Elliot T. Ryser

Antimicrobial Edible Films and Coatings

Increasing consumer demand for microbiologically safer foods, greater convenience, smaller packages, and longer product shelf life is forcing the industry to develop new food-processing, cooking, handling, and packaging strategies. Nonfluid ready-to-eat foods are frequently exposed to postprocess surface contamination, leading to a reduction in shelf life. The food industry has at its disposal a wide range of nonedible polypropylene- and polyethylene-based packaging materials and various biodegradable protein- and polysaccharide-based edible films that can potentially serve as packaging materials. Research on the use of edible films as packaging materials continues because of the potential for these films to enhance food quality, food safety, and product shelf life. Besides acting as a barrier against mass diffusion (moisture, gases, and volatiles), edible films can serve as carriers for a wide range of food additives, including flavoring agents, antioxidants, vitamins, and colorants. When antimicrobial agents such as benzoic acid, sorbic acid, propionic acid, lactic acid, nisin, and lysozyme have been incorporated into edible films, such films retarded surface growth of bacteria, yeasts, and molds on a wide range of products, including meats and cheeses. Various antimicrobial edible films have been developed to minimize growth of spoilage and pathogenic microorganisms, including Listeria monocytogenes, which may contaminate the surface of cooked ready-to-eat foods after processing. Here, we review the various types of protein-based (wheat gluten, collagen, corn zein, soy, casein, and whey protein), polysaccharide-based (cellulose, chitosan, alginate, starch, pectin, and dextrin), and lipid-based (waxes, acylglycerols, and fatty acids) edible films and a wide range of antimicrobial agents that have been or could potentially be incorporated into such films during manufacture to enhance the safety and shelf life of ready-to-eat foods.

A comparison of different chemical sanitizers for inactivating Escherichia coli O157:H7 and Listeria monocytogenes in solution and on apples, lettuce, strawberries, and cantaloupe.

Ozone (3 ppm), chlorine dioxide (3 and 5 ppm), chlorinated trisodium phosphate (100- and 200-ppm chlorine), and peroxyacetic acid (80 ppm) were assessed for reduction of Escherichia coli O157:H7 and Listeria monocytogenes in an aqueous model system and on inoculated produce. Initially, sanitizer solutions were inoculated to contain approximately 10(6) CFU/ml of either pathogen, after which aliquots were removed at 15-s intervals over a period of 5 min and approximately plated to determine log reduction times. Produce was dip inoculated to contain approximately 10(6) E. coli O157:H7 or L. monocytogenes CFU/g, held overnight, submerged in each sanitizer solution for up to 5 min, and then examined for survivors. In the model system study, both pathogens decreased > 5 log following 2 to 5 min of exposure, with ozone being most effective (15 s), followed by chlorine dioxide (19 to 21 s), chlorinated trisodium phosphate (25 to 27 s), and peroxyacetic acid (70 to 75 s). On produce, ozone and chlorine dioxide (5 ppm) were most effective, reducing populations approximately 5.6 log, with chlorine dioxide (3 ppm) and chlorinated trisodium phosphate (200 ppm chlorine) resulting in maximum reductions of approximately 4.9 log. Peroxyacetic acid was the least effective sanitizer (approximately 4.4-log reductions). After treatment, produce samples were stored at 4 degrees C for 9 days and quantitatively examined for E. coli O157:H7, L. monocytogenes, mesophilic aerobic bacteria, yeasts, and molds. Populations of both pathogens remained relatively unchanged, whereas numbers of mesophilic bacteria increased 2 to 3 log during storage. Final mold and yeast populations were significantly higher than initial counts for chlorine dioxide- and ozone-treated produce. Using the nonextended triangle test, whole apples exposed to chlorinated trisodium phosphate (200 ppm chlorine) and shredded lettuce exposed to peroxyacetic acid were statistically different from the other treated samples.

Stress, Sublethal Injury, Resuscitation, and Virulence of Bacterial Foodborne Pathogens

Environmental stress and food preservation methods (e.g., heating, chilling, acidity, and alkalinity) are known to induce adaptive responses within the bacterial cell. Microorganisms that survive a given stress often gain resistance to that stress or other stresses via cross-protection. The physiological state of a bacterium is an important consideration when studying its response to food preservation techniques. This article reviews the various definitions of injury and stress, sublethal injury of bacteria, stresses that cause this injury, stress adaptation, cellular repair and response mechanisms, the role of reactive oxygen species in bacterial injury and resuscitation, and the potential for cross-protection and enhanced virulence as a result of various stress conditions.

