Chapter 12
Inactivation of Microbial Contaminants in Fresh Produce Downloaded by CORNELL UNIV on June 26, 2012 | http://pubs.acs.org Publication Date: December 17, 2009 | doi: 10.1021/bk-2009-1020.ch012
Brendan A. Niemira, Bassam A. Annous, Xuetong Fan, Ching-Hsing Liao, Joseph Sites Food Safety Intervention Technologies Research Unit, Eastern Regional Research Center, U.S. Department of Agriculture, Agricultural Research Service, 600 E. Mermaid Lane, Wyndmoor, PA 19038 With the microbial safety of fresh produce of increasing concern, conventional sanitizing treatments need to be supplemented with effective new interventions to inactivate human pathogens. The Produce Safety research project at the United States Department of Agriculture’s Eastern Regional Research Center develops and validates new interventions to improve the safety of fresh and fresh-cut fruits and vegetables. Inoculation with suppressive microbial communities inhibits the growth of Salmonella on vegetable surfaces by up to 99% during the course of storage. Rapid thermal treatments and gaseous chlorine dioxide can achieve reductions of Salmonella on cantaloupe of more than 99.99%. Irradiation can reduce E. coli O157:H7, Salmonella and Listeria monocytogenes on leafy vegetables and other produce by more than 99.99% while preserving product quality. Chemical and sensory analysis has demonstrated the safety and wholesomeness of irradiated foods. A novel processing technology, cold plasma, has shown promising results, with 99.9% reductions of Salmonella and E. coli O157:H7 on apple surfaces. This chapter will summarize the advances made in these areas, as well as research results on the process of scaling up effective interventions from laboratory scale to pilot plant scale, including the critical process of evaluating the effects of the various interventions on sensory and nutritional quality attributes, yield, physiology, and shelf-life.
U.S. government work. Published 2009 American Chemical Society In Intentional and Unintentional Contaminants in Food and Feed; Al-Taher, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 0.
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184 Consumers in the United States have experienced increases in the incidence of foodborne illness (FBI) outbreaks associated with contaminated fruits, vegetables, salads, and juices in recent decades (1). While it is recognized that pre-harvest (good agricultural practices, GAP), post-harvest (good manufacturing practices, GMP) and supply-chain (good handling practices, GHP) controls can help to reduce risk, these practices have not been able to prevent product recalls of tomatoes, leafy greens, melons, sprouts and other fresh produce, and the associated repeated FBI outbreaks. The lack of a broadly applicable antimicrobial process (a “kill step”) is hampering the food safety efforts of the fresh produce industry (2, 3). In 2005, the U.S. Department of Agriculture initiated a new five year research project within the Agricultural Research Service to address this critical need (4). The project, entitled “Intervention Technologies for Enhancing the Safety and Security of Fresh and Minimally Processed Produce and Solid PlantDerived Foods”, builds on a notable history of produce safety research projects at the Eastern Regional Research Center in Wyndmoor, PA. The objective of the project is to develop more effective means for decontaminating organic and conventionally grown fresh and minimally processed fruits and vegetables, including sprout seeds. Food safety and security will be more completely assured by assessing the efficacy of new and/or improved intervention technologies. The researchers determine the effectiveness of treatment combinations (multiple hurdle approach), assess factors that might limit treatment efficacy, and transfer effective decontamination technology to the produce industry in order to reduce the risk of foodborne illness. The produce safety treatments being developed are broadly grouped into physical, chemical and biological interventions. This research group is a representative sample of the type of research activities being conducted across the entire field. In order to give an overview of the kind of research inquiries being pursued in this area at many different locations, the latest results from this particular research project will be highlighted in this chapter within the context of a broader review.
Cold Plasma Cold plasma is a relatively new sanitizing technology in the field of food processing. Various types of plasma-generating technologies have been used for non-food applications, such as lighting, electronics, and materials processing. As a food processing technology, however, a number of technological hurdles must be overcome before widespread implementation. For all practical purposes, cold plasma may be regarded as an energetic form of gas, although it is technically a distinct state of matter. As energy is added to materials, they change state, going from solid to liquid to gas, with large-scale inter-molecular structure breaking down. In general, as additional energy is added, the intra-atomic structures of the components of the gas break down, yielding plasmas - concentrated collections of ions, radical species and free electrons (5-8). According to Niemira and Sites (8), cold plasma technologies used to treat foods fall into three general categories: electrode contact (in which the target is in contact with or between electrodes), direct treatment (in which active plasma is deposited directly on the target) and remote treatment (in which active plasma is generated at some distance, and plasma is moved to the target).
