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Assessment of the effectiveness of antimicrobial packaging combined with high pressure to control Salmonella sp. in cooked ham
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Food Control 19 (2008) 634–638 www.elsevier.com/locate/foodcont
Short communication
Assessment of the effectiveness of antimicrobial packaging combined with high pressure to control Salmonella sp. in cooked ham Anna Jofre´, Teresa Aymerich, Margarita Garriga
*
IRTA, Finca Camps i Armet, E-17121 Monells, Spain Received 8 March 2007; received in revised form 30 May 2007; accepted 5 June 2007
Abstract The effectiveness of the application of interleavers containing enterocins A and B, sakacin K, nisin A, potassium lactate and nisin plus lactate alone or in combination with a 400 MPa high hydrostatic pressure treatment (HHP) was assessed in sliced cooked ham spiked with Salmonella spp. HHP produced an important reduction in Salmonella counts, which decreased from 104 CFU/g to <10 CFU/g, a value that was maintained for 3 months of storage at 6 C. However, the elimination of the pathogen could only be achieved by combining HHP and nisin-containing interleavers. Therefore, antimicrobial packaging, HHP and refrigerated storage appear as an effective combination of hurdles to obtain value added ready-to-eat products with a safe long-term storage. 2007 Elsevier Ltd. All rights reserved. Keywords: Active packaging; High pressure; Lactate; Nisin; Salmonella
1. Introduction Minimally processed ready-to-eat products challenge food safety and innovative ways to inhibit microbial growth, such as active packaging, are being developed. Antimicrobial packaging is a form of active packaging that reduces, inhibits or retards the growth of microorganisms present in packaged food through the incorporation of antimicrobials into the packaging, and is a promising tool for controlling the growth of food-borne pathogens or spoilage bacteria in ready-to-eat post-processed products (Appendini & Hotchkiss, 2002). Food products that have been submitted to an adequate heat treatment during processing are generally free of pathogens. However, epidemiological investigations into outbreaks related to several ready-to-eat products have demonstrated that the presence of Salmonella spp. in the *
Corresponding author. Tel.: +34 972630052; fax: +34 972 63 373. E-mail address: [email protected] (M. Garriga).
0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.06.007
consumed products was frequently due to post-process recontamination (Reij & Den Aantrekker, 2004). In these products, mainly affected by superficial recontamination, antimicrobial packaging could avoid the necessity to add large amounts of antimicrobials to the food and to diminish the reduction of antimicrobial activity due to interactions with food constituents (Appendini & Hotchkiss, 2002). A great variety of antimicrobial agents including organic acids and their salts, sulfites, nitrites, antibiotics, alcohols, enzymes and natural components such as bacteriocins, especially nisin, have been incorporated into active films ´ as, Agrasar, & Castro, 2005; Natrajan & (Guerra, Macı Sheldon, 2000a; Natrajan & Sheldon, 2000b; Pranoto, Salokhe, & Rakshit, 2005). Nisin is currently the only bacteriocin allowed to be used as a food additive (EC, 1995; FSIS, 2002). Enterocins A and B and sakacin K are bacteriocins whose antilisterial activity has been shown in a meat homogenate and have been applied experimentally as ingredients in several meat products (Aymerich et al.,
A. Jofre´ et al. / Food Control 19 (2008) 634–638
2000; Aymerich, Jofre´, Garriga, & Hugas, 2005; Garriga, Aymerich, Costa, Monfort, & Hugas, 2002; Hugas, Garriga, Pascual, Aymerich, & Monfort, 2002). Lactate salts have shown antimicrobial activity against several spoilage and food-borne pathogenic microorganisms and are widely used as ingredients in the meat industry (de Wit & Rombouts, 1990; Sallam, 2007; Vogel, Yin Ng, Hyldig, Mohr, & Gram, 2006). The antimicrobial activity of bacteriocins is generally limited to closely related gram-positive species and a cost-effective food biopreservation, especially against gram-negative bacteria, may require a ‘‘hurdle approach’’ (Leistner & Gorris, 1995). Bacteriocins could act synergistically with traditional or novel preservation systems which induce sublethal injury to bacteria such as high hydrostatic pressure (HHP). HHP is an emerging technology for microbial reduction in minimally processed foods with fresh-like quality and improved safety aspects (Rastogi, Raghavarao, Balasubramaniam, Niranjan, & Knorr, 2007). In gram-negative bacteria, HHP increases the antimicrobial efficiency of bacteriocins by permeabilization of the outer membrane (Kalchayanand, Sikes, Dunne, & Ray, 1994; Ter Steeg, Hellemons, & Kok, 1999). In food products, a limited number of studies have evaluated the combined effect of HHP and bacteriocins on gram-negative bacteria (Aymerich et al., 2005; Black, Kelly, & Fitzgerald, 2005; Garcı´a-Graells, Masschalck, & Michiels, 1999; Garriga et al., 2002; Patterson & Kilpatrick, 1998; Rodrı´guez, Arques, Nunez, Gaya, & Medina, 2005). To our knowledge, the combined effect of HHP and bacteriocins applied through antimicrobial packaging has not been previously evaluated. The objective of this study was to evaluate the application of active interleavers containing enterocins A and B, sakacin K, nisin and potassium lactate against Salmonella in sliced cooked ham. Furthermore, the effect of the application of an additional 400 MPa HHP treatment after slicing was evaluated during a 3-month refrigerated storage.