Fate of Listeria monocytogenes During the Manufacture and Ripening of Camembert Cheese

The ability of Listeria monocytogenes to survive the Camembert cheese-making process and grow during ripening of the cheese was examined. Pasteurized whole milk was inoculated to contain about 500 L. monocytogenes [strain Scott A, V7, California, (CA) or Ohio (OH)] CFU/ml and made into Camembert cheese according to standard procedures. All wheels of cheese were ripened at 6°C following 10 d of storage at 15-16°C to allow proper growth of Penicillium camemberti . Duplicate wedge (pie-shaped), surface and interior cheese samples were analyzed for numbers of L. monocytogenes by surface-plating appropriate dilutions made in Tryptose Broth (TB) on McBride Listeria Agar (MLA). Initial TB dilutions were stored at 3°C and surface-plated on MLA after 2, 4, 6 or 8 weeks if the organism was not quantitated in the original sample. Selected Listeria colonies from duplicate samples were confirmed biochemically. Results showed that numbers of Listeria in cheese increased 5- to 10-fold 24 h after its manufacture. Listeria counts for strains Scott A, CA and OH decreased to <10 to 100 CFU/g in all cheese samples taken during the first 18 d of ripening. In contrast, numbers of strain V7 remained unchanged during this period. All L. monocytogenes strains initiated growth in cheese after 18 d of ripening. Maximum Listeria counts of ca. 1 × 106 to 5 × 107 CFU/g were attained after 65 d of ripening. Generally, a 10- to 100-fold increase in numbers of Listeria occurred in wedge or surface as compared to interior cheese samples taken during the latter half of ripening. During this period, Listeria growth paralleled the increase in pH of the cheese during ripening.

Transfer of Listeria monocytogenes during mechanical slicing of Turkey Breast, Bologna, and Salami

A commercial delicatessen slicer was used as the vector for sequential quantitative transfer of Listeria monocytogenes (i) from an inoculated slicer blade (approximately 10(8), 10(5), or 10(3) CFU per blade) to 30 slices of uninoculated delicatessen turkey, bologna, and salami, (ii) from inoculated product (approximately 10(8) CFU/cm2) to the slicer, and (iii) from inoculated product (10(8), 10(5), or 10(3) CFU/cm2) to 30 slices of uninoculated product via the slicer blade. Cutting force and product composition also were assessed for their impact on L. monocytogenes transfer. Five product contact areas on the slicer, which were identified from residue of product bathed in Glow-Germ, were also sampled using a 1-ply composite tissue technique after inoculated product had been sliced. After being sliced with inoculated blades, each product slice was surface or pour plated on modified Oxford agar and/ or enriched in University of Vermont medium. Greater transfer (P < 0.05) occurred from inoculated turkey (10(8) CFU/cm2) to the five slicer contact areas from an application force of 4.5 kg as compared with 0 kg. On uninoculated product sliced with blades inoculated at 10(8) CFU per blade, L. monocytogenes populations decreased logarithmically to 10(2) CFU per slice after 30 slices. Findings for the inoculated slicer blade and product (10(5) CFU per blade or cm2) were similar; L. monocytogenes concentrations were 102 CFU per slice after 5 slices and enriched samples were generally negative for L. monocytogenes after 27 slices. For uninoculated product sliced with blades inoculated at 10(3) CFU per blade, the first 5 slices typically produced L. monocytogenes at approximately 10 CFU per slice by direct plating, and enrichments were negative for L. monocytogenes after 15 slices. The higher fat and lower moisture content of salami compared with turkey and bologna resulted in a visible fat layer on the blade that likely prolonged L. monocytogenes transfer. As a result of cross-contamination, those delicatessen-sliced meats that allow growth of L. monocytogenes during prolonged refrigerated storage likely pose an increased public health risk for certain consumers.

