Food Microbiology 38 (2014) 137e142
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Effectiveness of a bacteriophage in reducing Listeria monocytogenes on fresh-cut fruits and fruit juices M. Oliveira a, I. Viñas a, P. Colàs a, M. Anguera b, J. Usall b, M. Abadias b, * a b
Food Technology Department, University of Lleida, XaRTA-Postharvest, Agrotecnio Center, Rovira Roure 191, 25198 Lleida, Catalonia, Spain IRTA, XaRTA-Postharvest, Rovira Roure 191, 25198 Lleida, Catalonia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2013 Received in revised form 12 August 2013 Accepted 29 August 2013 Available online 11 September 2013
Listeria monocytogenes is a serious foodborne pathogen and new strategies to control it in food are needed. Among them, bacteriophages hold attributes that appear to be attractive. The objective of this study was to investigate the efficacy of the bacteriophage Listex P100 to control L. monocytogenes growth on melon, pear and apple products (juices and slices) stored at 10 C. L. monocytogenes grew well in untreated fruit slices. In juices, the pathogen grew in untreated melon, survived in untreated pear and decreased in untreated apple. Phage treatment was more effective on melon followed by pear, but no effect on apple products was observed. Reductions of about 1.50 and 1.00 log cfu plug1 for melon and pear slices were found, respectively. In juices, higher reductions were obtained in melon (8.00 log cfu mL1) followed by pear (2.10 log cfu mL1) after 8 days of storage. L. monocytogenes in apple juice was unaffected by phage treatment in which the phage decreased to almost undetectable numbers. These results highlight that Listex P100 could avoid pathogen growth on fresh-cut and in fruit juices with high pH during storage at 10 C. The combination with other technologies may be required to improve the phage application on high acidity fruits. Ó 2013 Published by Elsevier Ltd.
Keywords: Listex P100 Biocontrol Melon Apple Pear Biopreservation
1. Introduction Listeria monocytogenes is an ubiquitous organism in the environment, can be isolated from soil, water, vegetation, the faeces of livestock (Heaton and Jones, 2008), and is often associated with fresh or minimally processed fruits and vegetables (Abadias et al., 2008; Beuchat, 2002; Thunberg et al., 2002). The incidence of foodborne illness associated with consumption of minimally processed fruits and vegetables has consistently increased (Tauxe, 1997) possibly due to increased consumption of fresh-cut produce and better epidemiologic surveillance programs. L. monocytogenes has been associated with a number of serious foodborne outbreaks and recalls (Beuchat, 2002; Farber and Peterkin, 1991; Thunberg et al., 2002). The most recent (October 2011) affected a total of 146 persons and caused 30 deaths in the United States and was due to contaminated melon (CDC, 2011). This pathogen usually has low incidence. However, due to its high mortality rate (up to 30%), its ability to survive a wide range of environmental conditions and its ability to multiply at refrigeration
* Corresponding author. Tel.: þ34 973003430; fax: þ34 973238301. E-mail address: [email protected] (M. Abadias). 0740-0020/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fm.2013.08.018
temperatures, it is considered an important foodborne pathogen (Farber and Peterkin, 1991). Over the last years, a number of strategies to minimize the microbial load of raw products have been explored. A variety of disinfectants (including chlorine, hydrogen peroxide, organic acids and ozone) have been used to reduce initial bacterial populations on minimally processed produce (Beuchat, 1998). Chlorine is the most widely used sanitizer in the fresh produce industry. However, studies indicate that chlorine concentrations traditionally used (50e200 ppm) are not effective in reducing pathogen load on freshcut produce (Behrsing et al., 2000; Delaquis et al., 2002; Lee and Baek, 2008). Moreover, a prolonged exposure to chlorine vapor may cause irritation to the skin and respiratory tract of the workers, may affect the quality of foods and also adversely affect the environment (Beuchat, 1998). It is also known that the reaction of chlorine with organic matter results in the formation of carcinogenic products (trihalomethanes) for consumers (Nieuwenhuijsen et al., 2000). Thus, there is a need for better, safer and more environmental friendly methods to reduce the contamination. Bacteriophage (phage) prophylaxis is one possible alternative to achieve this goal. Phages are bacterial viruses that invade specific bacterial cells, disrupt bacterial metabolism, and cause the bacterium to lyse without compromising the viability of other flora in the
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habitat. They are the most abundant microorganisms in our environment (Brussow and Hendrix, 2002) and are present in high numbers in water and foods (Hsu et al., 2002; Kennedy et al., 1986). Phages are also part of gastrointestinal system (Greer, 2005), and may provide a natural, non-toxic, feasible approach for controlling several human pathogens. A study in humans with Escherichia colispecific phages also indicated that phages are safe for oral administration (Bruttin and Brüssow, 2005). This together with their specificity makes phages excellent tools for food safety purposes. Promising results using phage biocontrol have been reported for several pathogens, including Salmonella (Guenther et al., 2012; Kocharunchitt et al., 2009; Leverentz et al., 2001), Campylobacter (Goode et al., 2003), L. monocytogenes (Carlton et al., 2005; Dykes and Moorhead, 2002; Guenther et al., 2009; Leverentz et al., 2003) and E. coli O157:H7 (Abuladze et al., 2008; Sharma et al., 2009; Viazis et al., 2011). However, some studies reported that the effectiveness of many phages seems to decline at acidic pH (Leverentz et al., 2001, 2003). Moreover, the efficacy of phages in food also depends on the structure and chemical composition of the different food items and a homogeneous distribution and sufficient diffusion ability of the phage particles is necessary (Guenther et al., 2009). There are several phage preparations commercialized, such as ListShieldÔ and EcoShieldÔ (Intralytix, Inc., USA), AgriphageÔ (Omnilytics, Inc., USA) and ListexÔ P100 (Micreos Food Safety, formerly EBI Food Safety, The Netherlands). The approval of using phage product Listex P100 in food products by FDA and USDA provides the impetus to further investigate phage applications. Listex P100 is a culture of safe and natural bacteriophages, characterized by its broad spectrum towards L. monocytogenes strains. It can be used as a processing aid with all food products susceptible to L. monocytogenes. An oral toxicity study in rats receiving high doses of Listeria phage P100 did not reveal any side effects (Carlton et al., 2005). Little work is known about the efficacy of Listex P100 on fresh-cut fruits and fruit juices, which are characterized by its low pH. The aim of this study was to evaluate the effect of fruit pH (melon, pear and apple) and physical form of the food matrix (solid ¼ fruits slices vs liquid ¼ fruit juices) on the efficacy of the phage Listex P100 against a three strain cocktail of L. monocytogenes. 2. Material and methods 2.1. Fruits ‘Golden Delicious’ apples (Malus domestica cv. Golden Delicious) and ‘Conference’ pears (Pyrus communis cv. Conference) were obtained from local packinghouses in Lleida (Catalonia, Spain). ‘Piel de Sapo’ melons (Cucumis melo L. var. ‘Piel de sapo’) were purchased in a local supermarket. Prior to the experimental studies, fruits were washed in running tap water, surface disinfected with ethanol 70% and let to dry at room temperature.