2. Material and methods 2.1. Cooked ham Cooked ham with minced pork shoulder and the following composition (in g/kg) was prepared: water, 115; salt, 20.7; dextrose, 5.8; sodium tri-polyphosphate, 5.8; carragenate, 2.3; NaNO2, 0.1 and L-ascorbate, 0.6 (all additives from SKW Biosystems, Rubi, Spain). Minced meat (4 mm) was homogenised with other ingredients (Mixer 35P, Tecnotrip SA, Terrassa, Spain) for 30 min and stuffed (Stuffing machine H15, Tecnotrip SA) into impermeable plastic film (Prolan SV 150, 11 cm of diameter, Hispano Holandesa S.A. Sant Boi de Llobregat, Spain). Ham pieces of 2.5– 3 kg were cooked in an oven at 75 C until internal temperature reached 72 C (approximately 2.5 h). The pH of the cooked ham was 6.3 and the aw = 0.98.
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2.2. Bacteriocin extracts Sakacin K and enterocins A and B were obtained from overnight cultures of Lactobacillus sakei CTC494 and Enterococcus faecium CTC492, respectively, as described in Aymerich et al. (2000). 2.3. Preparation of interleavers containing antimicrobials Interleavers (11 · 11 cm and 133 lm thick) consisting of a perforated polypropylene layer, a non-woven polyamide layer in the middle and another non-perforated polypropylene layer (Gre`bol, 1995) were prepared by adding 200 or 2000 AU/cm2 of enterocins A and B (E200 and E2000), 200 or 2000 AU/cm2 of sakacin K (S200 and S2000), 200 AU/cm2 of nisin A (N200) (Nisaplin. Aplin & Barrett Ltd, UK), 1.8 g/100 g (1.8% w/w) ham of potassium lactate (L) (Purasal P/Hi Pure 60. Purac Biochem, Gorinchem, The Netherlands) and 200 AU/g nisin plus 1.8% potassium lactate (NL) and distilled water (control, C). Antimicrobials and water were spread on the perforated polypropylene layer, left to dry and stored overnight at 6 C. 2.4. Spiking, packaging and pressurization A cocktail of three Salmonella enterica subsp. enterica (S. London CTC1003, S. Schwarzengrund CTC1015 and S. Derby CTC1022) was prepared by properly diluting frozen stored stationary phase cultures of the strains. Each slice was inoculated with ca. 3 · 104 CFU/g and covered with an interleaver. Pairs of slices (11 cm diamenter and 1.5 mm thick) were vacuum packaged in plastic bags of PET/PE (O2 and water vapour permeability <50 cm3/m2/ 24 h and <15 mg/m2/24 h, respectively; Sacoliva S.L., Spain). Eight batches of packaged cooked ham were prepared: control, E200, E2000, S200, S2000, N200, L and NL. Half of the samples were pressurized in an industrial hydrostatic pressurization unit (Alstom, Nantes, France; chamber volume of 320 l and diameter of 280 mm) at 400 MPa for 10 min at 17 C. The come-up and release times and the adiabatic heating were 13.17 min, 1.33 min and <5 C, respectively. Pressurized and non-pressurized samples were subsequently stored at 6 C. 2.5. Microbiological analyses Three different packages of each treatment were analyzed on day 0, 1 and every 15 days for 3 months. Twenty five grams of ham were 1/10 diluted in buffered peptone water (BPW, AES Laboratories, Combourg, France) and homogenized in a Stomacher Labblender (model 400, Cooke Laboratory Products, Alexandria, USA). Brilliant Green Agar (BGA. Difco, Detroit, Michigan, USA) was used to count Salmonella (limit of detection of 10 CFU/ g). PCR as described in Aymerich et al. (2005) was used to confirm five colonies of each plate and to determine
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presence/absence of Salmonella sp. in BPW homogenates incubated at 37 C for 48 h. 2.6. Statistical analysis Data were subjected to ANOVA of the General Lineal Model procedure of SAS software (SAS system for Windows NT, release 8.1, SAS Inst. Cary NC, USA). The model included batch, treatment, storage time, and their interaction as fixed effects. Differences between effects were assessed by the Tukey test. 3. Results and discussion
1 log CFU/ml. Natrajan and Sheldon (2000b) evaluated the use of protein- and polysaccharide-based films containing a nisin formulation, including 5 mM EDTA, 3% citric acid and Tween 80, on the inhibition of S. typhimurium on poultry skin. Reductions up to 4.4 log cycles were shown after 96 h of exposure of the drumstick skin to films containing 500 lg/ml of nisin and stored at 4 C. In this regard, Deegan, Cotter, Hill, and Ross (2006) suggested that bacteriocins should not be used as the main processing step to prevent the growth or survival of pathogens but to provide an additional hurdle to reduce the likelihood of food-borne disease. The combination of antimicrobials with other inhibitory treatments such as HHP has also been proposed to achieve a higher inactivation of gramnegative food-borne pathogens.