In Intentional and Unintentional Contaminants in Food and Feed; Al-Taher, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 0.
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185 When applied to foods and inert surfaces, electrode contact systems have been shown to achieve reductions of Escherichia coli, Staphylococcus aureus, Bacillus subtillis, and Saccharomyces cerevisiae as high as 5 logs (9, 10). Direct treatment of E. coli placed within the 1 mm gap spacing of the plasma reactors reduced the pathogens populations by 4.6 and 5.1 log cfu/ml after treatment of 10 s and 60 s, respectively (11). As the space between the plasma emitter and the treated culture was increased, antimicrobial efficacy was reduced, until at 10 mm spacing, no reductions were observed at any power level tested. Remote treatment reactors reduced E. coli and St. aureus inoculated on polypropylene by 4 or 2 log cfu/ml, respectively, after a 10s treatment (12). The one atmosphere uniform glow discharge plasma system (OAUGDP) produced D-values of 22 s (Shigella flexneri and Vibrio parahaemolyticus) to 51s (E. coli O157:H7) for pathogens on agar (13). On food surfaces, a 2 min treatment with the OAUGDP reduced E. coli O157:H7 on red delicious apples by ca. 3 log cfu, reduced Salmonella Enteritidis on cantaloupe by ca. 3 log cfu, and reduced Listeria monocytogenes on Iceberg lettuce by ca. 2 log cfu (14). Cold plasma generated by a gliding arc emitter system (Figure 1) inactivated E. coli O157:H7 and Salmonella on agar plates and on the surface of golden delicious apples (8). Higher flow rates of plasma (30 or 40 L/min) were more effective than lower flow rates (10 or 20 L/min). Longer exposures also yielded greater reductions in pathogen population. With plasma flowing from the gliding arc emitter at 40 L/min, treatments of 3 minutes reduced Salmonella by 2.9 to 3.7 log cfu, and reduced E. coli O157:H7 by 3.4 - 3.6 log cfu. In that study, the plasma tended to increase the temperature of the treated apples. The maximum temperature of any plasma treated apple (50.8°C, 28°C above ambient) was obtained after a lower plasma flow rate (20 L/min) for 3 min. As this temperature is not high enough to kill either Salmonella or E. coli O157:H7, the antimicrobial effects obtained in this study were not the result of heat.
Figure 1. Gliding arc cold plasma applied as an antimicrobial treatment to Escherichia coli O157:H7 on the surface of a golden delicious apple.
In Intentional and Unintentional Contaminants in Food and Feed; Al-Taher, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 0.
186 Cold plasma is a promising new nonthermal process that can effectively reduce human pathogens on fresh produce. Research is ongoing to advance the state of the art in plasma emitter design, and to improve the operational application of the technology to fruits and vegetables.
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Irradiation of Fresh Produce Irradiation is a nonthermal process in which high-energy electrons or photons are applied to foods, resulting in the inactivation of associated pathogens (15). An extensive body of research has demonstrated that this technology is safe and effective. In 2008, the FDA approved the use of irradiation up to 4.0 kGy on fresh Iceberg lettuce and fresh spinach, to improve food safety and to extend shelf life (16). More recent research has focused on the ability of irradiation to inactivate pathogens within the interior spaces of a leaf, fruit or vegetable. These populations of internalized pathogens are isolated from conventional antimicrobial treatments. The inefficient uptake of bacteria via roots and vasculature make microbiological analysis problematic, complicating research aimed at developing effective interventions. Penetrating processes such as irradiation may be uniquely suited for dealing with this type of contamination, but the literature is as yet scant. In a study by Nthenge et al. (17), irradiation was shown to eliminate pathogenic bacteria internalized within leaf tissues as a result of root uptake. Lettuce plants grown in hydroponic solutions inoculated with E. coli O157:H7 contained the pathogen in the leaf tissue. Irradiation effectively killed the pathogen while a treatment with 200 ppm aqueous chlorine was ineffective. In other studies which used a direct inoculation method that introduces inoculum into the leaf intracellular spaces, irradiation was shown to be similarly effective in eliminating internalized E. coli O157:H7 from baby spinach and various types of lettuce (Romaine, Iceberg, Boston, green leaf, red leaf), while 300 or 600 ppm sodium hypochlorite was generally ineffective (18, 19). D10 values for internalized cells (0.30-0.45 kGy) were shown to be 2- to 3-fold higher than for