(TYSEB) at 37 1 C for 20e24 h. Bacterial cells were harvested by centrifugation at 9820 g for 10 min at 10 1 C and resuspended in sterile saline peptone (SP, 8.5 g L1 NaCl and 1 g L1 peptone). Equal volumes of each strain were combined to obtain the threestrain cocktail of L. monocytogenes. The concentration of L. monocytogenes was confirmed by plating duplicate serial suspension dilutions on Palcam medium (Palcam Agar Base with selective supplement, Biokar Diagnostics, Beauvais, France) followed by incubation at 37 1 C for 48 h. A suspension of 105 colony forming units (cfu) mL1 was prepared and confirmed by plating as described before. 2.3. Phage The bacteriophage Listex P100 (Micreos Food Safety, The Netherlands), characterized by its broad spectrum toward L. monocytogenes strains, was used in this study. The phage concentration was approximately 1011 plaque forming units (pfu) mL1 in buffered saline and was maintained at 5 C. 2.4. Preparation of fruit slices and fruit juices For fruit assays, apples and pears were peeled and cut into ten slices using a manual fruit slicer/corer. Melons were cut transversally in 14e16 mm slices, seeds and rind were removed and each slice was cut in trapezoidal pieces. In each fruit slice, a 6 mm diameter well was made on the center of each wedge to contain the inoculums. Three wedges were placed in commercial 500 mL food plastic bowls. The covers on the plastic bowls allowed sufficient air exchange to prevent modified atmosphere creation. Fruit juices were extracted by crushing peeled fruits wedges in a blender. Juice was filtered using glass wool, bottled and autoclaved at 115 C for 10 min. Thirty milliliters of sterile pear, apple and melon juices was individually transferred into 50 mL sterilized polypropylene containers (Deltalab, Barcelona, Spain). 2.5. Inoculation procedures The fruit slices were inoculated with the cocktail of L. monocytogenes by pipetting 15 mL of the bacterial suspension containing approximately 1 105 cfu mL1 onto the well of each wedge. Then, the phage was applied to the wells by pipetting 15 mL of the suspension at approximately 1 108 pfu mL1. There were 3 fruit slices per treatment at each recovery time. Samples of 30 mL of fruit juices were each inoculated with specific volume of the bacterial and phage suspensions to obtain final concentrations of about 1 105 cfu mL1 and 1 108 pfu mL1, respectively. There were 3 samples per treatment for each fruit juice. Juice samples were mixed thoroughly to ensure homogeneous distribution of the pathogen and phage. All samples (slices and juices) were stored at 10 C for 8 days. 2.6. Bacteria analysis
2.2. Bacteria and preparation of inocula Three strains of L. monocytogenes: serovar 1a (Spanish Type Culture Collection (CECT) 4031), serovar 4a (CECT 940) and serovar 4b (CECT 4032) were used in this study. L. monocytogenes strains were grown individually on tryptone soy agar (TSA, Oxoid, LTD, Basingstoke, Hampshire, England) supplemented with 6 g L1 yeast extract, 2.5 g L1 glucose and 2.5 g L1 dipotassium hydrogen phosphate (TYSEA) at 37 1 C for 20e24 h. An individual colony of each strain was transferred into a flask with 50 mL of tryptone soy broth (TSB, Oxoid, UK) supplemented with 6 g L1 yeast extract
Recovery of L. monocytogenes populations from fruit and juice samples were performed after 0, 2, 5 and 8 days of storage at 10 C. To recover the pathogen from the wedges, a cylinder (1.2 cm in diameter and 1 cm deep) containing the entire well was removed using a sterile cork borer and placed in a filtered sterile bag (Bagpage 80 mL, Interscience BagSystem, St Nom La Breteche, France) with 9 mL of buffered peptone water (BPW, Oxoid) and blended in a homogenizer for 120 s at high speed (Bagmixer 100 Minimix, Interscience). Aliquots of the mixture were then serially diluted in saline peptone (SP; 8.5 g L1 NaCl and 1 g L1 peptone)
M. Oliveira et al. / Food Microbiology 38 (2014) 137e142 Table 1 Determination of initial pH, soluble solids (SS,
Brix) and titratable acidity (TA, g malic or citric acid L1) of melon, pear and apple from fruits and juices used in the assays.
Melon
Pear
Fruit pH SS TA a
5.92 0.26 9.9 1.3 1.34 0.10
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a
Apple
Juice
Fruit
Juice
Fruit
Juice
5.77 0.12 10.2 0.5 1.32 0.26
4.91 0.14 15.7 1.5 1.19 0.22
4.61 0.02 14.5 0.2 1.62 0.07
3.76 0.17 13.6 0.5 3.41 0.08
3.70 0.10 14.2 0.1 3.00 0.49
Results expressed as mean the stander and deviation of the mean for each analysis.
and 100 mL was spread plated on Palcam agar. The plates were incubated at 37 1 C for 48 h and the data were plotted as log cfu plug1. Populations of L. monocytogenes in fruit juices were enumerated taking an aliquot of each juice and diluted in SP, followed by spread plating on Palcam agar as described previously. After colony counting, data were plotted as log cfu mL1. 2.7. Determination of phage titer In order to determine the phage titer from fruit and juice samples of phage treatments, the same samples obtained to determine L. monocytogenes populations were used. The phage titer was determined using the soft agar overlay method following the instructions given by the product supplier. Briefly, aliquots of 100 mL of the diluted sample were mixed with 100 mL of log-phase L. monocytogenes cocktail in 3.5 mL of molten brain-heart infusion soft agar (BHI containing 0.4% agar; Biokar Diagnostics). Immediately after, the soft agar mixture was poured onto solid BHI agar plates (1.2% agar) and incubated for 24 h at 30 C. At the end of incubation period, the visible plaques were counted and expressed as log pfu plug1 or log pfu mL1. 2.8. Fruit and juices quality parameters Quality analysis (pH, soluble solids and titratable acidity) of fruits and juices was performed before the experiment. Fruit flesh pH was determined using a penetration electrode (5231 Crison, and pH-meter Model GLP22, Crison Instruments S.A., Barcelona, Spain) for fruit slices assay. Juice pH was determined using pH-meter (5203 Crison, Model GLP22). There were three determinations per fruit and juice. Percent of soluble solids (SS, Brix) was measured at 20 C with a handheld refractometer (Atago Co. Ltd., Tokio, Japan) in juice extracted by crushing fruit pieces in a blender. There were three measurements per fruit and juice. To measure titratable acidity (TA), 10 mL of fruit juice (obtained by crushing fruit pieces) were diluted with 10 mL of distilled water and it was titrated with 0.1 N NaOH up to 8.1. The results were calculated as g of citric acid L1 for melons and g of malic acid L1 for pears and apples. There were three determinations per fruit and juice. 2.9. Statistical analysis All experiments were replicated three times with three different replications per treatment/day for each fruit sample. Therefore, reported populations represent the mean of nine values. The bacterial and phage data were transformed to log units. Population of L. monocytogenes recovered from fruit slices and juices treated with phage and the control were subjected to Statistical software JMP (SAS Institute, version 9.2, Cary, NC, USA). Significant differences between phage treatment and control were analyzed by t-student test at a significance level of P < 0.05.