3.1. Effect of interleavers containing antimicrobials on Salmonella
3.2. Combination of antimicrobial packaging and HHP
Interleavers containing the bacteriocins enterocin (E200 and E2000), sakacin (S200 and S2000) and nisin (N), and the potassium lactate salt applied alone (L) or in combination with nisin (NL), were evaluated for their ability to control or reduce the levels of Salmonella at 6 C. In all the batches the growth of Salmonella was inhibited by the temperature of storage and the counts experienced a progressive reduction of ca. 1.5 log units from the beginning to the end of the storage period (Fig. 1a). No additional reductions in the Salmonella levels were observed in the batches packaged with enterocin, sakacin or nisin after either 1 day or during 3 months of storage. Bacteriocins exert their mode of action by destabilization and permeabilisation of the membrane of sensitive cells. After formation of transitory poration complexes, through non-specific interaction with anionic polymers usually constituting the cell wall of gram-positive bacteria, pore formation and loss of membrane integrity provokes a passive efflux of small molecules, resulting in a loss of the proton motive force (Cintas, Casaus, Herranz, Nes, & Herna´ndez, 2001). In general, the activity of bacteriocins produced by lactic acid bacteria is against gram-positive bacteria but, while enterocins A and B and sakacin K are bacteriocins with an important anti-listeria activity, nisin, belonging to class I, has a broader spectrum of activity (Casaus et al., 1997; Stevens, Sheldon, Klapes, & Klaenhammer, 1991). To obtain a bacteriostatic or slightly bactericidal effect of bacteriocins against gram-negative bacteria, a previous destabilization of the cell envelope with the chelating agent EDTA or other destabilizing factors is necessary. Ukuku and Fett (2004) reported that the combination of nisin with EDTA, sodium lactate and potassium sorbate could control, through washing treatments, Salmonella in cantaloupe. Only a few studies have dealt with the effect of the application of antimicrobials through active films. Hoffman, Han, and Dawson (2001) incorporated nisin, lauric acid and EDTA to corn zein films that were exposed to broth cultures of S. enteritidis for 48 h and none of the combinations produced reductions of the pathogen greater than
The application of a 400 MPa HHP treatment to the active packaged slices of cooked ham produced a drastic reduction to the counts of Salmonella (Fig. 1b). One day after pressurization the levels were reduced from ca. 4.5 log CFU/g to levels below 10 CFU/g in the control batch. In antimicrobial-containing batches, neither enterocin (E200 and E2000), sakacin (S200 and S2000), L nor NL were shown to increase the inhibitory effect of the HHP compared with the control (P > 0.05). Besides, a lower decrease was observed in E2000 and S2000 batches compared with the control (P < 0.05). Only the combination of nisin and HHP recorded absence of Salmonella in 25 g of ham 24 h after pressurization. During refrigerated storage of pressurized ham at 6 C, a temperature that does not support the growth of Salmonella sp. (Roberts, Baird-Parker, & Tompkin, 1996), no further recovery of the pathogen was observed. The levels of the pathogen in C, N and NL batches continued below 10 CFU/g (P > 0.05) during the entire storage period. In L, E and S batches the initial reduction was lower but the counts progressively decreased during storage and after 60 days they were below 10 CFU/g in all the batches (P > 0.05). From day 60 to the end of the storage at 6 C, the counts continued at the same level and no significant differences were observed among batches (P > 0.05). Nisin was the only treatment that produced absence of Salmonella 24 h after pressurisation and the application of nisin through interleavers and combined with an HHP treatment appears as the most effective treatment to achieve absence when a high level inoculum is considered. The combined effect of HHP and antimicrobials on Salmonella has not been previously evaluated through antimicrobial packaging. Only a few studies have been performed in broth and bacteriocin-containing food products. Kalchayanand et al. (1994) reported in 0.1% peptone an increase in the inactivation of both S. typhimurium and E. coli by combining pressurization at 207–345 MPa and nisin, pediocin and both. Ter Steeg et al. (1999) showed in E. coli an increase in the synergism between nisin and HHP at
A. Jofre´ et al. / Food Control 19 (2008) 634–638
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a6
Log CFU/g
4
2
0 0
15
30
45
60
90
75
days
b
6 Enterocin200 Enterocin2000
Log ( C FU+1) /g
Sakacin200 Sakacin2000 4
Nisin200 Lactate Nisin+Lactate Control
2
0 0
15
30
45
60
75
90 days
Fig. 1. Evolution of Salmonella sp. in slices of cooked ham packaged with antimicrobial-containing interleavers and stored at 6 C. (a) Non-HHP treated ham; and (b) 400 MPa pressurized ham.
