certain basic preparation procedures such as slicing procedures, slice or chunk sizes, and sanitation methods will need to be agreed upon, especially by those participants working with the same or similar types of products. The participants will agree on standard anti-browning and texture stabilizing treatments for products being studied at different stations. Similarly, standard hedonic scoring systems and physical measurement methods for color and texture for each common product will be used as much as possible. Flavor-related factors will be measured upon sampling or removal from packages or storage containers after a standard, specified period of time at a specified temperature. Objective 1 Procedures: 1) We propose to compare the methods being used by project members for consumer and sensory panel testing of fresh-cut products and develop standard procedures. 2) We will conduct coordinated shelf life tests and develop standard procedures for measuring the shelf life of fresh-cut products in terms of retention of acceptable flavor versus physicochemical properties, including nutritionally important constituents. 3) We will relate instrumental with sensory measurements of fresh-cut product quality using newly developed instruments and statistical techniques. For comparison of techniques between laboratories, fresh-cut products will be shared among participants and quantitative descriptive analysis, flash profiling, ranking, or sensory discrimination analysis in conjunction with signal detection theory will be used, depending on respective resources. The same preparation protocol for each product being tested will be used at all participating locations, and comparison between laboratories made for descriptive analyses. Quality evaluations comparing sensory data with physicochemical analyses will be conducted with standard and novel instrumentation. Shelf life in terms of appearance, texture, nutritional value, and flavor will be compared and, specifically, interactions between volatile and non-volatile components of flavor will be determined. The chemical data for flavor compounds will be combined with sensory data to determine what type of aroma profile and sugar/acid ratios give the highest flavor quality (preference) or off-flavor (low preference) ratings for fresh-cut products. Standardized sensory evaluation methods and objective methods of measuring color, texture, and composition of fresh-cut products will be proposed. Compositional measurements will include constituents related to flavor (sugars, organic acids, aroma volatiles) and nutritional value [ascorbic acid (vitamin C), carotenoids, polyphenols (flavonoids), and other antioxidant phytochemicals]. Potential methods will be discussed, agreed upon, and revisions made, and the procedures finalized and distributed within and outside the multistate project. The updated sensory analysis guide will be a collaborative effort between, and not exclusive to, participants from BC, CA, FL, ARS-FL, LA, ARS-LA, ARS-MD, MI, ARS-PA, and TX. Objective 2 Procedures: 1) We will work with public and private entities to identify germplasm with outstanding sensory quality. 2) We will identify optimum initial (whole product) quality factors relating to improved flavor-based shelf life. 3) We will investigate improved processing and packaging strategies to better maintain fresh-cut product quality as compared to standard commercial practices. Genotype selections will be based on reduced or delayed ripening characteristics, better appearance and flavor, enhanced texture retention, and higher nutritional value. Sensory evaluations will be conducted by untrained panelists and by trained judges depending on the nature of the evaluation. Both intensity and overall acceptability characteristics of the fresh-cut products will be evaluated and later standardized. Work in this area will be conducted in BC, FL, ARS-FL, ARS-LA, ARS-MD, MI, and Spain. Selection of optimal quality produce for fresh-cut processing is another important consideration. Since fresh-cut products are intended for immediate consumption, fresh-cut fruit should be ripe or nearly ripe and vegetables should be fresh and showing no signs of senescence. Studies on initial product quality including optimal fruit maturity, preconditioning, and trait targeting will be emphasized; non-destructive instrumental measurements of vegetable and fruit quality will also be evaluated. Work in this area will be conducted in CA, FL, ARS-FL, MI, Italy and Spain. Pre-cutting treatments of intact produce destined for fresh-cut products will target ethylene synthesis or action, undesirable enzyme activities, and/or microbial loads of the fresh-cut products. We will evaluate 1-MCP and heat treatments and coordinate with the microbiologists in testing emerging sanitizers. Post-cutting treatments to be tested will include MAP; 1-MCP, natural products, and edible coatings (with or without food additives, antimicrobials and preservatives). Pectinesterase application with and without calcium in order to firm the tissue by creating pectin crosslinking will be evaluated for its effect on fresh-cut fruit tissue softening and watersoaking development