3. Results 3.1. Fruit quality parameters The pH, soluble solids and titratable acidity of fruits and juices used in the assay are summarized in Table 1. Apple products had the lowest pH (3.70e3.76) followed by pear (4.61e4.91). The highest pH (5.77e5.92) was measured in melon products. Pear and apple products had the highest soluble solids content, 14.5e15.7 and 13.6e14.2, respectively, and the lowest was observed in melon (9.9e10.2). The highest titratable acidity was determined in apple (3.00e3.41), followed by pear (1.19e1.62) and melon products (1.32e1.34). 3.2. Effect of phage treatment on survival of L. monocytogenes on fruit slices L. monocytogenes grew at 10 C on melon (pH 5.92), pear (pH 4.91) and apple (pH 3.76) slices throughout storage period (Fig. 1). Initial L. monocytogenes populations on untreated melon, pear and apple were 2.77, 2.94 and 2.48 log cfu plug1, respectively. The most remarkable bacterial growth in untreated samples was observed on melon, followed by pear and apple slices, reaching populations close to 8.00, 6.00 and 5.00 log cfu plug1, respectively, after 8 days of storage at 10 C. The largest statistical difference in L. monocytogenes populations on untreated and phage-treated slices was observed on melon with reductions of about 1.50 log cfu plug1 during all storage period (Fig. 1). L. monocytogenes populations on phage treated melon were close to 6.50 log cfu plug1 after 8 days at 10 C. Phage application on pear slices reduced 1.00, 1.15 and 0.62 log cfu plug1 the population of L. monocytogenes after 2, 5 and 8 days of storage, respectively. On treated apple slices significant reductions were not observed and the pathogen survived during all storage period at levels between 2.20 and 4.20 log cfu plug1. Phage populations on melon slices increased about 1.5-log units (Fig. 1) over a period of 8 days, reaching levels of about 6.90 log pfu plug1 at the end of storage. On pear slices the titer slightly decreased and remained between 5.79 and 4.82 log pfu plug1. In contrast, the phage titer declined rapidly after 2 days on apple slices and reached undetectable levels after 8 days at 10 C. 3.3. Effect of phage treatment on survival of L. monocytogenes on fruit juices L. monocytogenes grew in untreated melon (pH 5.77), survived in untreated pear (pH 4.61) and decrease in untreated apple (pH 3.70) juices throughout storage period at 10 C (Fig. 2). The initial concentration of L. monocytogenes in untreated fruit juices was around 5.30 log cfu mL1. In untreated melon juice, the pathogen showed a considerably increase during the storage period, reaching around 8.70 log cfu mL1 after 8 days of storage. L. monocytogenes populations in untreated pear juice remained
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Fig. 1. L. monocytogenes population (log cfu plug1) on melon (A), pear (B) and apple (C) slices and the titer of phage (log pfu plug1) stored at 10 C. Continuous line represent the titer of phage (Phage) and columns represent the growth of L. monocytogenes alone (Lm) or together with the phage (Lm þ Phage). Data represent the mean of three determinations and three experiment repetitions (n ¼ 9). Bars represent standard deviation of the mean and where not visible, they are smaller than symbol size. For each time, different letters indicate significant differences (P < 0.05) among treatments.
Fig. 2. L. monocytogenes population (log cfu mL1) on melon (A), pear (B) and apple (C) juices and the titer of phage (log pfu mL1) stored at 10 C. Continuous line represent the titer of phage (Phage) and columns represent the growth of L. monocytogenes alone (Lm) or together with the phage (Lm þ Phage). Data represent the mean of three determinations and three experiment repetitions (n ¼ 9). Bars represent standard deviation of the mean and where not visible, they are smaller than symbol size. For each time, different letters indicate significant differences (P < 0.05) among treatments.
constant during storage period with levels between 5.30 and 5.12 log cfu mL1. In untreated apple juice, there was a steady decline (almost 2-log units) of L. monocytogenes populations over time, approaching 3.26 log cfu mL1 after 8 days of storage. The bacterial populations were significantly (P < 0.05) lower on phage-treated melon and pear juices than on corresponding juices without phage, during storage period. Nevertheless, no significant reduction in the L. monocytogenes counts was observed in the apple juice treated with phage (Fig. 2). The largest difference in L. monocytogenes populations in untreated and phagetreated juices was observed on day 8 (8-log units) in melon juice, were L. monocytogenes reached final population around
0.60 log cfu mL1. Furthermore, the phage was also effective in pear juice. The reduction of pathogen was between 2.10 and 2.80 log cfu mL1 during storage, reaching populations close to 3.00 log cfu mL1 after 8 days at 10 C. There was no effect in L. monocytogenes populations in apple juice treated with phage, and the bacteria survived during storage period at levels between 5.00 and 3.30 log cfu mL1. Phage populations remained constant in melon and pear juices during storage period and counts were in the range of 7.90 and 7.40 log pfu mL1 (Fig. 2). In contrast, the phage titer declined rapidly in apple juice with a decrease of almost 7-log units after 8 days of storage.