150 and 200 MPa at reduced temperatures and a stronger inactivation when nisin was present during both pressurization and in the recovery medium than when it was only applied during either pressurization or recovery. They postulated that the rigidity of the membrane due to reduced temperature and binding of nisin increased the efficacy of the HHP treatment. In cooked ham, we previously showed (Aymerich et al., 2005) that the combination of nisin applied in the meat batter at 800 AU/g and HHP at 400 MPa also recorded the highest proportion of absences during a storage period of 3 months at 6 C. In contrast, other studies performed in a model meat system spiked with S. London CTC1003 and S. Schwarzengrund CTC1015 did not show significant differences among control and antimicrobial-containing (enterocins A and B, sakacin K, pediocin AcH and nisin A) meat homogenate after
pressurization at 400 MPa and during a storage of 61 days at 4 C (Garriga et al., 2002). Evaluation of the effectiveness of the assayed antimicrobial treatments to assure the absence of the pathogen after processing and during the entire shelf-life of the product is very important and required by the new European microbiological criteria (EC, 2005). Considering the inability of the pathogen to grow at refrigeration temperatures, the presumably low-level of post-processing recontaminations and the drastic reduction of Salmonella achieved by the HHP treatment recorded in this challenge test, pressurization of sliced cooked ham and storage at 66 C appears as an effective way of obtaining an added value product with a safe long-term storage. Furthermore, the addition of nisin through interleavers would provide an additional hurdle to reduce the likelihood of food-borne disease.
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Acknowledgement This work was supported by INIA Project CPE03-012C3. We thank R. Rubio for technical assistance. References Appendini, P., & Hotchkiss, J. H. (2002). Review of antimicrobial food packaging. Innovative Food Science & Emerging Technologies, 3, 113–126. Aymerich, M. T., Garriga, M., Ylla, J., Vallier, J., Monfort, J. M., & Hugas, M. (2000). Application of enterocins as biopreservatives against Listeria innocua in meat products. Journal of Food Protection, 63, 721–726. Aymerich, M. T., Jofre´, A., Garriga, M., & Hugas, M. (2005). Inhibition of Listeria monocytogenes and Salmonella by natural antimicrobials and high hydrostatic pressure in sliced cooked ham. Journal of Food Protection, 68, 173–177. Black, E. P., Kelly, A. L., & Fitzgerald, G. F. (2005). The combined effect of high pressure and nisin on inactivation of microorganisms in milk. Innovative Food Science & Emerging Technologies, 6, 286–292. Casaus, P., Nilsen, T., Cintas, L. M., Nes, I. F., Hernandez, P. E., & Holo, H. (1997). Enterocin B, a new bacteriocin from Enterococcus faecium T136 which can act synergistically with enterocin A. Microbiology, 143, 2287–2294. Cintas, L. M., Casaus, M. P., Herranz, C., Nes, I. F., & Herna´ndez, P. E. (2001). Bacteriocins of lactic acid bacteria. Food Science and Technology International, 7, 281–305. Deegan, L. H., Cotter, P. D., Hill, C., & Ross, P. (2006). Bacteriocins: Biological tools for bio-preservation and shelf-life extension. International Dairy Journal, 16, 1058–1071. de Wit, J. C., & Rombouts, F. M. (1990). Antimicrobial activity of sodium lactate. Food Microbiology, 7, 113–120. EC, European Parliament and Council of the European Community, (1995). European directive 95/2/CE relative to food additives other than colors and sweeteners. Official Journal of the European Union, Brussels, Belgium. EC, European Parliament and Council of the European Community, (2005). Commission Regulation (EC) No. 2073/2005 of 15 November 2005 on microbiological criteria for foodstuffs. Official Journal of the European Union, L338, 1-26. Brussels, Belgium. FSIS, Food Safety and Inspection Service (2002). Safe and suitable ingredients used in the production of meat and poultry products. Directive 7120.1, Washington, DC. Garcı´a-Graells, C., Masschalck, B., & Michiels, C. W. (1999). Inactivation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides. Journal of Food Protection, 62, 1248–1254. Garriga, M., Aymerich, M. T., Costa, S., Monfort, J. M., & Hugas, M. (2002). Bactericidal synergism through bacteriocins and high pressure in a meat model system during storage. Food Microbiology, 19, 509–518. Gre`bol, N. (1995). Procedimiento para la preparacio´n de productos loncheados y envasados y producto obtenido. Esteban Espun˜a S.A., Patent number 9500789. Guerra, N. P., Macı´as, C. L., Agrasar, A. T., & Castro, L. P. (2005). Development of bioactive packaging cellophane using nisaplin as biopreservative agent. Letters in Applied Microbiology, 40, 106– 110.