surface associated cells (0.12-0.14 kGy) (18). This suggests that the context of the intercellular space may provide a chemical or structural protection for these internalized bacteria. Additional information is necessary to develop a complete understanding of this phenomenon. Pathogen populations within the leaf are expected to be very low in a commercial setting; nearcomplete elimination of internalized pathogens may therefore be practically achieved using irradiation doses that do not cause undue sensory damage. Related research has recently begun to assess the ability of irradiation to inactivate biofilm-associated pathogens. Living within the tightly-knit exopolysaccharide matrix of a biofilm serves to protect pathogens from chemical antimicrobial treatments (20, 21). Irradiation is a penetrating process, but the data on the efficiency of irradiation in killing biofilm-associated pathogens is very limited. The particular isolate and the biofilm culture conditions (growth temperature, medium, time of cultivation, etc.) can influence irradiation efficacy. Biofilm-associated cells of Salmonella were as sensitive or significantly more sensitive to ionizing radiation than respective planktonic cells (22). Biofilms of L. monocytogenes and L. innocua grown at various temperatures were equally or more sensitive to irradiation as planktonic cells
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(23). For E. coli O157:H7, in vitro biofilms cultivated for varying times up to 72 h were either more sensitive or less sensitive to irradiation, depending on the isolate examined (24). That study concluded that the response of E. coli O157:H7 in the form of a biofilm was influenced by growth conditions, but did not extend the conclusion to address the probable response of in vivo biofilms. Thus, information is not yet available on the specific effect of leaf-surface biofilms on the efficacy of irradiation. The information available in this emerging field of inquiry suggests a complex difference between the two physiological states of these cells, planktonic and biofilm-associated, in their response to irradiation (24). Further research is expected to improve our understanding of how biofilms, in particular leaf-, fruit- or vegetable-surface biofilms, may alter the efficacy of irradiation.
Quality of Irradiated Fresh Produce Recent studies have demonstrated that most fresh-cut fruits and vegetables irradiated at doses of 1 kGy or less did not cause any significant change in appearance, texture, flavor or nutrient quality. Shelf-life of some fresh-cut fruits and vegetables can be extended by low dose irradiation due to the reduction of spoilage microorganisms. For example, Koorapati et al. (25) showed irradiation at doses above 0.5 kGy prevented microbial-induced browning and blotching of sliced mushroom. Studies have also shown that irradiated fresh produce may have higher antioxidant content than non-irradiated controls as irradiation increased synthesis of phenolic compounds (26). In some fresh-cut fruits and vegetables, irradiation may cause tissue softening and loss of ascorbic acid (27). The losses in quality due to irradiation can be minimized by combination with other sanitizers or techniques such as modified atmosphere packaging (MAP), heat treatment, calcium infiltration and antibrowning agents (15, 28). For example, Boynton et al. (29) showed that fresh-cut cantaloupes irradiated at 1 kGy in MAP of 4% O2, 10% CO2 had the highest rating in sweetness and cantaloupe flavor intensity and lowest in off-flavor after 17 days storage compared to the control and the 0.5 kGy samples.
Formation of Furan due to Irradiation Furan (C4H4O) is regarded as a possible carcinogen according to the Department of Health and Human Services and the International Agency for Research on Cancer, because it causes cancer in animals in studies where the animals are exposed to furan (30, 31). This compound is commonly found in foods that have been treated with traditional heating techniques, such as cooking, jarring, and canning (32, 33). As a result, both the U.S. Food and Drug Administration (FDA) and the European Food Safety Authority are requesting data and information about furan formation and its toxicity (34, 35). Studies showed that ionizing radiation can induce furan formation in solutions of simple sugars and ascorbic acid as well as in fruit juice (36, 37), suggesting that the sources of furan upon irradiation are simple sugars such as fructose, sucrose, glucose, and ascorbic acid. Many fruits and vegetables are rich in sugars and ascorbic acid. Fan and Sokorai (38) irradiated nineteen fruits and vegetables
In Intentional and Unintentional Contaminants in Food and Feed; Al-Taher, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 0.