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4. Discussion This study compared the effectiveness of phage Listex P100 in controlling the growth of three strain cocktail of L. monocytogenes on different fresh-cut fruits and in fruit juices. Three types of fruit with different pHs and acidities (melon, pear and apple) and also the physical form of food matrix (solid vs liquid) were tested. Our results demonstrated that both pH and physical form of fruit influenced on phage efficacy. We observed more effectiveness in controlling the growth of L. monocytogenes in fruit juices and also in fruits with higher pH. The growth of L. monocytogenes on melon stored at 10 C over 8 days was several log units higher than the growth on pear and apple. The pH of melon was approximately 5.77e5.92, 4.61 to 4.91 on pear, and 3.70 to 3.76 on apple. This pH difference may be a major factor contributing to the differences in the bacterial populations on the fruit slices and juices. Growth differences of pathogens on fruits and juices were also obtained in several studies (Abadias et al., 2012; Alegre et al., 2010a, 2010b; Leverentz et al., 2001, 2003; Raybaudi-Massilia et al., 2009). We also observed that L. monocytogenes grew on untreated apple slices but decreased in untreated apple juices stored at 10 C. According to Conway et al. (2000), the growth of L. monocytogenes on apple slices probably resulted from its ability to modify the immediate microenvironment. This type of environmental changes would not occur in apple juice, in which we observed that the population decreases during storage period. However, Yuste and Fung (2002) reported the survival but not the growth of L. monocytogenes in apple juice (pH 3.7) at 5 C. Phage treatment decreased L. monocytogenes populations on fruit slices and juices. However, these reductions were greater in fruit juices than on fruit slices. The detection of more pathogen counts in fruit slices than in fruit juices after phage treatment could suggest that phages are seemingly immobilized after addition to solid surfaces and therefore could not come into contact with the surviving bacteria through limited diffusion (Guenther et al., 2009). In liquids, even a very small initial number of phages can cause complete lysis of the bacteria in a relatively short time (Hagens and Loessner, 2010). On the other hand, in solid matrix a high concentration of phage is necessary to enable it to cover the entire space and infect the bacteria as it depends on the amount of fluid in the food. As a conclusion, the concentration of the phages must be sufficiently high to enable contact and subsequent infection, even when bacteria are present at very low numbers (Hagens and Loessner, 2010; Hagens and Offerhaus, 2008). Phage treatment was more effective on fresh-cut melon followed by pears with reductions about 1.50 and 1.00 log cfu plug1, respectively. Nevertheless, the phage treatment was not effective on apple slices. A study using the phage LMP-102 reduced by 2.0e 4.6 log cfu the population of L. monocytogenes on honeydew melons and no reduction was observed on apples (Leverentz et al., 2003). The efficacy of Listex P100 has also been tested in other foods. Guenther et al. (2009) observed reduction counts by up to 5-log units in ready-to-eat food. Carlton et al. (2005) reported approximately 2e3 log decrease on soft cheese. These reduction differences could be due to the different bacteriophage application and concentration, and also the different matrices used. Phages effectiveness is influenced by pH and other physiochemical properties of foods, activity on a solid substrate or biofilm, the emergence of resistant bacteria mutants, and the relative numbers of phages and host required to allow replication (Hudson et al., 2005). Similarly to what happened on fruit slices, a more pronounced reduction of L. monocytogenes was obtained in melon juice followed by pear juice. However, the numbers of L. monocytogenes in apple juice were unaffected by phage treatment. This result could be
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attributed to the increased sensitivity of phages to the acid environment of the apple juice (pH 3.70) which caused a marked phage titer decline. We observed a great tolerance of phage to survive during the storage on both slices and juice of melon and pear with a pH above 5.77 and 4.61, respectively. However, a rapid decline of phages to almost undetectable numbers on apple products with pH below 3.76 was observed. These results are in accordance with other studies that have shown a similar behavior of LMP-102 on melon and apple fruits (Leverentz et al., 2001, 2003). The inability of phages to tolerate an acidic pH is well documented (Leverentz et al., 2001, 2003; Smith et al., 1987) and is in agreement with our results. The application of bacteriophages on produce with a low pH could be improved by using greater numbers of phage or the development of low-pHtolerant phage mutants. Other alternative could be the combination of phages with other compounds, such as bacteriocins, antibiotics, chlorine compounds, antagonistic bacteria and essential oils. Phage persistence is an important parameter to render applied phages not just decontaminating agent but rather a protective or preservative or as a food additive to control growth of foodborne pathogens in food and beverage industries. Bacteriophage products are already in use in agricultural, food safety and diagnostic applications, demonstrating the utility and viability of such approaches. The application of bacteriophages represents a promising, safe, environmentally friendly and chemical-free alternative to the use of chemicals/sanitizers on produce. Acknowledgments The authors are grateful to the Spanish Government [AGL-201019385] for its financial support. References Abadias, M., Usall, J., Anguera, M., Solsona, C., Viñas, I., 2008. Microbiology quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail establishments. Int. J. Food Microbiol. 123, 151e158. Abadias, M., Alegre, I., Oliveira, M., Altisent, R., Viñas, I., 2012. Growth potential of Escherichia coli O157:H7 on fresh-cut fruits (melon and pineapple) and vegetables (carrot and escarole) stored under different conditions. Food Control 27, 37e44. Abuladze, T., Li, M., Menetrez, M.Y., Dean, T., Senecal, A., Sulakvelidze, A., 2008. Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl. Environ. Microbiol. 74, 6230e6238. Alegre, I., Abadias, M., Anguera, M., Oliveira, M., Viñas, I., 2010a. Factors affecting growth of foodborne pathogens on minimally processed apples. Food Microbiol. 27, 70e76. Alegre, I., Abadias, M., Anguera, M., Viñas, I., 2010b. Fate of Escherichia coli, Salmonella and Listeria innocua on minimally-processed peaches under different storage conditions. Food Microbiol. 27, 862e868. Behrsing, J., Winkler, S., Franz, P., Premier, R., 2000. Efficacy of chlorine for inactivation of Escherichia coli O157:H7 on vegetables. Postharvest Biol. Technol. 19 (2), 187e192. Beuchat, L.R., 1998. Surface Decontamination of Fruit and Vegetables Eaten Raw: A Review. Available at: http://www.who.int/foodsafety/publications/fs_management/ en/surface_decon.pdf (accessed 22.04.10.). Beuchat, L.R., 2002. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes Infect. 4, 413e423. Brussow, H., Hendrix, R.W., 2002. Phage genomics: small is beautiful. Cell 108, 13e16. Bruttin, A., Brüssow, H., 2005. Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy. Antimicrob. Agents Chemother. 49, 2874e 2878. Carlton, R.M., Noordman, W.H., Biswas, B., de Meester, E.D., Loessner, M.J., 2005. Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxixity study, and application. Regul. Toxicol. Pharmacol. 43, 301e312. CDC (Centers for Disease Control and Prevention), 2011. Investigation Update: Multistate Outbreak of Listeriosis Linked to Whole Cantaloupes from Jensen Farms, Colorado. http://www.cdc.gov/listeria/outbreaks/cantaloupes-jensenfarms/120811/index.html (accessed 16.04.20.). Conway, W.S., Leverentz, B., Saftner, R.A., 2000. Survival and growth of Listeria monocytogenes on fresh-cut apple slices and its interaction with Glomerella cingulata and Penicillium expansum. Plant Dis. 84, 177e181.
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Lee, S.Y., Baek, S.Y., 2008. Effect of chemical sanitizer combined with modified atmosphere packaging on inhibiting Escherichia coli O157:H7 in commercial spinach. Food Microbiol. 4, 582e587. Leverentz, B., Conway, W.S., Alavidze, Z., Janisiewicz, W.J., Fuchs, Y., Camp, M.J., Chighladze, E., Sulakvelidze, A., 2001. Examination of bacteriophage as a biocontrol method for Salmonella on fresh-cut fruit: a model study. J. Food Prot. 64, 1116e1121. Leverentz, B., Conway, W.S., Camp, M.J., Janisiewicz, W.J., Abuladze, T., Yang, M., Saftner, R., Sulakvelidze, A., 2003. Biocontrol of Listeria monocytogenes on freshcut produce by treatment with lytic bacteriophages and a bacteriocin. Appl. Environ. Microbiol. 69, 4519e4526. Nieuwenhuijsen, M.J., Toledano, M.B., Elliot, P., 2000. Uptake of chlorination disinfection by-products: a review and a discussion of its implications for exposure assessment in epidemiological studies. J. Expo. Anal. Environ. Epidemiol. 10, 586e599. Raybaudi-Massilia, R.M., Mosqueda-Melgar, J., Martín-Belloso, O., 2009. Antimicrobial activity of malic acid against Listeria monocytogenes, Salmonella Enteritidis and Escherichia coli O157:H7 in apple, pear and melon juices. Food Control 20, 105e112. Sharma, M., Patel, J.R., Conway, W.S., Ferguson, S., Sulakvelidze, A., 2009. Effectiveness of bacteriophages in reducing Escherichia coli O157:H7 on fresh-cut cantaloupes and lettuce. J. Food Prot. 72, 1481e1485. Smith, H.W., Huggins, M.B., Shaw, K.M., 1987. Factors influencing the survival and multiplication of bacteriophages in calves and their environment. J. Gen. Microbiol. 133, 1127e1135. Tauxe, R., 1997. Emerging foodborne diseases: an evolving public health challenge. Emerg. Infect. Dis. 3, 425e434. Thunberg, R.L., Tran, T.T., Bennett, R.W., Matthews, R.N., Belay, N., 2002. Microbial evaluation of selected fresh produce obtained at retail markets. J. Food Prot. 65, 677e682. Viazis, S., Akhtar, M., Feirtag, J., Diez-Gonzalez, F., 2011. Reduction of Escherichia coli O157:H7 viability on leafy green vegetables by treatment with a bacteriophage mixture and trans-cinnamaldehyde. Food Microbiol. 28, 149e157. Yuste, J., Fung, D.Y.C., 2002. Inactivation of Listeria monocytogenes Scott A 49594 in apple juice supplemented with cinnamon. J. Food Prot. 65, 1663e1666.
