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.

Application of an innovative water-assisted ultraviolet C light technology for the inactivation of microorganisms in tomato processing industries.

We aimed to study the efficacy of a water-assisted UVC light device (WUVC) as an innovative clean technology for the disinfection of fresh sound tomatoes and processing wash water and water turbidity was evaluated as a critical parameter. First, wash waters with different turbidities (from 0.4 to 828 NTU) were inoculated with Listeria innocua and treated in the WUVC device at different dosages. Secondly, fresh tomatoes, inoculated with L. innocua and non-inoculated ones, were treated using the WUVC device containing wash water of different turbidities for different times. The reduction of L. innocua populations on wash water and on the surface of tomato was influenced by turbidity; lower reduction values were observed at higher turbidities. Washing tomatoes with tap water with UVC lamps off (control treatment, TW) decreased L. innocua population on the surface of tomatoes but did not eliminate those bacteria that went into the water. Contrarily, when UVC lights were on, L. innocua population in wash water after treatment significantly decreased, those in clean water being the lowest populations. Reductions of native microbiota on the clean water treated with the highest UV-C radiation dose were lower than those obtained when tomatoes were artificially inoculated. We demonstrated that high reductions of L. innocua population on fresh tomatoes could be achieved using the WUVC system but some drawbacks related to the increase of turbidity should be solved for its implementation in real conditions.

Assessing water-assisted UV-C light and its combination with peroxyacetic acid and Pseudomonas graminis CPA-7 for the inactivation and inhibition of Listeria monocytogenes and Salmonella enterica in fresh-cut 'Iceberg' lettuce and baby spinach leaves.

The effectiveness of ultraviolet C light (UV-C) delivered in water (WUV) or in peroxyacetic acid (PAA) for the inactivation and inhibition of L. monocytogenes and S. enterica in ready-to-eat 'Iceberg lettuce' and baby spinach leaves, was evaluated throughout chilled storage in modified atmosphere packaging (MAP). The inhibition of pathogen's growth by sequential pretreatments with UV-C in PAA and then biocontrol using Pseudomonas graminis CPA-7 was assessed during MAP storage at 5 °C and upon a breakage of the cold-storage chain. In fresh-cut lettuce, 0 1 kJ/m2 UV-C, in water or in 40 mg/L PAA, inactivated both pathogens by up to 2.1 ±â€¯0.7 log10, which improved the efficacy of water-washing by up to 1.9 log10 and showed bacteriostatic effects on both pathogens. In baby spinach leaves, the combination of 0 3 kJ/m2 UV-C and 40 mg/L PAA reduced S. enterica and L. monocytogenes populations by 1.4 ±â€¯0.2 and 2.2 ±â€¯0.3 log10 respectively, which improved water-washing by 0.8 ±â€¯0.2 log10. Combined treatments (0.1 or 0 3 kJ/m2 WUV and 40 mg/L PAA) inactivated both pathogens in the process solution from lettuce or spinach single sanitation, respectively. Pretreating lettuce with UV-C in PAA reduced L. monocytogenes and S. enterica's growth by up to 0.9 ±â€¯0.1 log10 with respect to the PAA-pretreated control after 6 d at 5 °C in MAP. Upon a cold-chain breakage, CPA-7 prevented S. enterica growth in PAA-pretreated lettuce, whereas showed no effect on L. monocytogenes in any of both matrices. Low-dose UV-C in PAA is a suitable preservation strategy for improving the safety of ready-to-eat leafy greens and reducing the risk of cross contamination.

Effect of Pseudomonas graminis strain CPA-7 on the ability of Listeria monocytogenes and Salmonella enterica subsp. enterica to colonize Caco-2 cells after pre-incubation on fresh-cut pear.

To further gain insight into the mechanism by which the biopreservative bacterium Pseudomonas graminis CPA-7 develops its antimicrobial activity, we have examined the effect that the prior interaction stablished by this bacterium and two foodborne pathogens on fresh-cut pear, has on their capacity to colonize human epithelial cells (Caco-2 cell line) which is crucial for establishing infection. CPA-7 inhibited the growth of L. monocytogenes and S. enterica subsp. enterica ser. Enteritidis by 5.5 and 3.1 log10, respectively, after 7d of interaction at 10°C. Furthermore, CPA-7 attenuated the adherence of S. enterica to Caco-2 cells by 0.8 log10 regardless of the pre-adaptation on the fruit. Conversely, the adhesiveness of L. monocytogenes was not influenced by the interaction with the antagonist but it was reduced by 0.5 log10 after incubation on the food matrix. Pathogen-antagonist-food matrix interaction was associated to a significant reduction of the relative invasiveness of both pathogens, by 1.3 log10 in the case of L. monocytogenes and to an undetectable level (below 5CFU/g fruit) for S. enterica. CPA-7 can adhere to and internalize into intestinal epithelium which enables it for competition. Its adherence positively correlates to the multiplicity of infection (MOI) with respect to Caco-2 cells, increasing by 0.6 log10 in an MOI range of 0.1:1 to 100:1. For the same levels of inoculum, internalized cells could only be detected after 7d of pre-adaptation in the fruit (pH4.5-5.0). However, the combination of gastrointestinal digestion and habituation on the fruit resulted in a significant reduction of CPA-7 populations (by 2 log10 more after 7d of incubation than on inoculation day) as well as in the decrease of its adhesiveness (by 0.8 log10) and invasiveness (to undetectable levels).

Exposure to minimally processed pear and melon during shelf life could modify the pathogenic potential of Listeria monocytogenes.

Survival and virulence of foodborne pathogens can be influenced by environmental factors such as the intrinsic properties of food as well as the extrinsic properties that contribute to food shelf life (e.g., temperature and gas atmosphere). The direct contribution of food matrix characteristics on the survival of L. monocytogenes during fresh-cut fruit shelf life is not very well understood. In addition, the gastrointestinal tract is the primary route of listeriosis infection and penetration of the intestinal epithelial cell barrier is the first step in the infection process. Hence, the pathogenic potential of L. monocytogenes, measured as the capability for the organism to survive a simulated gastrointestinal tract and the proportion of cells able to subsequently adhere to and invade differentiated Caco-2 cells, subjected to fresh-cut pear and melon shelf life, was investigated. Samples were inoculated, stored at 10 °C for 7 days and evaluated after inoculation and again after 2 and 7 days of storage. A decrease in L. monocytogenes' capacity to survive a simulated gastrointestinal tract was observed with increasing storage time, regardless of the fruit matrix evaluated. Furthermore, L. monocytogenes placed on fresh-cut pear and melon was subjected to an attachment and invasion assay after crossing the simulated gastrointestinal tract. After inoculation, pathogen on fresh-cut pear showed 5-fold more capacity to adhere to Caco-2 cells than pathogen on fresh-cut melon. After 2 days of storage, L. monocytogenes grown on fresh-cut melon showed similar adhesive capacity (1.11%) than cells grown on pear (1.83%), but cells grown on melon had the higher invasive capacity (0.0093%). We can conclude that minimally processed melon could represent a more important hazard than pear under the studied shelf life.

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.