Hoffman, K. L., Han, I. Y., & Dawson, P. L. (2001). Antimicrobial effects of corn zein films impregnated with nisin, lauric acid and EDTA. Journal of Food Protection, 64, 885–889. Hugas, M., Garriga, M., Pascual, M., Aymerich, M. T., & Monfort, J. M. (2002). Enhancement of sakacin K activity against Listeria monocytogenes in fermented sausages with pepper or manganese as ingredients. Food Microbiology, 19, 519–528. Kalchayanand, N., Sikes, T., Dunne, C. P., & Ray, B. (1994). Hydrostatic pressure and electroporation have increased bactericidal efficiency in combination with bacteriocins. Applied and Environmental Microbiology, 60, 4174–4177. Leistner, L., & Gorris, L. G. M. (1995). Food preservation by hurdle technology. Trends in Food Science and Technology., 6, 2–46. Natrajan, N., & Sheldon, B. (2000a). Efficacy of nisin-coated polymer films to inactive Salmonella typhimurium of fresh broiler skin. Journal of Food Protection, 63, 1189–1196. Natrajan, N., & Sheldon, B. (2000b). Inhibition of Salmonella on poultry skin using protein- and polysaccharide-based films containing a nisin formulation. Journal of Food Protection, 63, 1268–1272. Patterson, M. F., & Kilpatrick, D. J. (1998). The combined effect of high hydrostatic pressure and mild heat on inactivation of pathogens in milk and poultry. Journal of Food Protection, 6, 432–436. Pranoto, Y., Salokhe, V. M., & Rakshit, S. K. (2005). Physical and antibacterial properties of alginate-based edible film incorporated with garlic oil. Food Research International, 38, 267–272. Rastogi, N. K., Raghavarao, K. S. M. S., Balasubramaniam, V. M., Niranjan, K., & Knorr, D. (2007). Opportunities and challenges in high pressure processing of foods. Critical Reviews in Food Science and Nutrition, 47, 69–112. Reij, M. W., & Den Aantrekker, E. D. (2004). Recontamination as a source of pathogens in processed foods. International Journal of Food Microbiology, 91, 1–11. Roberts, T. A., Baird-Parker, A. C., Tompkin, R. B. (1996). Microorganisms in foods. In B. A. Professional (Eds.), (pp. 126–140). London. Rodrı´guez, E., Arques, J. L., Nunez, M., Gaya, P., & Medina, M. (2005). Combined effect of high-pressure treatments and bacteriocin-producing lactic acid bacteria on inactivation of Escherichia coli O157:H7 in raw-milk cheese. Applied and Environmental Microbiology, 71, 3399–3404. Sallam, K. I. (2007). Antimicrobial and antioxidant effects of sodium acetate, sodium lactate, and sodium citrate in refrigerated sliced salmon. Food Control, 18(5), 566–575. Stevens, K. A., Sheldon, B. W., Klapes, N. A., & Klaenhammer, T. R. (1991). Nisin treatment for inactivation of Salmonella species and other gram-negative bacteria. Applied and Environmental Microbiology, 57, 3613–3615. Ter Steeg, P. F., Hellemons, J. C., & Kok, A. E. (1999). Synergistic actions of nisin, sublethal ultrahigh pressure, and reduced temperature on bacteria and yeast. Applied and Environmental Microbiology, 65, 4148–4154. Ukuku, D. O., & Fett, W. F. (2004). Effect of nisin in combination with EDTA, sodium lactate, and potassium sorbate for reducing Salmonella on whole and fresh-cut cantaloupe. Journal Food Protection, 67, 2143–2150. Vogel, B. F., Yin Ng, Y., Hyldig, G., Mohr, M., & Gram, L. (2006). Potassium lactate combined with sodium diacetate can inhibit growth of Listeria monocytogenes in vacuum-packed cold-smoked salmon and has no adverse sensory effects. Journal of Food Protection, 69, 2134–2142.
Food Control 19 (2008) 634–638 www.elsevier.com/locate/foodcont
Short communication
Assessment of the effectiveness of antimicrobial packaging combined with high pressure to control Salmonella sp. in cooked ham Anna Jofre´, Teresa Aymerich, Margarita Garriga
*
IRTA, Finca Camps i Armet, E-17121 Monells, Spain Received 8 March 2007; received in revised form 30 May 2007; accepted 5 June 2007
Abstract The effectiveness of the application of interleavers containing enterocins A and B, sakacin K, nisin A, potassium lactate and nisin plus lactate alone or in combination with a 400 MPa high hydrostatic pressure treatment (HHP) was assessed in sliced cooked ham spiked with Salmonella spp. HHP produced an important reduction in Salmonella counts, which decreased from 104 CFU/g to <10 CFU/g, a value that was maintained for 3 months of storage at 6 C. However, the elimination of the pathogen could only be achieved by combining HHP and nisin-containing interleavers. Therefore, antimicrobial packaging, HHP and refrigerated storage appear as an effective combination of hurdles to obtain value added ready-to-eat products with a safe long-term storage. 2007 Elsevier Ltd. All rights reserved. Keywords: Active packaging; High pressure; Lactate; Nisin; Salmonella
1. Introduction Minimally processed ready-to-eat products challenge food safety and innovative ways to inhibit microbial growth, such as active packaging, are being developed. Antimicrobial packaging is a form of active packaging that reduces, inhibits or retards the growth of microorganisms present in packaged food through the incorporation of antimicrobials into the packaging, and is a promising tool for controlling the growth of food-borne pathogens or spoilage bacteria in ready-to-eat post-processed products (Appendini & Hotchkiss, 2002). Food products that have been submitted to an adequate heat treatment during processing are generally free of pathogens. However, epidemiological investigations into outbreaks related to several ready-to-eat products have demonstrated that the presence of Salmonella spp. in the *
Corresponding author. Tel.: +34 972630052; fax: +34 972 63 373. E-mail address: [email protected] (M. Garriga).
