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Weekly UpdatesABSTRACT
The objective of the study was to determine the survival of Listeria monocytogenes and Salmonella sp. when inoculated in unwashed or washed whole and sliced mushrooms during postharvest storage at 12[degrees]C (54[degrees]F). White Agaricus bisporus mushrooms, obtained fresh, were washed using a two-stage wash process described by Beelman and Duncan (U.S. Patent: 5919507, 1999). The slicing operation was conducted following washing, drying, and inoculation of the foodborne pathogens over the mushroom cap surface. Inoculated mushrooms were kept in styrofoam containers over-wrapped with a commercial plasticized polyvinyl chloride (PVC) film. The packages were stored in a humidity controlled environmental chamber maintained at 12[degrees]C and 75-85% relative humidity. Mushroom packages were removed over a seven-day period to enumerate populations of L. monocytogenes and Salmonella sp. Whole mushrooms did not significantly support the growth of the foodborne pathogens. When inoculated at 4 log CFU/g, populations of L. monocytogenes increased by 0.5 log CFU/g during the first 24 h of storage at 12[degrees]C and then remained constant during a seven-day storage period at 12[degrees]C. However, sliced mushrooms significantly promoted the growth of the foodborne pathogens. Populations of L. monocytogenes in unwashed sliced mushrooms increased from 4 log CFU/g to 6.8 log CFU/g during five days of postharvest storage at 12[degrees]C. Populations of Salmonella sp. in unwashed sliced mushrooms increased from 4.5 log CFU/g to 6.7 log CFU/g during five days of postharvest storage at 12[degrees]C. Washing mushrooms using the two-stage wash process prior to inoculation and slicing significantly reduced the rate of growth of L. monocytogenes and Salmonella sp. during postharvest storage at 12[degrees]C (54[degrees]F).
Keywords: Agaricus, mushrooms, Listeria monocytogenes, Salmonella, minimal processing, washing, slicing, fresh-cut
INTRODUCTION
Mushroom farming is a large and economically important part of U.S. agriculture. Value of sales for the total 2004-05 U.S. mushroom crop including specialty mushrooms was $908 million. In 2004-05, 118 growers in the United States produced 838 million pounds of Agaricus mushrooms with a value of over $862.3 million (USDA, 2006). Within the last 10 years, Agaricus mushroom production has increased by 12.2%. In 2004-2005, U.S. fresh market Agaricus was 83 percent of total sales volume with processed Agaricus mushrooms making up the remaining 17 percent, compared to 1991-1992 figures where only 69 percent were grown for fresh market consumption and 31 percent of the mushrooms were processed. This change reflects the increasing consumer preference for fresh mushrooms. The market for sliced mushrooms is growing despite selling at a premium over whole mushrooms.
Washing mushrooms has recently gained commercial popularity as a means of improving appearance by removing casing soil particles and to increase shelf life. Significant research has been conducted to develop washing processes to achieve the objective (Sapers et al. 1994; Sapers et al. 1995, Sapers and Simmons, 1998; Sapers et al. 1999; Sapers et al. 2001). McConnell (1991) conducted a review of potential wash additives for mushrooms including sodium hypochlorite, hydrogen peroxide, potassium sorbate, and sodium salts of benzoate, EDTA and phosphoric acids. It was concluded that effective antioxidants, in addition to antimicrobial compounds, were required to enhance shelf life of fresh mushrooms by washing. Beelman and Duncan (1999) developed a two-stage mushroom wash process (U.S. Patent 5,919, 507). The method employed a first stage high pH (pH of 9.0 or above) antibacterial wash followed by a neutralizing wash containing browning inhibitors. The neutralizing wash contained a buffered solution of erythorbic acid and sodium erythorbate. Other browning inhibitors such as ascorbates, EDTA, or calcium chloride were identified as suitable ingredients for addition to the neutralizing solution. The two-stage washing process helped remove soil residue and significantly delayed microbial spoilage of fresh mushrooms during postharvest storage.
Sliced mushrooms represent a significant part of the fresh mushroom market. For instance, the mushroom industry in Ireland supplies up to 25% of its fresh output as slices (Brennan and Gormley 1998) and the trend in the United States is similar. Consumer demand for convenience foods has rapidly increased in recent years, and the growth in market share for sliced mushrooms reflects this trend. There is a high demand for sliced mushrooms among pizza producing companies and supermarkets are selling an increasing number of overwrapped packages of sliced fresh mushrooms (Brennan and Gormley 1998). Sliced fresh mushrooms are marketed either unwashed or sliced following a wash process.