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Food Microbiology journal homepage: www.elsevier.com/locate/fm
Effectiveness of a bacteriophage in reducing Listeria monocytogenes on fresh-cut fruits and fruit juices M. Oliveira a, I. Viñas a, P. Colàs a, M. Anguera b, J. Usall b, M. Abadias b, * a b
Food Technology Department, University of Lleida, XaRTA-Postharvest, Agrotecnio Center, Rovira Roure 191, 25198 Lleida, Catalonia, Spain IRTA, XaRTA-Postharvest, Rovira Roure 191, 25198 Lleida, Catalonia, Spain
a r t i c l e i n f o
a b s t r a c t
Article history: Received 13 June 2013 Received in revised form 12 August 2013 Accepted 29 August 2013 Available online 11 September 2013
Listeria monocytogenes is a serious foodborne pathogen and new strategies to control it in food are needed. Among them, bacteriophages hold attributes that appear to be attractive. The objective of this study was to investigate the efficacy of the bacteriophage Listex P100 to control L. monocytogenes growth on melon, pear and apple products (juices and slices) stored at 10 C. L. monocytogenes grew well in untreated fruit slices. In juices, the pathogen grew in untreated melon, survived in untreated pear and decreased in untreated apple. Phage treatment was more effective on melon followed by pear, but no effect on apple products was observed. Reductions of about 1.50 and 1.00 log cfu plug1 for melon and pear slices were found, respectively. In juices, higher reductions were obtained in melon (8.00 log cfu mL1) followed by pear (2.10 log cfu mL1) after 8 days of storage. L. monocytogenes in apple juice was unaffected by phage treatment in which the phage decreased to almost undetectable numbers. These results highlight that Listex P100 could avoid pathogen growth on fresh-cut and in fruit juices with high pH during storage at 10 C. The combination with other technologies may be required to improve the phage application on high acidity fruits. Ó 2013 Published by Elsevier Ltd.
Keywords: Listex P100 Biocontrol Melon Apple Pear Biopreservation
1. Introduction Listeria monocytogenes is an ubiquitous organism in the environment, can be isolated from soil, water, vegetation, the faeces of livestock (Heaton and Jones, 2008), and is often associated with fresh or minimally processed fruits and vegetables (Abadias et al., 2008; Beuchat, 2002; Thunberg et al., 2002). The incidence of foodborne illness associated with consumption of minimally processed fruits and vegetables has consistently increased (Tauxe, 1997) possibly due to increased consumption of fresh-cut produce and better epidemiologic surveillance programs. L. monocytogenes has been associated with a number of serious foodborne outbreaks and recalls (Beuchat, 2002; Farber and Peterkin, 1991; Thunberg et al., 2002). The most recent (October 2011) affected a total of 146 persons and caused 30 deaths in the United States and was due to contaminated melon (CDC, 2011). This pathogen usually has low incidence. However, due to its high mortality rate (up to 30%), its ability to survive a wide range of environmental conditions and its ability to multiply at refrigeration
* Corresponding author. Tel.: þ34 973003430; fax: þ34 973238301. E-mail address: [email protected] (M. Abadias). 0740-0020/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.fm.2013.08.018
temperatures, it is considered an important foodborne pathogen (Farber and Peterkin, 1991). Over the last years, a number of strategies to minimize the microbial load of raw products have been explored. A variety of disinfectants (including chlorine, hydrogen peroxide, organic acids and ozone) have been used to reduce initial bacterial populations on minimally processed produce (Beuchat, 1998). Chlorine is the most widely used sanitizer in the fresh produce industry. However, studies indicate that chlorine concentrations traditionally used (50e200 ppm) are not effective in reducing pathogen load on freshcut produce (Behrsing et al., 2000; Delaquis et al., 2002; Lee and Baek, 2008). Moreover, a prolonged exposure to chlorine vapor may cause irritation to the skin and respiratory tract of the workers, may affect the quality of foods and also adversely affect the environment (Beuchat, 1998). It is also known that the reaction of chlorine with organic matter results in the formation of carcinogenic products (trihalomethanes) for consumers (Nieuwenhuijsen et al., 2000). Thus, there is a need for better, safer and more environmental friendly methods to reduce the contamination. Bacteriophage (phage) prophylaxis is one possible alternative to achieve this goal. Phages are bacterial viruses that invade specific bacterial cells, disrupt bacterial metabolism, and cause the bacterium to lyse without compromising the viability of other flora in the
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habitat. They are the most abundant microorganisms in our environment (Brussow and Hendrix, 2002) and are present in high numbers in water and foods (Hsu et al., 2002; Kennedy et al., 1986). Phages are also part of gastrointestinal system (Greer, 2005), and may provide a natural, non-toxic, feasible approach for controlling several human pathogens. A study in humans with Escherichia colispecific phages also indicated that phages are safe for oral administration (Bruttin and Brüssow, 2005). This together with their specificity makes phages excellent tools for food safety purposes. Promising results using phage biocontrol have been reported for several pathogens, including Salmonella (Guenther et al., 2012; Kocharunchitt et al., 2009; Leverentz et al., 2001), Campylobacter (Goode et al., 2003), L. monocytogenes (Carlton et al., 2005; Dykes and Moorhead, 2002; Guenther et al., 2009; Leverentz et al., 2003) and E. coli O157:H7 (Abuladze et al., 2008; Sharma et al., 2009; Viazis et al., 2011). However, some studies reported that the effectiveness of many phages seems to decline at acidic pH (Leverentz et al., 2001, 2003). Moreover, the efficacy of phages in food also depends on the structure and chemical composition of the different food items and a homogeneous distribution and sufficient diffusion ability of the phage particles is necessary (Guenther et al., 2009). There are several phage preparations commercialized, such as ListShieldÔ and EcoShieldÔ (Intralytix, Inc., USA), AgriphageÔ (Omnilytics, Inc., USA) and ListexÔ P100 (Micreos Food Safety, formerly EBI Food Safety, The Netherlands). The approval of using phage product Listex P100 in food products by FDA and USDA provides the impetus to further investigate phage applications. Listex P100 is a culture of safe and natural bacteriophages, characterized by its broad spectrum towards L. monocytogenes strains. It can be used as a processing aid with all food products susceptible to L. monocytogenes. An oral toxicity study in rats receiving high doses of Listeria phage P100 did not reveal any side effects (Carlton et al., 2005). Little work is known about the efficacy of Listex P100 on fresh-cut fruits and fruit juices, which are characterized by its low pH. The aim of this study was to evaluate the effect of fruit pH (melon, pear and apple) and physical form of the food matrix (solid ¼ fruits slices vs liquid ¼ fruit juices) on the efficacy of the phage Listex P100 against a three strain cocktail of L. monocytogenes. 2. Material and methods 2.1. Fruits ‘Golden Delicious’ apples (Malus domestica cv. Golden Delicious) and ‘Conference’ pears (Pyrus communis cv. Conference) were obtained from local packinghouses in Lleida (Catalonia, Spain). ‘Piel de Sapo’ melons (Cucumis melo L. var. ‘Piel de sapo’) were purchased in a local supermarket. Prior to the experimental studies, fruits were washed in running tap water, surface disinfected with ethanol 70% and let to dry at room temperature.