0956-7135/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodcont.2007.06.007
consumed products was frequently due to post-process recontamination (Reij & Den Aantrekker, 2004). In these products, mainly affected by superficial recontamination, antimicrobial packaging could avoid the necessity to add large amounts of antimicrobials to the food and to diminish the reduction of antimicrobial activity due to interactions with food constituents (Appendini & Hotchkiss, 2002). A great variety of antimicrobial agents including organic acids and their salts, sulfites, nitrites, antibiotics, alcohols, enzymes and natural components such as bacteriocins, especially nisin, have been incorporated into active films ´ as, Agrasar, & Castro, 2005; Natrajan & (Guerra, Macı Sheldon, 2000a; Natrajan & Sheldon, 2000b; Pranoto, Salokhe, & Rakshit, 2005). Nisin is currently the only bacteriocin allowed to be used as a food additive (EC, 1995; FSIS, 2002). Enterocins A and B and sakacin K are bacteriocins whose antilisterial activity has been shown in a meat homogenate and have been applied experimentally as ingredients in several meat products (Aymerich et al.,
A. Jofre´ et al. / Food Control 19 (2008) 634–638
2000; Aymerich, Jofre´, Garriga, & Hugas, 2005; Garriga, Aymerich, Costa, Monfort, & Hugas, 2002; Hugas, Garriga, Pascual, Aymerich, & Monfort, 2002). Lactate salts have shown antimicrobial activity against several spoilage and food-borne pathogenic microorganisms and are widely used as ingredients in the meat industry (de Wit & Rombouts, 1990; Sallam, 2007; Vogel, Yin Ng, Hyldig, Mohr, & Gram, 2006). The antimicrobial activity of bacteriocins is generally limited to closely related gram-positive species and a cost-effective food biopreservation, especially against gram-negative bacteria, may require a ‘‘hurdle approach’’ (Leistner & Gorris, 1995). Bacteriocins could act synergistically with traditional or novel preservation systems which induce sublethal injury to bacteria such as high hydrostatic pressure (HHP). HHP is an emerging technology for microbial reduction in minimally processed foods with fresh-like quality and improved safety aspects (Rastogi, Raghavarao, Balasubramaniam, Niranjan, & Knorr, 2007). In gram-negative bacteria, HHP increases the antimicrobial efficiency of bacteriocins by permeabilization of the outer membrane (Kalchayanand, Sikes, Dunne, & Ray, 1994; Ter Steeg, Hellemons, & Kok, 1999). In food products, a limited number of studies have evaluated the combined effect of HHP and bacteriocins on gram-negative bacteria (Aymerich et al., 2005; Black, Kelly, & Fitzgerald, 2005; Garcı´a-Graells, Masschalck, & Michiels, 1999; Garriga et al., 2002; Patterson & Kilpatrick, 1998; Rodrı´guez, Arques, Nunez, Gaya, & Medina, 2005). To our knowledge, the combined effect of HHP and bacteriocins applied through antimicrobial packaging has not been previously evaluated. The objective of this study was to evaluate the application of active interleavers containing enterocins A and B, sakacin K, nisin and potassium lactate against Salmonella in sliced cooked ham. Furthermore, the effect of the application of an additional 400 MPa HHP treatment after slicing was evaluated during a 3-month refrigerated storage.
2. Material and methods 2.1. Cooked ham Cooked ham with minced pork shoulder and the following composition (in g/kg) was prepared: water, 115; salt, 20.7; dextrose, 5.8; sodium tri-polyphosphate, 5.8; carragenate, 2.3; NaNO2, 0.1 and L-ascorbate, 0.6 (all additives from SKW Biosystems, Rubi, Spain). Minced meat (4 mm) was homogenised with other ingredients (Mixer 35P, Tecnotrip SA, Terrassa, Spain) for 30 min and stuffed (Stuffing machine H15, Tecnotrip SA) into impermeable plastic film (Prolan SV 150, 11 cm of diameter, Hispano Holandesa S.A. Sant Boi de Llobregat, Spain). Ham pieces of 2.5– 3 kg were cooked in an oven at 75 C until internal temperature reached 72 C (approximately 2.5 h). The pH of the cooked ham was 6.3 and the aw = 0.98.