during storage. Rinsing the cut fruit with buffered alkaline solution will also be tested to determine if hyper-acidification of the cut surface due to vacuole rupture might play a role in the development of watersoaking and softening due to activation of hydrolases in the cell wall and membrane. New alternatives for fresh-cut produce packaging will be developed based on testing and evaluating the effect of emerging packaging materials (bio-based films and others), and technologies (active and intelligent packaging) on fresh-cut product quality and safety. New and current packaging systems will be compared to evaluate improvements in quality and safety of the fresh-cut products. The behavior of different packaging systems in the cold chain will be evaluated for improvement of safety and quality of fresh-cut fruits and vegetables. Quality evaluations to assess the effects of product selection, pre- and post-cutting treatments, packaging technologies, and changes during storage include physicochemical analyses and sensory evaluations. Our strategy will be to first evaluate the effects of chemical and physical treatments on visual quality to determine whether the treatments are worth further study in terms of more complex physicochemical analyses and sensory evaluations during post-treatment storage periods at 1 to10°C depending on commodity and commercial practices. Physicochemical techniques will include mostly instrumental measurements of surface pH, phytonutrient levels, surface color, firmness, sugar and acid levels, aromatic volatile abundance, microbial loads, and dissolved solid/electrolyte contents. Standardized methods developed in our previous project or in Objective 1 will be used. The effects of various pre- and post-cutting treatments on fresh-cut vegetable and fruit quality attributes including microbe levels is an area that will involve interaction between the physiologists and microbiologists in the project. Work in those areas described above will be conducted in AL, BC, CA, ARS-FL, FL, GA, IA, LA, ARS-LA, MI, ARS-MD, NS, OR, ARS-PA, TX, Italy and Spain. Objective 3 Procedures: In this objective, we will concentrate on investigating the role of ethylene and stress physiology in fresh-cut product quality and shelf life. We propose to determine the effects of wounding and heat stress on the tissue antioxidative capacity and concentrations of bioactive components during storage of fresh-cut vegetables and fruits. We will measure the levels of active oxygen species and other free radicals in response to fresh-cut processing and investigate how postharvest treatments such as MAP and heat can be used to enhance the plant antioxidant system in order to prevent the accumulation of those damaging compounds (BC and FL). Ethylene-dependent and ethylene-independent wound responses in fresh-cut vegetables and fruits will be investigated in CA and FL by utilizing inhibitors of ethylene binding such as 1-MCP. The role of membrane deterioration in terms of electrolyte efflux (FL) and analysis of lipoxygenase and phospholipase action (FL and ARS-MD) will also be investigated. Standard spectrophotometric and HPLC analyses of chlorophyll, anthocyanins, carotenoids, flavonols, tocopherols, ascorbates, and phenolic pigments (Tomás-Barberán et al., 1997) will be used. Antioxidant capacity will be measured using the oxygen radical absorbance capacity (ORAC) assay. Contents of cysteine, reduced and oxidized glutathione and activities of dehydroascorbate reductase, ascorbate peroxidase, peroxidase, catalase, superoxide dismutase, glutathione reductase, and glutathione S-transferase, will be measured as previously described (Soto-Zamora et al., 2005). Lipid class analysis will be conducted as previously described by Picchioni et al. (1996), and lipid degrading enzyme activity as described by Todd et al. (1992). Objective 4 Procedures: 1) We propose to develop research best-practice guidance and standardized methods for food safety risk assessments of fresh-cut product quality enhancing treatments or efficacy of disinfection measures. 2) We will evaluate the influence of inoculum production, method of application, rate of drying, potential for sub-lethal injury on quantitative and qualitative recovery of pathogens, duration and condition of storage, biofilm development, and method of viable recovery. 3) We will validate culture-based and non-cultural methods for improved sensitivity and specificity of detecting fecal contaminants on fresh-cut products and processing environments.4) We will determine the potential for fresh-cut product:modified atmosphere packaging combinations and conditions to influence pathogen gene expression related to virulence following consumption. Real-time RT-PCR methods for the detection of human noroviruses from intact and fresh-cut produce (leafy greens, tomatoes, green onions, blueberries) will be developed and optimized (TN). Real-time RT-PCR methods and rapid alternatives to direct nucleic acid sequencing for the detection or fingerprinting of Salmonella spp., Escherichia coli O157:H7, Listeria monocytogenes and other human pathogens from intact or fresh-cut produce, including leafy greens, tomatoes, and