(TYSEB) at 37 1 C for 20e24 h. Bacterial cells were harvested by centrifugation at 9820 g for 10 min at 10 1 C and resuspended in sterile saline peptone (SP, 8.5 g L1 NaCl and 1 g L1 peptone). Equal volumes of each strain were combined to obtain the threestrain cocktail of L. monocytogenes. The concentration of L. monocytogenes was confirmed by plating duplicate serial suspension dilutions on Palcam medium (Palcam Agar Base with selective supplement, Biokar Diagnostics, Beauvais, France) followed by incubation at 37 1 C for 48 h. A suspension of 105 colony forming units (cfu) mL1 was prepared and confirmed by plating as described before. 2.3. Phage The bacteriophage Listex P100 (Micreos Food Safety, The Netherlands), characterized by its broad spectrum toward L. monocytogenes strains, was used in this study. The phage concentration was approximately 1011 plaque forming units (pfu) mL1 in buffered saline and was maintained at 5 C. 2.4. Preparation of fruit slices and fruit juices For fruit assays, apples and pears were peeled and cut into ten slices using a manual fruit slicer/corer. Melons were cut transversally in 14e16 mm slices, seeds and rind were removed and each slice was cut in trapezoidal pieces. In each fruit slice, a 6 mm diameter well was made on the center of each wedge to contain the inoculums. Three wedges were placed in commercial 500 mL food plastic bowls. The covers on the plastic bowls allowed sufficient air exchange to prevent modified atmosphere creation. Fruit juices were extracted by crushing peeled fruits wedges in a blender. Juice was filtered using glass wool, bottled and autoclaved at 115 C for 10 min. Thirty milliliters of sterile pear, apple and melon juices was individually transferred into 50 mL sterilized polypropylene containers (Deltalab, Barcelona, Spain). 2.5. Inoculation procedures The fruit slices were inoculated with the cocktail of L. monocytogenes by pipetting 15 mL of the bacterial suspension containing approximately 1 105 cfu mL1 onto the well of each wedge. Then, the phage was applied to the wells by pipetting 15 mL of the suspension at approximately 1 108 pfu mL1. There were 3 fruit slices per treatment at each recovery time. Samples of 30 mL of fruit juices were each inoculated with specific volume of the bacterial and phage suspensions to obtain final concentrations of about 1 105 cfu mL1 and 1 108 pfu mL1, respectively. There were 3 samples per treatment for each fruit juice. Juice samples were mixed thoroughly to ensure homogeneous distribution of the pathogen and phage. All samples (slices and juices) were stored at 10 C for 8 days. 2.6. Bacteria analysis
2.2. Bacteria and preparation of inocula Three strains of L. monocytogenes: serovar 1a (Spanish Type Culture Collection (CECT) 4031), serovar 4a (CECT 940) and serovar 4b (CECT 4032) were used in this study. L. monocytogenes strains were grown individually on tryptone soy agar (TSA, Oxoid, LTD, Basingstoke, Hampshire, England) supplemented with 6 g L1 yeast extract, 2.5 g L1 glucose and 2.5 g L1 dipotassium hydrogen phosphate (TYSEA) at 37 1 C for 20e24 h. An individual colony of each strain was transferred into a flask with 50 mL of tryptone soy broth (TSB, Oxoid, UK) supplemented with 6 g L1 yeast extract
Recovery of L. monocytogenes populations from fruit and juice samples were performed after 0, 2, 5 and 8 days of storage at 10 C. To recover the pathogen from the wedges, a cylinder (1.2 cm in diameter and 1 cm deep) containing the entire well was removed using a sterile cork borer and placed in a filtered sterile bag (Bagpage 80 mL, Interscience BagSystem, St Nom La Breteche, France) with 9 mL of buffered peptone water (BPW, Oxoid) and blended in a homogenizer for 120 s at high speed (Bagmixer 100 Minimix, Interscience). Aliquots of the mixture were then serially diluted in saline peptone (SP; 8.5 g L1 NaCl and 1 g L1 peptone)
M. Oliveira et al. / Food Microbiology 38 (2014) 137e142 Table 1 Determination of initial pH, soluble solids (SS,
Brix) and titratable acidity (TA, g malic or citric acid L1) of melon, pear and apple from fruits and juices used in the assays.
Melon
Pear
Fruit pH SS TA a
5.92 0.26 9.9 1.3 1.34 0.10
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a
Apple
Juice
Fruit
Juice
Fruit
Juice
5.77 0.12 10.2 0.5 1.32 0.26
4.91 0.14 15.7 1.5 1.19 0.22
4.61 0.02 14.5 0.2 1.62 0.07
3.76 0.17 13.6 0.5 3.41 0.08
3.70 0.10 14.2 0.1 3.00 0.49
Results expressed as mean the stander and deviation of the mean for each analysis.
