The impact of a cold chain break on the survival of Salmonella enterica and Listeria monocytogenes on minimally processed ‘Conference’ pears during their shelf life

BACKGROUND: In recent years, improved detection methods and increased fresh-cut processing of produce have led to an increased number of outbreaks associated with fresh fruits and vegetables. During fruit and vegetable processing, natural protective barriers are removed and tissues are cut, causing nutrient rich exudates and providing attachment sites for microbes. Consequently, fresh-cut produce is more susceptible to microbial proliferation than whole produce. RESULTS: The aim of this study was to examine the impact of storage temperature on the growth and survival of Listeria monocytogenes and Salmonella enterica on a fresh-cut ‘Conference’ pear over an eight day storage period. Pears were cut, dipped in antioxidant solution, artificially inoculated with L. monocytogenes and S. enterica, packed under modified atmospheric conditions simulating commercial applications and stored in properly refrigerated conditions (constant storage at 4 °C for 8 days) or in temperature abuse conditions (3 days at 4 °C plus 5 days at 8 °C). After 8 days of storage, both conditions resulted in a significant decrease of S. enterica populations on pear wedges. In contrast, when samples were stored at 4 °C for 8 days, L. monocytogenes populations increased 1.6 logarithmic units, whereas under the temperature abuse conditions, L. monocytogenes populations increased 2.2 logarithmic units. CONCLUSION: Listeria monocytogenes was able to grow on fresh-cut pears processed under the conditions described here, despite low pH, refrigeration and use of modified atmosphere.


INTRODUCTION
Processed products attractive to consumers could be a way to increase consumption of fruits and vegetables, with a positive impact on consumer health if processing does not alter the nutritional benefits of the raw products. 1 Minimally processed pears could satisfy consumer demand because pears have low protein and lipid contents and are rich in sugars, including fructose, sorbitol, and sucrose, and are low in glucose. Pears also contain micronutrients, including vitamins (vitamin C, vitamin E, and niacin) and minerals (potassium, phosphorous, calcium, and magnesium). 2 However, fruit processing promotes faster deterioration due to tissue damage, which leads to increased physiological activity and major physicochemical changes, including enzymatic browning, softening, and tissue degradation. 3 Several investigators have developed technologies to minimize these processing effects. [4][5][6][7][8][9][10][11][12][13][14][15] Use of antioxidant solutions and edible coatings together with a modified atmosphere package (MAP) can reduce surface browning and water loss. 16 In addition to improving processing techniques to maintain quality, precautions should be taken to ensure product safety. The potential for microbiological contamination of fruits and vegetables is high because of the wide variety of conditions to which produce is exposed during growth, harvest, processing, and distribution. It is well established that fresh produce may contain high contamination levels after harvest. During processing, spoilage and pathogenic microorganisms can also contaminate the product surface, and the nutrients inside the fruit contribute to their growth. 3 Thus, disinfection is one of the most important processing steps affecting the quality, safety, and shelf life of the end product. 17 Safety requirements related to fresh-cut produce include good agricultural practices (GAP) and good processing practices (GMF) that result in the absence of pathogens, mycotoxins, pesticide residues, and any other chemical or physical contamination that might risk consumer health. 16 In the Europe Union, food safety criteria for the presence of microorganisms in food is regulated by EC N° 2073/2005 and subsequent amendments. The food safety criteria for L. monocytogenes are of particular interest on ready-to-eat (RTE) foods. In RTE foods that may support the growth of L. monocytogenes, the limit is 100 CFU g -1 during the shelf life and the absence of L. monocytogenes in 25 g of the food just before it has left the immediate control of the food business operator who produced it. In RTE foods unable to support the growth of L. monocytogenes, the limit is also 100 CFU g -1 during the shelf life. 18 In recent years, improved detection methods and increased fresh-cut processing of produce have led to an increased number of outbreaks associated with fresh fruits and vegetables. 19 The storage temperature is an important factor affecting the growth of microorganisms. Thus, effective cold chain management is critical for maintaining the quality and shelf life of the product. The aim of this study was to examine the impact of storage temperature on the growth and survival of Listeria monocytogenes and Salmonella enterica on a fresh-cut 'Conference' pear during its shelf life.