There have been no reported outbreaks associated with consumption of fresh mushrooms. However, there have been several reports of human pathogens isolated from fresh mushrooms. The foodborne pathogens Campylo-bacter jejuni, Listeria monocytogenes and Salmonella spp. have been found on fresh mushrooms. Doyle and Schoeni (1986) isolated C. jejuni in three of 200 (1.5%) retail mushroom packages from several Midwestern U.S. grocery stores. In a survey in the Pacific Northwest, 1% of retail samples tested positive for L. monocytogenes and 5% tested positive for Salmonella spp (Samadpour et al. 1999; Samadpour et al. 2006). In the Netherlands, L. monocytogenes was recovered in 10% of mushrooms (2/20) purchased at a grocery store (van Netten et al. 1989). In 2001, a health alert was issued in Ireland by government authorities after Salmonella Kedougou was detected on mushrooms. The pathogen was found in the mushroom compost, the mushroom casing soil and in several mushroom samples. It was concluded that the most likely source of the pathogen was the dried sugar beet lime used as a mushroom casing soil amendment (FSAI 2001; Meikle 2001). The most recent report related to food safety and fresh mushrooms was in August 2003 when the Georgia Department of Agriculture recalled a brand of fresh sliced mushrooms which tested positive for L. monocytogenes (FDA 2003).
Fresh produce may serve as a vehicle for the transmission of disease unless all measures are taken to prevent contamination and growth of human pathogens at all points in the farm to fork continuum. Because it is possible for microbial populations on fresh mushrooms to include human pathogens, either from natural sources or human activity, research is needed to determine survival and growth characteristics of foodborne pathogens on fresh whole and sliced mushrooms. The objective of this study was to evaluate the survival of L. monocytogenes and Salmonella sp. in whole and sliced mushrooms (unwashed or washed before slicing) during postharvest storage at 12[degrees]C (54[degrees]F). The packages were stored at 12[degrees]C to simulate a temperature-abuse condition. Washed mushrooms were inoculated with the foodborne pathogens following washing and prior to the slicing operation.
MATERIAL & METHODS
Cultures of Foodborne Pathogen
Chicken litter isolates of Salmonella serotypes Enteriditis (03-8145), and Typhimurium (03-8970) were obtained from the National Veterinary Services Laboratory (Ames, IA). L. monocyto-genes cultures Scott A (serotype 4b) and FSLR2-499 (serotype 1/2a, human epidemic origin) was obtained from the PSU Food Microbiology Laboratory Collection. L. monocytogenes serotypes 4b and 1/2a are most frequently associated with foodborne illness (FDA/USDA 2003).
Working cultures of each of the above strains were maintained on Tryptic Soy Agar supplemented with 0.6% Yeast Extract (TSAYE; Difco-Bec-ton, Dickinson and Co, Sparks, MD) with monthly transfers. The inoculum was prepared by individually growing the strains in Tryptic Soy Broth supplemented with 0.6% Yeast Extract (TSBYE; Difco). An isolated colony was added to 10 mL of TSBYE and incubated for 18-22 h at 33[degrees]C. A 0.1 mL aliquot was then subcultured into 10 mL of TSBYE and incubated for 18-22 h at 33[degrees]C, producing stationary phase populations. Cells were harvested by centrifugation at 8000 x g for 10 min at 4[degrees]C. The cell pellets from individual strains were washed and resuspended in 10 mL of 0.85% saline. Suspensions of Salmonella strains or L. monocytogenes strains were combined separately (cocktail strain mixes of foodborne pathogens are generally used in food process validation studies). The composite Salmonella stock suspension contained approximately 1 x 108 CFU/mL. The composite L. mono-cytogenes stock suspension contained approximately 2 x 108 CFU/mL.
Washing fresh mushrooms
All mushrooms used in this study were a hybrid off-white (U-1) strain of Agaricus bisporus and were obtained from the Penn State Mushroom Test Demonstration Facility (MTDF), University Park, PA. The mushrooms were used in the experiments immediately after harvesting. Freshly harvested mushrooms (15 kg) were obtained from the MTDF. The mushrooms stipes were trimmed to within 5mm of the mushroom cap and then split into two batches. One batch was used as the control (unwashed lot). The other lot of mushrooms was washed using a two-stage process (PSU wash) described by Beelman and Duncan (1999) and shown below:
Stage I: 0.05 M Bicarbonate (NaH-CO3) buffer, pH 10.5, 30[degrees]C, 45 s wash
Stage II: Erythorbate buffer (0.6% erythorbic acid + 2.4% Sodium erythorbate) + CaCl2 (1000 ppm) + EDTA (1000 ppm), pH 4.5, 20[degrees]C, 45 s wash
Stipe-trimmed mushrooms (2 kg lots) were dumped into a stainless steel bucket containing 15 gal of stage I wash solution. The mushrooms were stirred gently in the wash solutions for 45 s and then drained in plastic colanders. Immediately after draining, the Stage I washed mushrooms were transferred to the Stage II wash solution. The mushrooms were stirred gently in the Stage II wash solution for 45 s, removed and drained using the colanders. The wash process was repeated using three sets of 2 kg mushroom batches to yield 6 kg of washed mushrooms. After draining, the mushrooms were surface dried by placing them in a laminar flow biological safety cabinet (Class II, Type A/B3, Nuaire Inc., Plymouth, MN) with continuous airflow for 2 h. Following the drying protocol, the mushrooms were placed in 8-oz. styrofoam containers. Eight mushrooms of approximately equal size were aseptically placed using sterilized forceps in each styrofoam container to yield between 190 and 210 g per container.
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