and 100 mL was spread plated on Palcam agar. The plates were incubated at 37 1 C for 48 h and the data were plotted as log cfu plug1. Populations of L. monocytogenes in fruit juices were enumerated taking an aliquot of each juice and diluted in SP, followed by spread plating on Palcam agar as described previously. After colony counting, data were plotted as log cfu mL1. 2.7. Determination of phage titer In order to determine the phage titer from fruit and juice samples of phage treatments, the same samples obtained to determine L. monocytogenes populations were used. The phage titer was determined using the soft agar overlay method following the instructions given by the product supplier. Briefly, aliquots of 100 mL of the diluted sample were mixed with 100 mL of log-phase L. monocytogenes cocktail in 3.5 mL of molten brain-heart infusion soft agar (BHI containing 0.4% agar; Biokar Diagnostics). Immediately after, the soft agar mixture was poured onto solid BHI agar plates (1.2% agar) and incubated for 24 h at 30 C. At the end of incubation period, the visible plaques were counted and expressed as log pfu plug1 or log pfu mL1. 2.8. Fruit and juices quality parameters Quality analysis (pH, soluble solids and titratable acidity) of fruits and juices was performed before the experiment. Fruit flesh pH was determined using a penetration electrode (5231 Crison, and pH-meter Model GLP22, Crison Instruments S.A., Barcelona, Spain) for fruit slices assay. Juice pH was determined using pH-meter (5203 Crison, Model GLP22). There were three determinations per fruit and juice. Percent of soluble solids (SS, Brix) was measured at 20 C with a handheld refractometer (Atago Co. Ltd., Tokio, Japan) in juice extracted by crushing fruit pieces in a blender. There were three measurements per fruit and juice. To measure titratable acidity (TA), 10 mL of fruit juice (obtained by crushing fruit pieces) were diluted with 10 mL of distilled water and it was titrated with 0.1 N NaOH up to 8.1. The results were calculated as g of citric acid L1 for melons and g of malic acid L1 for pears and apples. There were three determinations per fruit and juice. 2.9. Statistical analysis All experiments were replicated three times with three different replications per treatment/day for each fruit sample. Therefore, reported populations represent the mean of nine values. The bacterial and phage data were transformed to log units. Population of L. monocytogenes recovered from fruit slices and juices treated with phage and the control were subjected to Statistical software JMP (SAS Institute, version 9.2, Cary, NC, USA). Significant differences between phage treatment and control were analyzed by t-student test at a significance level of P < 0.05.
3. Results 3.1. Fruit quality parameters The pH, soluble solids and titratable acidity of fruits and juices used in the assay are summarized in Table 1. Apple products had the lowest pH (3.70e3.76) followed by pear (4.61e4.91). The highest pH (5.77e5.92) was measured in melon products. Pear and apple products had the highest soluble solids content, 14.5e15.7 and 13.6e14.2, respectively, and the lowest was observed in melon (9.9e10.2). The highest titratable acidity was determined in apple (3.00e3.41), followed by pear (1.19e1.62) and melon products (1.32e1.34). 3.2. Effect of phage treatment on survival of L. monocytogenes on fruit slices L. monocytogenes grew at 10 C on melon (pH 5.92), pear (pH 4.91) and apple (pH 3.76) slices throughout storage period (Fig. 1). Initial L. monocytogenes populations on untreated melon, pear and apple were 2.77, 2.94 and 2.48 log cfu plug1, respectively. The most remarkable bacterial growth in untreated samples was observed on melon, followed by pear and apple slices, reaching populations close to 8.00, 6.00 and 5.00 log cfu plug1, respectively, after 8 days of storage at 10 C. The largest statistical difference in L. monocytogenes populations on untreated and phage-treated slices was observed on melon with reductions of about 1.50 log cfu plug1 during all storage period (Fig. 1). L. monocytogenes populations on phage treated melon were close to 6.50 log cfu plug1 after 8 days at 10 C. Phage application on pear slices reduced 1.00, 1.15 and 0.62 log cfu plug1 the population of L. monocytogenes after 2, 5 and 8 days of storage, respectively. On treated apple slices significant reductions were not observed and the pathogen survived during all storage period at levels between 2.20 and 4.20 log cfu plug1. Phage populations on melon slices increased about 1.5-log units (Fig. 1) over a period of 8 days, reaching levels of about 6.90 log pfu plug1 at the end of storage. On pear slices the titer slightly decreased and remained between 5.79 and 4.82 log pfu plug1. In contrast, the phage titer declined rapidly after 2 days on apple slices and reached undetectable levels after 8 days at 10 C. 3.3. Effect of phage treatment on survival of L. monocytogenes on fruit juices L. monocytogenes grew in untreated melon (pH 5.77), survived in untreated pear (pH 4.61) and decrease in untreated apple (pH 3.70) juices throughout storage period at 10 C (Fig. 2). The initial concentration of L. monocytogenes in untreated fruit juices was around 5.30 log cfu mL1. In untreated melon juice, the pathogen showed a considerably increase during the storage period, reaching around 8.70 log cfu mL1 after 8 days of storage. L. monocytogenes populations in untreated pear juice remained
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Fig. 1. L. monocytogenes population (log cfu plug1) on melon (A), pear (B) and apple (C) slices and the titer of phage (log pfu plug1) stored at 10 C. Continuous line represent the titer of phage (Phage) and columns represent the growth of L. monocytogenes alone (Lm) or together with the phage (Lm þ Phage). Data represent the mean of three determinations and three experiment repetitions (n ¼ 9). Bars represent standard deviation of the mean and where not visible, they are smaller than symbol size. For each time, different letters indicate significant differences (P < 0.05) among treatments.
Fig. 2. L. monocytogenes population (log cfu mL1) on melon (A), pear (B) and apple (C) juices and the titer of phage (log pfu mL1) stored at 10 C. Continuous line represent the titer of phage (Phage) and columns represent the growth of L. monocytogenes alone (Lm) or together with the phage (Lm þ Phage). Data represent the mean of three determinations and three experiment repetitions (n ¼ 9). Bars represent standard deviation of the mean and where not visible, they are smaller than symbol size. For each time, different letters indicate significant differences (P < 0.05) among treatments.
constant during storage period with levels between 5.30 and 5.12 log cfu mL1. In untreated apple juice, there was a steady decline (almost 2-log units) of L. monocytogenes populations over time, approaching 3.26 log cfu mL1 after 8 days of storage. The bacterial populations were significantly (P < 0.05) lower on phage-treated melon and pear juices than on corresponding juices without phage, during storage period. Nevertheless, no significant reduction in the L. monocytogenes counts was observed in the apple juice treated with phage (Fig. 2). The largest difference in L. monocytogenes populations in untreated and phagetreated juices was observed on day 8 (8-log units) in melon juice, were L. monocytogenes reached final population around
0.60 log cfu mL1. Furthermore, the phage was also effective in pear juice. The reduction of pathogen was between 2.10 and 2.80 log cfu mL1 during storage, reaching populations close to 3.00 log cfu mL1 after 8 days at 10 C. There was no effect in L. monocytogenes populations in apple juice treated with phage, and the bacteria survived during storage period at levels between 5.00 and 3.30 log cfu mL1. Phage populations remained constant in melon and pear juices during storage period and counts were in the range of 7.90 and 7.40 log pfu mL1 (Fig. 2). In contrast, the phage titer declined rapidly in apple juice with a decrease of almost 7-log units after 8 days of storage.