Behavior of Listeria monocytogenes During the Manufacture and Ripening of Cheddar Cheese

The ability of Listeria monocytogenes to survive the Cheddar cheesemaking process and persist during ripening of cheese was examined. Pasteurized whole milk inoculated to contain 5 ×102 cells of L. monocytogenes [strain Scott A, V7 or California (CA)]/ml was made into stirred-curd Cheddar cheese in a pilot-plant-sized vat. Cheese was ripened at 6 or 13°C. Listeria counts were obtained by surface-plating samples diluted in Tryptose Broth (TB) on McBride Listeria Agar (MLA). Initial TB dilutions were stored at 3°C and plated on MLA after 2, 4, 6 and 8 weeks if the organism was not detected with the original plating on MLA. Selected Listeria colonies from each sample were confirmed biochemically. During Cheddar cheese manufacture, Listeria counts remained relatively constant at ca. 5 x 102/ml of milk. After pressing the curd overnight, numbers of L. monocytogenes increased to about 1 × 103/g. Generally, greatest numbers of Listeria , about 5 × 103 cells/g, were detected in cheese after 14 d of ripening. Listeria counts for all 3 strains decreased during further ripening and except for strain V7, no appreciable difference in survival occurred in cheese aged at 6 or 13°C. Strains Scott A, CA and V7 survived for as long as 224, 154 and at least 434 d, respectively, in Cheddar cheese of normal composition. Strains V7 and CA were uniformly distributed throughout another set of cheese blocks and numbers of Listeria decreased uniformly throughout blocks of cheese during 98 d of storage.

A mathematical risk model for Escherichia coli O157:H7 cross-contamination of lettuce during processing.

A stochastic simulation modelling approach was taken to determine the extent of Escherichia coli O157:H7 contamination in fresh-cut bagged lettuce leaving the processing plant. A probabilistic model was constructed in Excel to account for E. coli O157:H7 cross contamination when contaminated lettuce enters the processing line. Simulation of the model was performed using @Risk Palisade© Software, providing an estimate of concentration and prevalence in the final bags of product. Three different scenarios, named S1, S2, and S3, were considered to represent the initial concentration on the contaminated batch entering the processing line which corresponded to 0.01, 1 and 100 cfu/g, respectively. The model was satisfactorily validated based on Standard Error of Prediction (SEP), which ranged from 0.00-35%. ANOVA analysis performed on simulated data revealed that the initial concentration in the contaminated batch (i.e., S1, S2, and S3) did not influence significantly (p=0.4) the E. coli O157:H7 levels in bags derived from cross contamination. In addition, significantly different (p<0.001) prevalence was observed at the different levels simulated (S1; S2 and S3). At the lowest contamination level (0.01 cfu/g), bags were cross-contaminated sporadically, resulting in very low E. coli O157:H7 populations (mean: ≤2 cfu/bag) and prevalence levels (<1%). In contrast, higher average prevalence levels were obtained for S2 and S3 corresponding to 3.05 and 13.39%, respectively. Furthermore, the impact of different interventions on E. coli O157:H7 cross-contamination (e.g., pathogen testing, chlorination, irradiation, and cleaning and disinfection procedures) was evaluated. Model showed that the pathogen was able to survive and be present in the final bags in all simulated interventions scenarios although irradiation (0.5 KGy) was a more effective decontamination step in reducing prevalence than chlorination or pathogen testing under the same simulated conditions.

Impact of bacterial stress and biofilm-forming ability on transfer of surface-dried Listeria monocytogenes during slicing of delicatessen meats.

