Occurrence of Alicyclobacillus acidoterrestris in pasteurized and high hydrostatic pressure-treated fruit juices and isolates' characterization.

In recent years, acidophilic, heat-resistant, and spore-forming spoilage bacteria have been identified in pasteurized or treated by high hydrostatic pressure (HPP) fruit juices. Alicyclobacillus acidoterrestris is the bacteria more frequently linked to the spoilage of this type of product because its spores can survive conventional pasteurization and HPP treatments. Under favourable conditions, such as an acidic pH, its spores can germinate and multiply, with the consequent production of guaiacol. Guaiacol is a compound with an undesirable odour ("medicinal", "smoked" or "antiseptic"). In this context, our objective was to determine the prevalence of A. acidoterrestris in 150 Spanish pasteurized and HPP-treated fruit juices purchased from supermarkets or received from manufacturers. Then, the isolates and the reference strain (CECT 7094 T) were characterized to establish differences in terms of (i) growth capacity at different pH and temperatures, and in (ii) guaiacol production capacity. The results showed a high incidence of A. acidoterrestris (18.0 %) in the analysed juices. The 44.4 % of the isolates came from blends of fruit juices. Within juice blends, 9 juices contained apple juice among their ingredients. This represents a 18.8 % of incidence with respect to the total of blended juices with apple. A high incidence in monovarietal apple juices was also observed (3 out of 14 samples). Regarding the characterization of the isolates, EC1 (isolated from an apple concentrate) showed the highest growth capacity at pH 4.0 at temperatures from 20 to 55 °C. Besides, three strains (R42, EC10, and EZ13, isolated from clementine, plum and white grape juice, respectively) could grow at room temperatures (20 and 25 °C). For pH, only EZ13, isolated from white grape juice, was able to grow significantly at pH 2.5. Finally, the production of guaiacol ranged from 74.1 to 145.6 ppm, being the isolate EC1 the one that produced more guaiacol after 24 h of incubation at 45 °C (145.6 ppm). As we have observed, there is a high incidence of A. acidoterrestris in marketed juices and intermediate products despite the treatments received (pasteurization or HPP). Under favourable conditions for the development of this microorganism, it could produce enough guaiacol to spoil the juices before their consumption. Therefore, in order to improve the quality of fruit juices it is necessary to investigate in more detail the origin of this microorganism and to find strategies to reduce its presence in final products.

Evaluation of Commercial Anti-Listerial Products for Improvement of Food Safety in Ready-to-Eat Meat and Dairy Products.

In ready-to-eat products, such as cooked ham, fresh cheese, and fuet in which Listeria monocytogenes is a concern, the use of biopreservation techniques represents an additional hurdle to inhibit pathogen growth during storage. The objective of this study was to apply several biopreservation techniques in three different food matrices to reduce the growth of Listeria innocua, used as a surrogate of L. monocytogenes. Several lactic acid bacteria, the bacteriocin nisin, the bacteriophage PhageGuard ListexTM P100, and the enzyme lysozyme were evaluated. Cooked ham treated with the bacteriophage PhageGuard ListexTM at 0.5% or with the lactic acid bacteria SafePro® B-SF-43 (25 g/100 kg) reduced L. innocua population to below the detection limit after 7 days of storage (4 °C plus modified atmosphere packaging). In fresh cheese, the application of PhageGuard ListexTM at 0.2 and 0.5% reduced L. innocua counts by more than 3.4 logarithmic units after 6 days at 4 °C. In fuet, the 1.0% of PhageGuard ListexTM reduced L. innocua population by 0.7 ± 0.2 logarithmic units in front of control with no significant differences to other evaluated biopreservative agents. The present results confirm that the application of biopreservation techniques was able to inhibit L. innocua in fuet, cooked ham, and fresh cheese, and suggest that the type of food matrix and its physicochemical characteristics influence the biopreservative efficacy.

Inactivation of Escherichia coli, Salmonella enterica and Listeria monocytogenes on apple peel and apple juice by ultraviolet C light treatments with two irradiation devices.