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2.2. Bacteriocin extracts Sakacin K and enterocins A and B were obtained from overnight cultures of Lactobacillus sakei CTC494 and Enterococcus faecium CTC492, respectively, as described in Aymerich et al. (2000). 2.3. Preparation of interleavers containing antimicrobials Interleavers (11 · 11 cm and 133 lm thick) consisting of a perforated polypropylene layer, a non-woven polyamide layer in the middle and another non-perforated polypropylene layer (Gre`bol, 1995) were prepared by adding 200 or 2000 AU/cm2 of enterocins A and B (E200 and E2000), 200 or 2000 AU/cm2 of sakacin K (S200 and S2000), 200 AU/cm2 of nisin A (N200) (Nisaplin. Aplin & Barrett Ltd, UK), 1.8 g/100 g (1.8% w/w) ham of potassium lactate (L) (Purasal P/Hi Pure 60. Purac Biochem, Gorinchem, The Netherlands) and 200 AU/g nisin plus 1.8% potassium lactate (NL) and distilled water (control, C). Antimicrobials and water were spread on the perforated polypropylene layer, left to dry and stored overnight at 6 C. 2.4. Spiking, packaging and pressurization A cocktail of three Salmonella enterica subsp. enterica (S. London CTC1003, S. Schwarzengrund CTC1015 and S. Derby CTC1022) was prepared by properly diluting frozen stored stationary phase cultures of the strains. Each slice was inoculated with ca. 3 · 104 CFU/g and covered with an interleaver. Pairs of slices (11 cm diamenter and 1.5 mm thick) were vacuum packaged in plastic bags of PET/PE (O2 and water vapour permeability <50 cm3/m2/ 24 h and <15 mg/m2/24 h, respectively; Sacoliva S.L., Spain). Eight batches of packaged cooked ham were prepared: control, E200, E2000, S200, S2000, N200, L and NL. Half of the samples were pressurized in an industrial hydrostatic pressurization unit (Alstom, Nantes, France; chamber volume of 320 l and diameter of 280 mm) at 400 MPa for 10 min at 17 C. The come-up and release times and the adiabatic heating were 13.17 min, 1.33 min and <5 C, respectively. Pressurized and non-pressurized samples were subsequently stored at 6 C. 2.5. Microbiological analyses Three different packages of each treatment were analyzed on day 0, 1 and every 15 days for 3 months. Twenty five grams of ham were 1/10 diluted in buffered peptone water (BPW, AES Laboratories, Combourg, France) and homogenized in a Stomacher Labblender (model 400, Cooke Laboratory Products, Alexandria, USA). Brilliant Green Agar (BGA. Difco, Detroit, Michigan, USA) was used to count Salmonella (limit of detection of 10 CFU/ g). PCR as described in Aymerich et al. (2005) was used to confirm five colonies of each plate and to determine
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presence/absence of Salmonella sp. in BPW homogenates incubated at 37 C for 48 h. 2.6. Statistical analysis Data were subjected to ANOVA of the General Lineal Model procedure of SAS software (SAS system for Windows NT, release 8.1, SAS Inst. Cary NC, USA). The model included batch, treatment, storage time, and their interaction as fixed effects. Differences between effects were assessed by the Tukey test. 3. Results and discussion
1 log CFU/ml. Natrajan and Sheldon (2000b) evaluated the use of protein- and polysaccharide-based films containing a nisin formulation, including 5 mM EDTA, 3% citric acid and Tween 80, on the inhibition of S. typhimurium on poultry skin. Reductions up to 4.4 log cycles were shown after 96 h of exposure of the drumstick skin to films containing 500 lg/ml of nisin and stored at 4 C. In this regard, Deegan, Cotter, Hill, and Ross (2006) suggested that bacteriocins should not be used as the main processing step to prevent the growth or survival of pathogens but to provide an additional hurdle to reduce the likelihood of food-borne disease. The combination of antimicrobials with other inhibitory treatments such as HHP has also been proposed to achieve a higher inactivation of gramnegative food-borne pathogens.