M. Oliveira et al. / Food Microbiology 38 (2014) 137e142
4. Discussion This study compared the effectiveness of phage Listex P100 in controlling the growth of three strain cocktail of L. monocytogenes on different fresh-cut fruits and in fruit juices. Three types of fruit with different pHs and acidities (melon, pear and apple) and also the physical form of food matrix (solid vs liquid) were tested. Our results demonstrated that both pH and physical form of fruit influenced on phage efficacy. We observed more effectiveness in controlling the growth of L. monocytogenes in fruit juices and also in fruits with higher pH. The growth of L. monocytogenes on melon stored at 10 C over 8 days was several log units higher than the growth on pear and apple. The pH of melon was approximately 5.77e5.92, 4.61 to 4.91 on pear, and 3.70 to 3.76 on apple. This pH difference may be a major factor contributing to the differences in the bacterial populations on the fruit slices and juices. Growth differences of pathogens on fruits and juices were also obtained in several studies (Abadias et al., 2012; Alegre et al., 2010a, 2010b; Leverentz et al., 2001, 2003; Raybaudi-Massilia et al., 2009). We also observed that L. monocytogenes grew on untreated apple slices but decreased in untreated apple juices stored at 10 C. According to Conway et al. (2000), the growth of L. monocytogenes on apple slices probably resulted from its ability to modify the immediate microenvironment. This type of environmental changes would not occur in apple juice, in which we observed that the population decreases during storage period. However, Yuste and Fung (2002) reported the survival but not the growth of L. monocytogenes in apple juice (pH 3.7) at 5 C. Phage treatment decreased L. monocytogenes populations on fruit slices and juices. However, these reductions were greater in fruit juices than on fruit slices. The detection of more pathogen counts in fruit slices than in fruit juices after phage treatment could suggest that phages are seemingly immobilized after addition to solid surfaces and therefore could not come into contact with the surviving bacteria through limited diffusion (Guenther et al., 2009). In liquids, even a very small initial number of phages can cause complete lysis of the bacteria in a relatively short time (Hagens and Loessner, 2010). On the other hand, in solid matrix a high concentration of phage is necessary to enable it to cover the entire space and infect the bacteria as it depends on the amount of fluid in the food. As a conclusion, the concentration of the phages must be sufficiently high to enable contact and subsequent infection, even when bacteria are present at very low numbers (Hagens and Loessner, 2010; Hagens and Offerhaus, 2008). Phage treatment was more effective on fresh-cut melon followed by pears with reductions about 1.50 and 1.00 log cfu plug1, respectively. Nevertheless, the phage treatment was not effective on apple slices. A study using the phage LMP-102 reduced by 2.0e 4.6 log cfu the population of L. monocytogenes on honeydew melons and no reduction was observed on apples (Leverentz et al., 2003). The efficacy of Listex P100 has also been tested in other foods. Guenther et al. (2009) observed reduction counts by up to 5-log units in ready-to-eat food. Carlton et al. (2005) reported approximately 2e3 log decrease on soft cheese. These reduction differences could be due to the different bacteriophage application and concentration, and also the different matrices used. Phages effectiveness is influenced by pH and other physiochemical properties of foods, activity on a solid substrate or biofilm, the emergence of resistant bacteria mutants, and the relative numbers of phages and host required to allow replication (Hudson et al., 2005). Similarly to what happened on fruit slices, a more pronounced reduction of L. monocytogenes was obtained in melon juice followed by pear juice. However, the numbers of L. monocytogenes in apple juice were unaffected by phage treatment. This result could be
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attributed to the increased sensitivity of phages to the acid environment of the apple juice (pH 3.70) which caused a marked phage titer decline. We observed a great tolerance of phage to survive during the storage on both slices and juice of melon and pear with a pH above 5.77 and 4.61, respectively. However, a rapid decline of phages to almost undetectable numbers on apple products with pH below 3.76 was observed. These results are in accordance with other studies that have shown a similar behavior of LMP-102 on melon and apple fruits (Leverentz et al., 2001, 2003). The inability of phages to tolerate an acidic pH is well documented (Leverentz et al., 2001, 2003; Smith et al., 1987) and is in agreement with our results. The application of bacteriophages on produce with a low pH could be improved by using greater numbers of phage or the development of low-pHtolerant phage mutants. Other alternative could be the combination of phages with other compounds, such as bacteriocins, antibiotics, chlorine compounds, antagonistic bacteria and essential oils. Phage persistence is an important parameter to render applied phages not just decontaminating agent but rather a protective or preservative or as a food additive to control growth of foodborne pathogens in food and beverage industries. Bacteriophage products are already in use in agricultural, food safety and diagnostic applications, demonstrating the utility and viability of such approaches. The application of bacteriophages represents a promising, safe, environmentally friendly and chemical-free alternative to the use of chemicals/sanitizers on produce. Acknowledgments The authors are grateful to the Spanish Government [AGL-201019385] for its financial support. References Abadias, M., Usall, J., Anguera, M., Solsona, C., Viñas, I., 2008. Microbiology quality of fresh, minimally-processed fruit and vegetables, and sprouts from retail establishments. Int. J. Food Microbiol. 123, 151e158. Abadias, M., Alegre, I., Oliveira, M., Altisent, R., Viñas, I., 2012. Growth potential of Escherichia coli O157:H7 on fresh-cut fruits (melon and pineapple) and vegetables (carrot and escarole) stored under different conditions. Food Control 27, 37e44. Abuladze, T., Li, M., Menetrez, M.Y., Dean, T., Senecal, A., Sulakvelidze, A., 2008. Bacteriophages reduce experimental contamination of hard surfaces, tomato, spinach, broccoli, and ground beef by Escherichia coli O157:H7. Appl. Environ. Microbiol. 74, 6230e6238. Alegre, I., Abadias, M., Anguera, M., Oliveira, M., Viñas, I., 2010a. Factors affecting growth of foodborne pathogens on minimally processed apples. Food Microbiol. 27, 70e76. Alegre, I., Abadias, M., Anguera, M., Viñas, I., 2010b. Fate of Escherichia coli, Salmonella and Listeria innocua on minimally-processed peaches under different storage conditions. Food Microbiol. 27, 862e868. Behrsing, J., Winkler, S., Franz, P., Premier, R., 2000. Efficacy of chlorine for inactivation of Escherichia coli O157:H7 on vegetables. Postharvest Biol. Technol. 19 (2), 187e192. Beuchat, L.R., 1998. Surface Decontamination of Fruit and Vegetables Eaten Raw: A Review. Available at: http://www.who.int/foodsafety/publications/fs_management/ en/surface_decon.pdf (accessed 22.04.10.). Beuchat, L.R., 2002. Ecological factors influencing survival and growth of human pathogens on raw fruits and vegetables. Microbes Infect. 4, 413e423. Brussow, H., Hendrix, R.W., 2002. Phage genomics: small is beautiful. Cell 108, 13e16. Bruttin, A., Brüssow, H., 2005. Human volunteers receiving Escherichia coli phage T4 orally: a safety test of phage therapy. Antimicrob. Agents Chemother. 49, 2874e 2878. Carlton, R.M., Noordman, W.H., Biswas, B., de Meester, E.D., Loessner, M.J., 2005. Bacteriophage P100 for control of Listeria monocytogenes in foods: genome sequence, bioinformatic analyses, oral toxixity study, and application. Regul. Toxicol. Pharmacol. 43, 301e312. CDC (Centers for Disease Control and Prevention), 2011. Investigation Update: Multistate Outbreak of Listeriosis Linked to Whole Cantaloupes from Jensen Farms, Colorado. http://www.cdc.gov/listeria/outbreaks/cantaloupes-jensenfarms/120811/index.html (accessed 16.04.20.). Conway, W.S., Leverentz, B., Saftner, R.A., 2000. Survival and growth of Listeria monocytogenes on fresh-cut apple slices and its interaction with Glomerella cingulata and Penicillium expansum. Plant Dis. 84, 177e181.
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