peppers, will be developed (IA, TN). A multiplexed system for simultaneous detection of human noroviruses and Salmonella spp. will be developed for detection of these pathogens on fresh produce such as leafy greens and tomatoes (TN). New approaches for selective concentration of target pathogens from fresh produce or processing effluents will be developed, including exploration of alternative binders for selective pathogen capture (IA). Additional approaches for pre-analytical sample preparation (capture, physical or chemical detachment and concentration of pathogens, removal of inhibitors, etc.) will be developed for use on intact and fresh-cut produce (IA, TN). Objective 5 Procedures: 1) test novel intervention strategies and compare to standard commercial practices Commodity tolerance at different maturity stages to hot water immersion over a range of temperatures and effects on pathogen populations will be determined (FL, ARS-PA).Whole produce (tomatoes, pineapples, mango) will be inoculated with the appropriate pathogen of interest or its surrogate to give an initial microbial load of 106 CFU/g. The potential for reducing the transfer of microorganisms of interest from the peel to the flesh of produce during fresh-cut preparation will be evaluated by testing of fresh-cut pieces for pathogen contamination. The potential for injured cells of interest to grow following storage will be also evaluated after 1 to 21 days in storage by plating to selective and nonselective media. Subtracting populations enumerated on selective media from those on nonselective media gives an estimate of the number of injured cells present. The impact of hot water immersion on the produce quality will be evaluated using sensory evaluation and chemical and flavor analyses in conjunction with physiologists and food scientists working on Objectives 1-3 (CA, FL, ARS-FL, ARS-LA). Studies will be conducted to evaluate ClO2 and O3 gas on whole as well as fresh-cut fruits and vegetables (CA, FL, ARS-PA, CA). Whole produce (initially melons and tomatoes) will be inoculated as described above. Gas treatment will be conducted in specially designed fumigation chambers for produce. Various times of exposure and ClO2 gas concentrations will first be tested for the effect on produce sensory qualities and shelf-life. Exposure times and concentrations will be optimized at 4 or 20°C at various relative humidities for reduction or elimination of pathogens on inoculated fresh produce. Chlorine dioxide gas will be generated on-site using different available generation technologies. Residual microbial populations will be enumerated as described above. ClO2 in the packages of fresh-cut lettuce, strawberries, and apples will be generated by controlled released sachets. The fruits inoculated with Salmonella or E. coli O157:H7 will be subjected to various amounts of ClO2 and release rates at different temperatures. Microbial populations will be enumerated during storage at 4°C. In-package ozonation will be evaluated for tomatoes and cut-lettuce in modified atmosphere packages (MAP). Ozone will be produced inside MAP by PlasmaLabel technology in which thousands of ppm of O3 can be produced within 1 min. In addition to O3, other reactive species and UV light that are capable of destroying pathogens are also generated in the package. The efficacy of the in-package ozonation system in inactivating human pathogens and maintaining quality of tomatoes and lettuce will be evaluated during storage at 10°C and 4°C, respectively. Common chemical sanitizers (acids, chlorine, peroxiacetic acid, and hydrogen peroxide) will be combined with surfactants and other treatments (hurdle technology), such as ultrasonic and UV to increase the efficacy of chemical washes (GA, ARS-PA). Novel chemical approaches, including potentially synergistic systems containing mixtures of functional food ingredients, will be evaluated for the control of human noroviral surrogates and bacterial pathogens on intact fresh produce such as leafy greens and tomatoes (IA, TN). Use of natural antimicrobial compounds (phyto and bacto antimicrobials) to inactivate food borne pathogens in fresh and fresh-cut produce will be evaluated (OR, IA, ARS-PA). Value-added materials from agricultural waste streams, such as grape seed extract and other phenolic-rich plant extracts, will be evaluated as potential antimicrobial treatments for control of human pathogens on fresh produce (IA). Novel antimicrobial packaging and coating for fresh-cut fruits and vegetables will be developed (FL, MI, ARS-PA). Use of chitosan and its water-soluble derivatives will be evaluated as an ecofriendly approach for the control of human noroviral surrogates on intact or fresh-cut produce including lettuce and tomatoes (TN) The impact of food safety intervention treatments and technologies on quality of fresh and fresh-cut produce will be determined, and means to minimize the loss of quality and accumulation of chemical by-products will be evaluated (ARS-PA, ARS-MD). Possible use of nonthermal processing (ionizing radiation and UV) to enhance microbial safety and maintaining the freshness of fresh-cut fruits and vegetables will be studied (ARS-PA).

