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Spray application of liquid smoke to reduce or eliminate Listeria monocytogenes surface inoculated on frankfurters
Meat Science 85 (2010) 640–644
Contents lists available at ScienceDirect
Meat Science journal homepage: www.elsevier.com/locate/meatsci
Spray application of liquid smoke to reduce or eliminate Listeria monocytogenes surface inoculated on frankfurters Elizabeth M. Martin a, Corliss A. O’Bryan b,*, Robert Y. Lary Jr. c, Carl L. Griffis a, Katherine L.S. Vaughn a, John A. Marcy d, Steven C. Ricke b,d, Philip G. Crandall b a
Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72704, USA Department of Food Science and Center for Food Safety, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704, USA Sysco Corporation, 1390 Enclave Parkway, Houston, TX 77077, USA d Department of Poultry Science, Division of Agriculture, University of Arkansas, Fayetteville, AR 72704, USA b c
a r t i c l e
i n f o
Article history: Received 30 April 2009 Received in revised form 6 November 2009 Accepted 11 March 2010
Keywords: Liquid smoke Listeria monocytogenes Frankfurters
a b s t r a c t In a simulated post process contamination scenario liquid smoke was sprayed on the frankfurters after peeling, and then inoculated with Listeria monocytogenes (Lm). Samples that did not receive a liquid smoke spray remained at approximately 2 log cfu/cm2 during the 48 h of storage while the levels on the liquid smoke treated frankfurters continued to decline until they were below detection level (1 cfu/100 cm2). A shelf-life study lasting 140 days indicated that liquid smoke suppressed the growth of Lm for up to 130 days. An application of 2 or 3 ml liquid smoke at packaging resulted in at least a 1 log reduction of Lm within 12 h post packaging. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Listeria monocytogenes (Lm) is a Gram-positive, non spore forming rod that can cause a disease in humans known as listeriosis. Although listeriosis can occur in healthy adults, most (80%) of the cases occur in immunocompromised persons, pregnant women, newborns, or the elderly (Schuchat, Swaminathan, & Broome, 1991). A pregnant woman is likely to experience only mild discomfort, but the Listeria infection can cause a miscarriage, premature birth or stillbirth; in addition newborns infected during pregnancy are at increased risk of dying (Painter & Slutsker, 2007). Although there is considerable data obtained from outbreaks of listeriosis, the vast majority of the cases are isolated and sporadic (Painter & Slutsker, 2007). Mead et al. (1999) estimated that twice as many cases of listeriosis actually occurred as were reported and projected that 2518 cases of listeriosis occur each year, with 504 cases resulting in death. The realization that Listeria infections could be considered as a foodborne illness stems from the 1981 investigation of a large number of cases of listeriosis in Canada which was subsequently traced to coleslaw (Schlech et al., 1983). Since this recognition, investigations of Listeria outbreaks as well as sporadic cases of listeriosis have implicated a variety of foods, but a common characteristic of the foods is that the consumer eats them without * Corresponding author. Tel.: +1 479 575 5328; fax: +1 479 575 6936. E-mail address: [email protected] (C.A. O’Bryan). 0309-1740/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2010.03.017
cooking (Norton & Braden, 2007). Many of these ‘‘ready to eat” foods are cooked, cured or pasteurized during manufacturing but are susceptible to post processing contamination before packaging (Wenger et al., 1990). The United States Department of Agriculture’s Food Safety and Inspection Service (USDA/FSIS) has designated frankfurters and hot dogs to be at the top of their ‘‘high risk” foods list (USDA/FSIS, 2003) because of their long shelf life, being marketed as ready to eat, and their high levels of consumption by children or immunocompromised persons such as the elderly. USDA issued a ruling in 2003 that affects foods that are