Following the market trends, the consumption of fresh and cold-pressed juice in Europe is increasing. However, a primary concern - particularly in apple juice - is the related outbreaks caused by food-borne pathogens. One of the challenges is to find methods able to reduce pathogenic loads while avoiding deterioration of nutritional properties and bioactive compounds that occur in thermal pasteurization processes. In this study, the inactivation of Escherichia coli, Salmonella enterica and Listeria monocytogenes was evaluated under different ultraviolet C (UVC254nm) light treatments (up to 10,665.9 ± 28.1 mJ/cm2), in two different steps of the production chain (before and after juice processing): on apple peel discs and in apple juice. The systems proposed were a horizontal chamber with UVC254nm emitting lamps treating the product disposed at a distance of 12 cm, and a tank containing UVC254nm lamps and in which the product is immersed and agitated. Final reductions ranged from 3.3 ± 0.5 to 5.3 ± 0.4 logarithmic units, depending on the microorganism, matrix and used device. The survival curves were adjusted to Weibull and biphasic models (R2-adj ≥ 0.852), and UVC doses needed for the first decimal reduction were calculated, being lower for the apple peel discs (0.20 to 83.83 mJ/cm2) than they were for apple juice (174.60 to 1273.31 mJ/cm2), probably for the low transmittance of the apple juice compared to the surface treatment occurring on the peels. Within the treatments evaluated, the UVC254nm irradiation of apple peels immersed in water was the best option as it resulted in a reduction of the tested microorganisms of ca. 2-3 log units at lower UVC254nm doses (< 500 mJ/cm2) when compared to those occurring in apple peel treated with the UVC chamber and in juice. As contamination can proceed from apples, the sanitization of these fruit prior to juice production may be helpful in reducing the safety risks of the final product, reducing the drawbacks related to the poor transmittance of the fruit juices.

Bacterial Spore Inactivation in Orange Juice and Orange Peel by Ultraviolet-C Light.

Spore-forming bacteria are a great concern for fruit juice processors as they can resist the thermal pasteurization and the high hydrostatic pressure treatments that fruit juices receive during their processing, thus reducing their microbiological quality and safety. In this context, our objective was to evaluate the efficacy of Ultraviolet-C (UV-C) light at 254 nm on reducing bacterial spores of Alicyclobacillus acidoterrestris, Bacillus coagulans and Bacillus cereus at two stages of orange juice production. To simulate fruit disinfection before processing, the orange peel was artificially inoculated with each of the bacterial spores and submitted to UV-C light (97.8-100.1 W/m2) with treatment times between 3 s and 10 min. The obtained product, the orange juice, was also tested by exposing the artificially inoculated juice to UV-C light (100.9-107.9 W/m2) between 5 and 60 min. A three-minute treatment (18.0 kJ/m2) reduced spore numbers on orange peel around 2 log units, while more than 45 min (278.8 kJ/m2) were needed to achieve the same reduction in orange juice for all evaluated bacterial spores. As raw fruits are the main source of bacterial spores in fruit juices, reducing bacterial spores on fruit peels could help fruit juice processors to enhance the microbiological quality and safety of fruit juices.

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 8 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. © 2016 Society of Chemical Industry.

Effect of ripeness stage during processing on Listeria monocytogenes growth on fresh-cut 'Conference' pears.