Measurement of Progress and Results

Outputs

• The S-294 Technical Committee will issue annual project reports highlighting the results for the previous year, which will be made generally available on the project website.
• Participants will submit research findings for publication in peer reviewed and non-refereed journals and trade publications.
• Standard sensory and instrumental measures of flavor quality will be developed
• Standard procedures for measuring the shelf life of fresh-cut products in terms of retention of acceptable flavor will be developed.
• Fruit and vegetable germplasm with outstanding sensory quality for use as fresh-cut products will be identified.
• Optimum initial (whole product) quality factors relating to improved flavor-based shelf life of fresh-cut products will be identified for various fruits and vegetables.
• Improved processing and packaging strategies to better maintain fresh-cut product quality will be identified or developed.
• Effects of wounding and heat stress on the tissue antioxidative capacity and concentrations of bioactive components during storage of fresh-cut vegetables and fruits will be determined.
• Ethylene-dependent and ethylene-independent wound responses in fresh-cut vegetables and fruits will be identified.
• Research best-practice guidance and standardized methods for food safety risk assessments of fresh-cut product treatments will be developed.; We will determine the influence of inoculum production, method of application, rate of drying, potential for sub-lethal injury on quantitative and qualitative recovery of pathogens, duration and condition of storage, biofilm development, and method of viable recovery.
• We will validate culture-based and non-cultural methods for improved sensitivity and specificity of detecting fecal contaminants on fresh-cut products and processing environments.
• We will determine the potential for fresh-cut product:modified atmosphere packaging combination's and conditions to influence pathogen gene expression related to virulence following consumption.
• The tolerance at different whole and fresh-cut products to hot water immersion and effects on pathogen populations will be determined.
• The efficacy of ClO2 and O3 gas in reducing microbial populations on whole as well as fresh-cut fruits and vegetables will be determined.
• The efficacy of common chemical sanitizers when combined with surfactants and ultrasonic and UV will be determined.
• Potentially synergistic systems containing mixtures of functional food ingredients will be evaluated for the control of human noroviral surrogates and bacterial pathogens on leafy greens and tomatoes.
• Natural antimicrobial compounds to inactivate food borne pathogens in fresh and fresh-cut produce will be evaluated.
• Novel antimicrobial packaging and coating for fresh-cut fruits and vegetables will be developed.
• Chitosan and its water-soluble derivatives will be evaluated as an ecofriendly approach for the control of human noroviral surrogates on intact or fresh-cut produce.
• The potential for use of ionizing radiation, UV, and high hydrostatic pressure to enhance microbial safety and maintaining the freshness of fresh-cut fruits and vegetables will be determined.
• A final report will be issued at the conclusion of the project.

Outcomes or Projected Impacts

• Relevant information will be available to fresh-cut processors to assist them in making decisions on harvest maturity, processing procedures, handling and packaging conditions to best maintain fresh-cut product quality and safety.
• The fresh-cut industry will achieve considerable savings (potentially millions of dollars a year) from reductions of product losses and recalls.
• Consumers will benefit from increased availability of fresh-cut products with improved sensory quality and higher nutritional value through improvements in cultivars and more effective preparation and handling practices.
• Incidence of fresh-cut products at retail with insufficient shelf life for consumer satisfaction will decrease.
• Human health will be improved as a result of increased consumption of vegetables and fruits.
• Availability of best-practice guidance and standardized methods for food safety risk assessments of fresh-cut product treatments will reduce the likelihood of food safety events by replacing ineffective food safety practices with science-based procedures.
• Food safety risk will be reduced through availability of new, more efficacious, strategies for controlling human pathogens.
• Researchers will have standard protocols for quantifying flavor-based shelf life and standard microbiological methods.
• Longer-term scientific benefits will be derived from obtaining a better understanding of ethylene and stress physiology of wounded plant tissues.