ready to eat and that might be exposed to Lm in the food processing plant after cooking, but before or during packaging (Anonymous, 2003). These USDA guidelines give processors three alternative methods of Lm control. Alternative 1 dictates the use of a ‘‘post-lethality treatment” that reduces or eliminates Listeria on the product and an antimicrobial agent that suppresses or limits the growth of Listeria throughout the shelf life of the product. Alternative 2 allows either a post-lethality treatment or an agent that suppresses or limits growth of Listeria throughout shelf life. Alternative 3 allows the use of sanitation measures only. Extensive research has been done on post-lethality treatments and agents that suppress Lm growth, including the use of organic salts and acids. Sodium lactate, sodium diacetate, lactic acid, and acetic acid have been reported to have antimicrobial effects on Lm when used alone or in combination (Barmpalia et al., 2004; Geornaras et al., 2006; Lu, Sebranek, Dickson, Mendonca, & Bailey, 2005; Porto et al., 2002; Samelis &
E.M. Martin et al. / Meat Science 85 (2010) 640–644
Metaxopoulos, 1999). Lauric arginate, a newly introduced Generally Recognized as Safe (GRAS) antimicrobial agent has proven to be effective as a post-lethality treatment in ham and frankfurters (Bakal & Diaz, 2005; Luchansky et al., 2005; Martin et al., 2008). Smoking meats for preservation is a traditional method of preservation and smoking is still practiced (Maga, 1988). Commercially manufactured liquid smoke extracts have been approved as GRAS and are being used as antimicrobial agents by the ready to eat (RTE) industry. Recent studies have indicated reductions of Lm utilizing commercially available liquid smoke extracts (Zesti-B and AM3) as a surface application on frankfurters that were formulated with or without lactate/diacetate (Gedela, Escoubas, & Muriana, 2007). Frankfurters without lactate/diacetate allowed growth of Lm to the level of 9 log CFU in 10 weeks, while Lm levels on frankfurters treated with liquid smoke remained below 1 log CFU during this time. Murphy et al. (2005) combined the use of liquid smoke (Select 23P, Red Arrow Intl., Manitowoc, Wis., USA) with steam pasteurization on surface inoculated frankfurters and found no growth of Lm during 47 days of storage. These studies all present data for controlling Listeria in RTE foods. However, there is still a need for additional research to address an effective mechanical application of liquid smoke, the optimum time period for application, and a way devised to integrate the antimicrobial application equipment onto existing plant machinery, which were the objectives of this research.
641
The specifications of this fraction were titratable acidity 1.8–2.1%, specific gravity 1.095–1.120 at 25 °C, phenol level 0.3–0.8 mg/ml, pH 4.25–4.85 and color light amber to yellow. The fraction was stored at room temperature and used as received. Liquid smoke was sprayed onto frankfurters using either a hand held spray bottle (Fig. 1) or an AutoJet PWM control panel with a Pulsajet 10000AUH-03 spray nozzle (Spraying Systems Co., Wheaton, IL) (Fig. 2). The pressure to the spray gun was maintained at 30 psig and controller was set to ‘‘one shot mode”. Spray time was calibrated with a gram scale to deliver 1.5 ml per shot. One shot was dispensed in the sterile weigh boat, frankfurters were added to the boat and one shot dispensed over the top of the frankfurters. For simulated post processing contamination frankfurters were individually sprayed with liquid smoke using a bottle sprayer, placed in a sterile stainless steel pan and inoculated (see Section 2.6). For shelf-life studies, frankfurters were individually sprayed with liquid smoke using a bottle sprayer, inoculated with Lm, placed in a sterile pouch and vacuum sealed (see Section 2.7). For the simulated two-stage application process, the AutoJet sprayer was used as described (see Section 2.7). 2.4. Microbiological analysis At set times (as described in each experimental design) vacuum pouches (3 mil Barrier Pouches, Ultravac Solutions) containing