There are several factors that affect the shelf life of fresh-cut fruit, including the cultivar, the ripeness stage of the fruit during processing and the fruit's storage atmosphere and temperature. The effect of fruit ripeness during processing on the survival and growth of Listeria monocytogenes on fresh-cut 'Conference' pear slices at different temperatures (5, 10 and 20 °C) was studied. The four ripeness stages studied in this work (assessed by a fruit's firmness) were mature-green (54-60 N), partially ripe (43-53 N), ripe (31-42 N) and overripe (<31 N). In our studies, pH, acidity and soluble solids content did not significantly change during conditioning at 20 °C. L. monocytogenes grew under all experimental conditions, showing an increase of approximately 2 log CFU g(-1) after 8 days of storage at 5 °C. There were significant differences in the L. monocytogenes population between different ripeness stages at the end of the experiments at 10 and 20 °C. Regardless of the ripeness stage of a fresh-cut pear, the growth potential of L. monocytogenes increased with increasing temperature. A pear's ripeness stage during processing is an important consideration to ensure the quality of a fresh-cut pear, but it is not as important for preventing L. monocytogenes growth at common storage temperatures.

Control of foodborne pathogens on fresh-cut fruit by a novel strain of Pseudomonas graminis.

The consumption of fresh-cut fruit has substantially risen over the last few years, leading to an increase in the number of outbreaks associated with fruit. Moreover, consumers are currently demanding wholesome, fresh-like, safe foods without added chemicals. As a response, the aim of this study was to determine if the naturally occurring microorganisms on fruit are "competitive with" or "antagonistic to" potentially encountered pathogens. Of the 97 and 107 isolates tested by co-inoculation with Escherichia coli O157:H7, Salmonella and Listeria innocua on fresh-cut apple and peach, respectively, and stored at 20 °C, seven showed a strong antagonistic capacity (more than 1-log unit reduction). One of the isolates, CPA-7, achieved the best reduction values (from 2.8 to 5.9-log units) and was the only isolate able to inhibit E. coli O157:H7 at refrigeration temperatures on both fruits. Therefore, CPA-7 was selected for further assays. Dose-response assays showed that CPA-7 should be present in at least the same amount as the pathogen to adequately reduce the numbers of the pathogen. From the results obtained in in vitro assays, competition seemed to be CPA-7's mode of action against E. coli O157:H7. The CPA-7 strain was identified as Pseudomonas graminis. Thus, the results support the potential use of CPA-7 as a bioprotective agent against foodborne pathogens in minimally processed fruit.

Antagonistic effect of Pseudomonas graminis CPA-7 against foodborne pathogens in fresh-cut apples under simulated commercial conditions.

Recently, we reported that the application of the strain CPA-7 of Pseudomonas graminis, previously isolated from apple, could reduce the population of foodborne pathogens on minimally processed (MP) apples and peaches under laboratory conditions. Therefore, the objective of the present work was to find an antioxidant treatment and a packaging atmosphere condition to improve CPA-7 efficacy in reducing a cocktail of four Salmonella and five Listeria monocytogenes strains on MP apples under simulated commercial processing. The effect of CPA-7 application on apple quality and its survival to simulated gastric stress were also evaluated. Ascorbic acid (2%, w/v) and N-acetyl-l-cysteine (1%, w/v) as antioxidant treatments reduced Salmonella, L. monocytogenes and CPA-7 recovery, meanwhile no reduction was observed with NatureSeal(®) AS1 (NS, 6%, w/v). The antagonistic strain was effective on NS-treated apple wedges stored at 10 °C with or without modified atmosphere packaging (MAP). Then, in a semi-commercial assay, efficacy of CPA-7 inoculated at 10(5) and 10(7) cfu mL(-1) against Salmonella and L. monocytogenes strains on MP apples with NS and MAP and stored at 5 and 10 °C was evaluated. Although high CPA-7 concentrations/populations avoided Salmonella growth at 10 °C and lowered L. monocytogenes population increases were observed at both temperatures, the effect was not instantaneous. No effect on apple quality was detected and CPA-7 did not survived to simulated gastric stress throughout storage. Therefore, CPA-7 could avoid pathogens growth on MP apples during storage when use as part of a hurdle technology in combination with disinfection techniques, low storage temperature and MAP.

Determination of free chlorine concentrations needed to prevent Escherichia coli O157:H7 cross-contamination during fresh-cut produce wash.