3.1. Effect of interleavers containing antimicrobials on Salmonella
3.2. Combination of antimicrobial packaging and HHP
Interleavers containing the bacteriocins enterocin (E200 and E2000), sakacin (S200 and S2000) and nisin (N), and the potassium lactate salt applied alone (L) or in combination with nisin (NL), were evaluated for their ability to control or reduce the levels of Salmonella at 6 C. In all the batches the growth of Salmonella was inhibited by the temperature of storage and the counts experienced a progressive reduction of ca. 1.5 log units from the beginning to the end of the storage period (Fig. 1a). No additional reductions in the Salmonella levels were observed in the batches packaged with enterocin, sakacin or nisin after either 1 day or during 3 months of storage. Bacteriocins exert their mode of action by destabilization and permeabilisation of the membrane of sensitive cells. After formation of transitory poration complexes, through non-specific interaction with anionic polymers usually constituting the cell wall of gram-positive bacteria, pore formation and loss of membrane integrity provokes a passive efflux of small molecules, resulting in a loss of the proton motive force (Cintas, Casaus, Herranz, Nes, & Herna´ndez, 2001). In general, the activity of bacteriocins produced by lactic acid bacteria is against gram-positive bacteria but, while enterocins A and B and sakacin K are bacteriocins with an important anti-listeria activity, nisin, belonging to class I, has a broader spectrum of activity (Casaus et al., 1997; Stevens, Sheldon, Klapes, & Klaenhammer, 1991). To obtain a bacteriostatic or slightly bactericidal effect of bacteriocins against gram-negative bacteria, a previous destabilization of the cell envelope with the chelating agent EDTA or other destabilizing factors is necessary. Ukuku and Fett (2004) reported that the combination of nisin with EDTA, sodium lactate and potassium sorbate could control, through washing treatments, Salmonella in cantaloupe. Only a few studies have dealt with the effect of the application of antimicrobials through active films. Hoffman, Han, and Dawson (2001) incorporated nisin, lauric acid and EDTA to corn zein films that were exposed to broth cultures of S. enteritidis for 48 h and none of the combinations produced reductions of the pathogen greater than
The application of a 400 MPa HHP treatment to the active packaged slices of cooked ham produced a drastic reduction to the counts of Salmonella (Fig. 1b). One day after pressurization the levels were reduced from ca. 4.5 log CFU/g to levels below 10 CFU/g in the control batch. In antimicrobial-containing batches, neither enterocin (E200 and E2000), sakacin (S200 and S2000), L nor NL were shown to increase the inhibitory effect of the HHP compared with the control (P > 0.05). Besides, a lower decrease was observed in E2000 and S2000 batches compared with the control (P < 0.05). Only the combination of nisin and HHP recorded absence of Salmonella in 25 g of ham 24 h after pressurization. During refrigerated storage of pressurized ham at 6 C, a temperature that does not support the growth of Salmonella sp. (Roberts, Baird-Parker, & Tompkin, 1996), no further recovery of the pathogen was observed. The levels of the pathogen in C, N and NL batches continued below 10 CFU/g (P > 0.05) during the entire storage period. In L, E and S batches the initial reduction was lower but the counts progressively decreased during storage and after 60 days they were below 10 CFU/g in all the batches (P > 0.05). From day 60 to the end of the storage at 6 C, the counts continued at the same level and no significant differences were observed among batches (P > 0.05). Nisin was the only treatment that produced absence of Salmonella 24 h after pressurisation and the application of nisin through interleavers and combined with an HHP treatment appears as the most effective treatment to achieve absence when a high level inoculum is considered. The combined effect of HHP and antimicrobials on Salmonella has not been previously evaluated through antimicrobial packaging. Only a few studies have been performed in broth and bacteriocin-containing food products. Kalchayanand et al. (1994) reported in 0.1% peptone an increase in the inactivation of both S. typhimurium and E. coli by combining pressurization at 207–345 MPa and nisin, pediocin and both. Ter Steeg et al. (1999) showed in E. coli an increase in the synergism between nisin and HHP at
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a6
Log CFU/g
4
2
0 0
15
30
45
60
90
75
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b
6 Enterocin200 Enterocin2000
Log ( C FU+1) /g
Sakacin200 Sakacin2000 4
Nisin200 Lactate Nisin+Lactate Control
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Fig. 1. Evolution of Salmonella sp. in slices of cooked ham packaged with antimicrobial-containing interleavers and stored at 6 C. (a) Non-HHP treated ham; and (b) 400 MPa pressurized ham.
150 and 200 MPa at reduced temperatures and a stronger inactivation when nisin was present during both pressurization and in the recovery medium than when it was only applied during either pressurization or recovery. They postulated that the rigidity of the membrane due to reduced temperature and binding of nisin increased the efficacy of the HHP treatment. In cooked ham, we previously showed (Aymerich et al., 2005) that the combination of nisin applied in the meat batter at 800 AU/g and HHP at 400 MPa also recorded the highest proportion of absences during a storage period of 3 months at 6 C. In contrast, other studies performed in a model meat system spiked with S. London CTC1003 and S. Schwarzengrund CTC1015 did not show significant differences among control and antimicrobial-containing (enterocins A and B, sakacin K, pediocin AcH and nisin A) meat homogenate after
pressurization at 400 MPa and during a storage of 61 days at 4 C (Garriga et al., 2002). Evaluation of the effectiveness of the assayed antimicrobial treatments to assure the absence of the pathogen after processing and during the entire shelf-life of the product is very important and required by the new European microbiological criteria (EC, 2005). Considering the inability of the pathogen to grow at refrigeration temperatures, the presumably low-level of post-processing recontaminations and the drastic reduction of Salmonella achieved by the HHP treatment recorded in this challenge test, pressurization of sliced cooked ham and storage at 66 C appears as an effective way of obtaining an added value product with a safe long-term storage. Furthermore, the addition of nisin through interleavers would provide an additional hurdle to reduce the likelihood of food-borne disease.
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