Milestones

(2012): Redesign, expand and update the content of the project website. Compile the methods being used by project members for consumer and sensory panel testing of fresh-cut products in order to develop guidelines for standard sensory and instrumental measures of flavor quality for use in further project research. Organize a task force to develop the procedures for assessing food safety risks in fresh-cut processing. Identify the most promising intervention strategies to reduce microbial populations on fresh-cut products.

(2013): Identify fruit and vegetable cultivars that possess the highest levels of flavor-based quality factors and nutritionally important components for use in further project research efforts. Identify the critical factors in controlled inoculation studies with human pathogens and surrogates. Initiate collaborations between participating institutions for multi-station coordinated testing of sanitizers for fresh-cut products and for measuring shelf life of fresh-cut products. Obtain stakeholder feedback on quality and safety procedures and activities planned so far.

(2014): Establish collaborations between participating institutions for research on new pre-cutting and post-cutting treatments including packaging to better maintain fresh-cut product quality.

(2015): Identify additional or alternative intervention strategies to investigate for controlling human pathogens on fresh-cut produce; prioritize the approaches and conduct collaborative studies. Identify other pre-cutting and post-cutting treatments to better maintain fresh-cut product quality; prioritize the approaches and conduct collaborative studies.

(2016): Develop research best-practice guidance and standardized methods for food safety risk assessments of fresh-cut product, quality enhancing treatments, and efficacy of disinfection measures; present the recommendations to the industry. Issue final project report.

Projected Participation

View Appendix E: Participation

Outreach Plan

Results from the proposed research activities will be disseminated via presentations at scientific and industry meetings and conventions (especially the United Fresh Produce Association), and will be published as project reports posted to the S-294 web site, as well as in peer-reviewed and non-refereed (popular) publications. Participation by project members involved in undergraduate teaching, graduate student advisement, and extension activities associated with Land Grant Universities will promote the general dissemination of knowledge developed in the proposed project.

Organization/Governance

The offices of the Technical Committee will be the chair, vice-chair, secretary, and past-chair and will serve as the Executive Committee. The first three officers will be elected for one-year terms at the organizational meeting for the Technical Committee. Thereafter, a secretary will be elected annually at the Technical Committee meeting. All voting members of the Technical Committee will be eligible for office. The officers will be promoted biennially in the following sequence: secretary to vice-chair, vice-chair to chair, chair to past-chair. These four officers will then constitute the Executive Committee to provide leadership and continuity and/or immediate action. The duties of the chair, vice-chair, and secretary will be as prescribed in the Guidelines for Multistate Research Activities; that of the past-chair will be to serve as a resource for the other committee members in carrying out their duties.

The project will have two subcommittees with chairs appointed by the Executive Committee chair. These will be the 'Quality and Physiology Subcommittee' and the 'Microbiology Subcommittee'. Members of each subcommittee will be those Technical Committee members whose research falls under the subcommittee area. The purpose of the subcommittees is to coordinate research activities within each research area, foster grant-writing activities, and create reports as directed by the Executive Committee.

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Land Grant Participating States/Institutions

AL, AR, CA, FL, GA, IA, IL, LA, MD, MI, MS, NJ, NY, TX

Non Land Grant Participating States/Institutions

Agriculture & Agri-Food Canada, Agriculture and Agri-Food Canada, Agriculture Canada, Kentville, Centro de Investigación y Tecnología Agroalimentaria de Aragón, CSIC, Pacific Agri-Food Research Centre, Pennsylvania (ERRC), University of Foggia, USDA ARS, USDA-ARS-SSRC, USDA-ARS/Florida, USDA-ARS/Maryland