188 and measured furan formation in those irradiated produce. Their results showed that irradiation produced low levels of furan in grape and pineapple. In all other fruits and vegetables, furan levels were not detectable or below 1 ng/g. Soluble solids content, titratable acidity and pH varied among the fruits. It appears that the presence of high amount of sugars and low pH are prerequisites for furan formation in fresh-cut produce. Considering the low ng/g of furan detected in grape and pineapple as well as the volatility of furan, irradiation-induced furan is unlikely to be a concern for fresh-cut produce.
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Use of Irradiation and Hot Water Treatment on Cantaloupes Consumption of fresh produce has been linked to outbreaks of foodborne illness and recalls in the U.S. due to contamination with human pathogens. Melons (mostly cantaloupes) are one of the groups of produce that are most frequently associated with outbreaks and contamination with foodborne pathogens (39, 40). Between 1990 and 2000, more than 700 cases of salmonellosis were reported in the U.S. and Canada (41). The high rates of pathogen contamination associated with melon highlight the need for effective interventions for both whole and cut melons. Over the last decade, many chemical antimicrobials have been investigated for their effectiveness against human pathogens. However, most chemical interventions have limited effectiveness for reducing the microbiological population on the surface of cantaloupes (42, 43), partially due to the rough surface (netting), which provides a protective environment to microbes. Certain hot water treatments have been shown to effectively reduce human pathogens and native microflora on whole cantaloupes (44, 45). Ukuku et al. (46) demonstrated that immersion of inoculated cantaloupe in hot water or 5% hydrogen peroxide solution at 70 °C for 1 min resulted in up to a 3.8 log cfu/cm2 reduction in Salmonella. Annous et al. (44) reported that surface pasteurization with hot water at 76 °C for 3 min resulted in more than 5 log CFU/cm2 reduction in S. enterica serovar Poona and E. coli populations with inoculated cantaloupes. Fan et al. (47) submerged whole cantaloupes into water in the following three conditions: 10 °C water for 20 min (control), 20 ppm chlorine at 10 °C for 20 min, and 76 °C water for 3 min. The hot water significantly reduced both total plate count (TPC) and yeast and mold counts on rind of whole fruits while chlorine or cold water wash did not result in a significant reduction of microbial population. Fresh-cut pieces prepared from hot water-treated cantaloupes had lower TPC than the other two treatments in the later storage periods (day 13-20) (Table I). Lower yeast and mold count on rind of whole fruits due to hot water treatment did not always result in lower count of the microorganism on fresh-cut fruit prepared from the hot-water-treated cantaloupes. Soluble solids content, ascorbic acid content, fluid loss, and aroma and appearance scores were not consistently affected by either hot water or chlorine treatment. Therefore, hot water treatment of whole fruit was superior to chlorine in reducing microbial population of both whole and fresh-cut cantaloupe.
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189 Table I. The Effect of Hot Water Pasteurization of Whole Cantaloupe on Total Plate Count (Log CFU/g) of Fresh-Cut Cantaloupes Stored at 4 °C Storage Treatments time (day) Cold water control Chlorine Hot water Trial 1 1 3.1±0.6 a 2.9±0.7 a 2.9±0.6 a 6 3.8±0.8 a 3.5±0.3 ab 2.3±0.7 b 8 4.2±0.6 a 4.3±0.2 a 3.9±0.5 a 10 4.9±0.5 a 4.8±0.7 a 2.9±0.8 b 13 6.3±0.7 a 5.6±0.8 a 3.6±0.3 b 16 6.9±0.7 a 6.9±0.7 a 2.8±0.3 b 20 7.9±0.7 a 7.8±0.7 a 5.0±1.3 b LSD 1.1 1.1 1.2 Trial 2 1 2.8±1.0 a 2.6±0.9 a 3.2±1.7 a 6 4.3±0.8 a 3.9±1.1 a 2.4±2.1 a 8 4.8±0.9 a 4.9±2.2 a 3.0±0.6 a 10 5.8±0.9 a 6.0±2.4 a 2.7±2.0 a 13 7.3±0.5 a 6.5±1.6 a 3.2±0.8 b 16 7.4±0.5 a 7.4±0.8 a 3.8±0.4 b 20 8.0±0.6 a 7.3±0.5 ab 4.6±2.8 b LSD 1.4 2.5 2.8 a
Means with same letters within the same rows are not significantly different (P>0.05). The least significant difference at P