This study was conducted to investigate the effect of free chlorine concentrations in wash water on Escherichia coli O157:H7 reduction, survival, and transference during washing of fresh-cut lettuce. The effectiveness of rewashing for inactivation of E. coli O157:H7 on newly cross-contaminated produce previously washed with solutions containing an insufficient amount of chlorine also was assessed. Results indicate that solutions containing a minimum of 0.5 mg/liter free chlorine were effective for inactivating E. coli O157:H7 in suspension to below the detection level. However, the presence of 1 mg/liter free chlorine in the wash solution before washing was insufficient to prevent E. coli O157:H7 survival and transfer during washing because the introduction of cut lettuce to the wash system quickly depleted the free chlorine. Although no E. coli O157:H7 was detected in the wash solution containing 5 mg/liter free chlorine before washing a mix of inoculated and uninoculated lettuce, low numbers of E. coli O157:H7 cells were detected on uninoculated lettuce in four of the seven experimental trials. When the prewash free chlorine concentration was increased to 10 mg/liter or greater, no E. coli O157:H7 transfer was detected. Furthermore, although rewashing newly cross-contaminated lettuce in 50 mg/liter free chlorine for 30 s significantly reduced (P = 0.002) the E. coli O157:H7 populations, it failed to eliminate E. coli O157:H7 on lettuce. This finding suggests that rewashing is not an effective way to correct for process failure, and maintaining a sufficient free chlorine concentration in the wash solution is critical for preventing pathogen cross-contamination.

Microbiological and physicochemical quality of fresh-cut apple enriched with the probiotic strain Lactobacillus rhamnosus GG.

The effectiveness as protective culture of the probiotic Lactobacillus rhamonosus GG (L. rham. GG) against Salmonella and Listeria monocytogenes on minimally-processed apples throughout storage as well as its effect on apple quality and natural microflora was evaluated. Survival to subsequent exposure to gastric stress was also reported. Apples were cut into wedges and dipped in a solution containing Salmonella and L. monocytogenes (10(5) cfu mL(-1)) and/or L. rham. GG (10(8) cfu mL(-1)). Apple wedges were packed and stored at 5 and 10 °C. Periodically, microbial population, bacterial survival to gastric stress and quality of apple wedges were evaluated. Although Salmonella was not affected by co-inoculation with L. rham. GG, L. monocytogenes population was 1-log units lower in the presence of L. rham. GG. L. rham. GG population maintained over recommended levels for probiotic action (10(6) cfu g(-1)) along storage, however, viable cells after gastric stress were only above this level during the first 14 days. Pathogen survival after gastric stress was <1% after 7 days at 5 °C. Moreover, apple wedges quality was not affected by L. rham. GG addition. Thus, L. rham. GG could be a suitable probiotic for minimally-processed apples capable to reduce L. monocytogenes growth; nevertheless shelf life should not be higher to 14 days to guarantee the probiotic effect.

Fate of Escherichia coli O157:H7, Salmonella and Listeria innocua on minimally-processed peaches under different storage conditions.

Consumption of fresh-cut produce has sharply increased recently causing an increase of foodborne illnesses associated with these products. As generally, acidic fruits are considered 'safe' from a microbiological point of view, the aim of this work was to study the growth and survival of Escherichia coli O157:H7, Salmonella and Listeria innocua on minimally-processed peaches. The three foodborne pathogens population increased more than 2 log(10)units on fresh-cut peach when stored at 20 and 25 degrees C after 48 h. At 10 degrees C only L. innocua grew more than 1 log(10)unit and it was the only pathogen able to grow at 5 degrees C. Differences in growth occurred between different peach varieties tested, with higher population increases in those varieties with higher pH ('Royal Glory' 4.73+/-0.25 and 'Diana' 4.12+/-0.18). The use of common strategies on extending shelf life of fresh-cut produce, as modified atmosphere packaging and the use of the antioxidant substance, ascorbic acid (2%w/v), did not affect pathogens' growth at any of the temperatures tested (5 and 25 degrees C). Minimally-processed peaches have shown to be a good substrate for foodborne pathogens' growth regardless use of modified atmosphere and ascorbic acid. Therefore, maintaining cold chain and avoiding contamination is highly necessary.