Fruit
'Conference' pears (Pyrus communis cv. Conference) were obtained from local packing houses in Lleida (Catalonia, Spain). The guide for minimally processing 'Conference' pears used in this paper was based on previous work. 20 Prior to processing, the pears were disinfected by immersion in a 0.1 g L -1 sodium hypochlorite (NaClO) solution (pH 6.5) for 2 min, rinsed in running tap water and allowed to dry at room temperature. Each pear was peeled and cut into 10 wedges using a handheld apple corer and slicer. In some fruit pieces, a 6 mm diameter well was made at the centre of each wedge for the inoculum. All the 'Conference' pear wedges were treated with an optimum antioxidant solution (20 g L -1 calcium ascorbate plus 10 g L -1 calcium chloride solution) by immersion for 2 min in the solution (1:2 w/v), which was maintained on a rotating platform at 150 rpm. The treated pear wedges were allowed to dry in a laminar flow biosafety cabinet for a short time. Physicochemical characteristics (soluble solids content and titratable acidity) of the pear wedges were evaluated in triplicate after the antioxidant treatment.
The pear wedges were squeezed, and the soluble solids content (SCC) was determined using a handheld refractometer (ATAGO CO., LTD, Japan) at 20 °C. Results were reported as °Brix.
To measure titratable acidity (TA), triplicate samples of 10 mL of fruit extract were diluted with 10 mL of distilled water, and 2 drops of phenolphthalein solution, 0.1 mL L -1 (Panreac, Barcelona, Spain) were added. The mixture was titrated with sodium hydroxide solution (NaOH, 0.1 N) until the pH indicator changed colour. The results were calculated as g of malic acid per litre of juice.

Foodborne pathogens
The bacterial strains used in this work included the serovars of Salmonella enterica subsp. Initially, samples were stored in conditions simulating a commercial application (constant storage at 4 ± 1 °C). Three trays per pathogen were examined at day 0 and three more after 3 days of storage at 4 ± 1 °C. Then, the rest of the samples were divided into two lots, one was stored at 8 ± 1 °C for 5 days (temperature abuse conditions) simulating more realistic conditions during transport and in a refrigerated display window while the other was maintained at a constant temperature of 4 ± 1 °C for 5 days.

Bacterial analysis
Recovery of pathogen populations were performed at day 0 and after 3 and 8 days of storage under the two conditions. Before opening the trays, the headspace gas composition was determined using a handheld gas analyser (CheckPoint O2/CO2, PBI Dansensor, Denmark).
To recover the pathogens from the wedges, a plug (1.2 cm in diameter and 1 cm deep, approximately 1 g plug -1 ) containing the entire well was removed using a sterile cork borer. One plug per repetition was placed in a sterile filter bag (80 mL, BagPage®, Interscience BagSystem, Saint Nom, France) and diluted with 9 mL of buffered peptone water (BPW, Oxoid). The mixture was homogenized in paddle blender for 2 min at high speed (MiniMix, Interscience, France), and aliquots of the mixture were then serially diluted in SP and plated on XLD for enumerating S. enterica or on Palcam agar for L. monocytogenes. The agar plates were incubated at 37 ± 1 °C for 24 h (S. enterica) or 48 h (L. monocytogenes). The data were transformed to CFU g -1 pear. Three determinations per pathogen were made at each sampling point in duplicate.

Statistical analysis
All data were checked for significant differences by applying variance analysis (ANOVA) using the JMP8 (SAS, Statistical Analysis System) statistical package. They were subjected to mean separation by least significant differences by Tukey's test (p< 0.05).

RESULTS AND DISCUSSION
Several authors have reported that foodborne pathogens, including L. monocytogenes and Salmonella spp., may often be able to grow on the flesh of some fruits, including apples, peaches, strawberries, melons, watermelons, papayas, persimmons and pears. 22 The initial populations of L. monocytogenes and S. enterica on fresh-cut pears were 3.2 ± 0.9 and 3.6 ± 0.3 log CFU g -1 pear, respectively (Fig. 2). After 3 days of storage at 4 °C, L. monocytogenes and S. enterica populations remained at the initial levels. After 8 days of storage, both constant storage (4 °C) and temperature abuse conditions caused a significant decrease of S. enterica populations on pear wedges, reaching 3.1 ± 0.2 and 3.0 ± 0.3 log CFU g -1 pear, respectively. In contrast, L. monocytogenes grew in both storage conditions. The values were 4.9 ± 0.5 log CFU g -1 pear (1.6 log increase) in samples stored at 4 °C and 5.4 ± 1.0 log CFU g -1 pear (2.20 log increase) in the temperature abuse conditions. Thus, the temperature increase for 5 days caused a greater increase in the L. monocytogenes populations on the freshcut pear product even in the highest CO 2 levels (22 kPa Previously, we observed that L. monocytogenes was able to grow on fresh-cut pears processed (without antioxidant treatment and without MAP) at different ripeness stages and that the growth rate of L. monocytogenes increased with increasing temperature. 24 Alegre et al. 22,23 determined the survival and growth of E. coli O157:H7, Salmonella and Listeria innocua on some minimally processed fruits, including peaches and apples stored at different temperatures. This study demonstrated that L. monocytogenes was able to grow on fresh-cut pears processed under the conditions described here, despite low pH, refrigeration and use of modified atmosphere. Thus, minimally processed pears should be protected from contamination by this pathogen during preparation, and food business operators should guarantee the absence of L. monocytogenes in 25 g of fruit before the food has left its control area.