Listeria monocytogenes contamination of delicatessen slicer blades can lead to cross-contamination of luncheon meats. A cocktail of 3 strong or 3 weak biofilm-forming strains of L. monocytogenes suspended in turkey slurry was used to inoculate stainless steel delicatessen slicer blades at a level of 6 log CFU/blade. The cocktails were used with or without injury (cold-shocked at 4 degrees C for 2 h, or chlorine-injured at 100 ppm for 1 min). Inoculated blades were held at 22 degrees C/78+/-2% relative humidity for 6 and 24 h, before being used to generate 30 slices from chubs of roast turkey breast or Genoa salami. Slices (25 g) were diluted 1:5 in University of Vermont Medium, homogenized by stomaching and then pour-plated using tryptose phosphate agar supplemented with esculin and ferric ammonium citrate. Greater cumulative transfer to the 30 slices was seen for the strong (3.62 log CFU) as opposed to weak biofilm-forming cocktails (3.12 log CFU) with transfer also significantly greater to turkey (3.61 log CFU) than to salami (3.12 log CFU). Among the three treatments, cold-shock significantly increased subsequent L. monocytogenes transfer (3.69 log CFU) compared to the uninjured control (3.30 log CFU) and chlorine-injury (3.12 log CFU). Significantly greater transfer was also seen for blades used after 6 as opposed to 24 h of incubation. Differences in product composition and survival of L. monocytogenes, as seen via viability staining, are likely reasons for these observed differences in transfer.

Diversity of Listeria ribotypes recovered from dairy cattle, silage, and dairy processing environments

Listeria strains isolated over the past 10 years from farms and dairy processing environments were subjected to strain-specific ribotyping using the automated Riboprinter microbial characterization system, alpha version (E. I. du Pont de Nemours & Co., Inc.). A total of 388 Listeria isolates from 20 different dairy processing facilities were examined along with 44 silage, 14 raw milk bulk tank, and 29 dairy cattle (26 udder quarter milk, 1 brain, 1 liver, and 1 aborted fetus) isolates. These 475 isolates included 93 L. monocytogenes , 362 L. innocua , 11 L. welshimeri , 6 L. seeligeri , 2 L. grayi , and 1 L. ivanovii strains. Thirty-seven different Listeria ribotypes (RTs) comprising 16 L. monocytogenes (including five known clinical RTs responsible for foodborne listeriosis), 12 L. innocua , 5 L. welshimeri , 2 L. seeligeri , 1 L. ivanovii , and 1 L. grayi were identified. Greatest diversity was seen among isolates from dairy processing facilities with 14 of 16 (87.5%) of the L. monocytogenes RTs (including five clinical RTs) and 19 of 21 (90.5%) of the non- L. monocytogenes RTs detected. Sixty-five of the 93 L. monocytogenes isolates belonged to a group of five clinical RTs. These five clinical RTs included one RT unique to dairy processing environments, two RTs common to dairy processing environments and silage, and one RT common to dairy processing environments, silage, and dairy cattle with the last RT appearing in dairy processing environments, silage, raw milk bulk tanks, and dairy cattle. These findings, which support the link between on-farm sources of Listeria contamination (dairy cattle, raw milk, silage) and subsequent contamination of dairy processing environments, stress the importance of farm-based HACCP programs for controling listeriae.

Transfer of surface-dried Listeria monocytogenes from stainless steel knife blades to roast turkey breast.

Listeria contamination of food contact surfaces can lead to cross-contamination of ready-to-eat foods in delicatessens. Recognizing that variations in Listeria biofilm-forming ability exist, the goal of this study was to determine whether these differences in biofilm formation would affect the Listeria transfer rate during slicing of delicatessen turkey meat. In this study, six previously identified strong and weak biofilm-forming strains of Listeria monocytogenes were grown at 22 degrees C for 48 h on Trypticase soy agar containing 0.6% yeast extract and harvested in 0.1% peptone. Thereafter, the strains were combined to obtain two 3-strain cocktails, resuspended in turkey slurry, and inoculated onto flame-sterilized AISI grade 304 stainless steel knife blades that were subjected to 6 and 24 h of ambient storage at approximately 78% relative humidity. After mounting on an Instron Universal Testing Machine, these blades were used to obtain 16 slices of retail roast turkey breast. Based on an analysis of the slices by direct plating, Listeria populations decreased 3 to 5 log CFU per slice after 16 slices. Overall, total transfer to turkey was significantly greater for strong (4.4 log CFU total) as opposed to weak (3.5 log CFU total; P < 0.05) biofilm formers. In addition, significantly more cells were transferred at 6 (4.6 log CFU total) than at 24 h (3.3 log CFU total; P < 0.05) with Listeria quantifiable to the 16th slice, regardless of the inoculation level. Increased survival by the strong biofilm formers, as evidenced by viability staining, suggests that these strains are better adapted to survive stressful conditions than their weak biofilm-forming counterparts.