Factors affecting growth of foodborne pathogens on minimally processed apples.

Escherichia coli O157:H7, Salmonella and Listeria innocua increased by more than 2 log(10) units over a 24 h period on fresh-cut 'Golden Delicious' apple plugs stored at 25 and 20 degrees C. L. innocua reached the same final population level at 10 degrees C meanwhile E. coli and Salmonella only increased 1.3 log(10) units after 6 days. Only L. innocua was able to grow at 5 degrees C. No significant differences were observed between the growth of foodborne pathogens on fresh-cut 'Golden Delicious', 'Granny Smith' and 'Shampion' apples stored at 25 and 5 degrees C. The treatment of 'Golden Delicious' and 'Granny Smith' apple plugs with the antioxidants, ascorbic acid (2%) and NatureSeal (6%), did not affect pathogen growth. The effect of passive modified atmosphere packaging (MAP) on the growth of E. coli, Salmonella and L. innocua on 'Golden Delicious' apple slices was also tested. There were no significant differences in growth of pathogens in MAP conditions compared with air packaging of 'Golden Delicious' apple plugs, but the growth of mesophilic and psychrotrophic microorganisms was inhibited. These results highlight the importance of avoiding contamination of fresh-cut fruit with foodborne pathogens and the maintenance of the cold chain during storage until consumption.

Efficacy of neutral electrolyzed water (NEW) for reducing microbial contamination on minimally-processed vegetables.

Consumption of minimally-processed, or fresh-cut, fruit and vegetables has rapidly increased in recent years, but there have also been several reported outbreaks associated with the consumption of these products. Sodium hypochlorite is currently the most widespread disinfectant used by fresh-cut industries. Neutral electrolyzed water (NEW) is a novel disinfection system that could represent an alternative to sodium hypochlorite. The aim of the study was to determine whether NEW could replace sodium hypochlorite in the fresh-cut produce industry. The effects of NEW, applied in different concentrations, at different treatment temperatures and for different times, in the reduction of the foodborne pathogens Salmonella, Listeria monocytogenes and Escherichia coli O157:H7 and against the spoilage bacterium Erwinia carotovora were tested in lettuce. Lettuce was artificially inoculated by dipping it in a suspension of the studied pathogens at 10(8), 10(7) or 10(5) cfu ml(-1), depending on the assay. The NEW treatment was always compared with washing with deionized water and with a standard hypochlorite treatment. The effect of inoculum size was also studied. Finally, the effect of NEW on the indigenous microbiota of different packaged fresh-cut products was also determined. The bactericidal activity of diluted NEW (containing approximately 50 ppm of free chlorine, pH 8.60) against E. coli O157:H7, Salmonella, L. innocua and E. carotovora on lettuce was similar to that of chlorinated water (120 ppm of free chlorine) with reductions of 1-2 log units. There were generally no significant differences when treating lettuce with NEW for 1 and 3 min. Neither inoculation dose (10(7) or 10(5) cfu ml(-1)) influenced the bacterial reduction achieved. Treating fresh-cut lettuce, carrot, endive, corn salad and 'Four seasons' salad with NEW 1:5 (containing about 50 ppm of free chlorine) was equally effective as applying chlorinated water at 120 ppm. Microbial reduction depended on the vegetable tested: NEW and sodium hypochlorite treatments were more effective on carrot and endive than on iceberg lettuce, 'Four seasons' salad and corn salad. The reductions of indigenous microbiota were smaller than those obtained with the artificially inoculated bacteria tested (0.5-1.2 log reduction). NEW seems to be a promising disinfection method as it would allow to reduce the amount of free chlorine used for the disinfection of fresh-cut produce by the food industry, as the same microbial reduction as sodium hypochlorite is obtained. This would constitute a safer, 'in situ', and easier to handle way of ensuring food safety.