2. Materials and methods 2.1. Frankfurters Frankfurters were formulated without lactate/diacetate and thermally processed in a commercial processing plant using the following formulation: mechanically separated chicken (55.7%), high fat pork trim (14.7%), 50% fat beef (2.0%), modified food starch (2.7%), dextrose (2.0%), corn syrup solids (1.5%), salt (2.35%), spices (0.75%), phosphate (0.32%), nitrite (0.039%), and water (19%). After stuffing, the frankfurters were thermally processed in a continuous run oven, cooked to a minimum internal temperature of 170 °F and held at that temperature for 10–15 min. Frankfurters were delivered to our lab, refrigerated and held at 4 °C prior to the study. 2.2. Bacterial cultures Six strains of Lm were used in these studies; five strains (ARS V67, ARS V72, ARS V113, ARS V125, and ARS 105) were obtained from Dr. M.E. Berrang at USDA Agricultural Research Service, Athens, GA. Lm strain LCDC 81-861 (4b) was obtained from Dr. M. Johnson at the Department of Food Science, University of Arkansas, Fayetteville, AR. Stock cultures were maintained frozen at 80 °C. One loop of the frozen stock cultures of each strain of Lm was inoculated into tryptic soy broth with 0.06% yeast extract (TSBYE; Becton Dickinson, Sparks, MD) and incubated at 37 °C for 18–20 h. One loop of each strain was streaked for isolated colonies onto Modified Oxford agar (MOX: Becton Dickinson, Sparks, MD). Individual colonies were picked and grown in TSBYE for two or three additional passages to build up inoculum levels. One ml was taken from each six cultured strains and placed in a tube and vortexed to form the inoculum ‘‘cocktail”. The cocktail was diluted with sterile 0.1% peptone water (PW) to a concentration of 105/ml. 2.3. Liquid smoke A refined liquid smoke fraction (AM-3, Mastertaste, Inc., Crossville, TN) with reduced color and flavor was used in these studies.
Fig. 1. Spray bottles for hand delivery of antimicrobials, control sterile water on the left, liquid smoke (AM3) on the right.
Fig. 2. Spray technique for frankfurters, using an AutoJet PWM control panel with a Pulsajet 10000AUH-03 spray nozzle (Spraying Systems Co., Wheaton, IL). Half of the liquid smoke was dispensed in the weigh boat, frankfurters added, and other half of liquid smoke sprayed on top of frankfurters.
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control or treated frankfurters were removed from refrigeration (4 °C) and aseptically opened. Sterile 0.1% PW was added to the pouch at the rate of 9 ml per frankfurter and the pouches were hand massaged to release attached Lm. Serial 10-fold dilutions were made with 0.1% sterile PW and plated onto MOX for Lm counts. Plates were incubated at 37 °C for 72 h and enumerated. Sampling was done in triplicate. Total aerobic plate counts were done on Petrifilm™ aerobic plate count which was incubated at 37 °C for 48–72 h. Lactic acid bacteria were enumerated on Man– Rogosa–Sharp agar plates (MRS: Becton Dickinson and Co., Sparks, MD) incubated at 30 °C for 5 days, using the MGC AnaeroPack System (Mitsubishi Gas Chemical Company of America, Inc., New York, NY 10017). 2.5. Antimicrobial activity on post application contaminated frankfurters All manipulations were carried out in a Labconco Class II Biosafety Cabinet (Labconco Corporation, 8811 Prospect Avenue, Kansas City, Missouri 64132-2696). To simulate application just after peeling the frankfurters from the casings the liquid smoke was applied to the frankfurters prior to inoculation. Frankfurters were individually hand-sprayed to simulate in process plant application of the liquid smoke with either 3 ml of liquid smoke or 3.0 ml sterile deionized water. Frankfurters were then placed in sterile stainless steel pans, and surface inoculated with 105 CFU/ml of the six strain Lm ‘‘cocktail” to provide 1 ml of inoculum per three frankfurters, and the inoculum was dispersed over the frankfurters by gently shaking the pans. Containers were sealed with sterile lids, covered with aluminum foil to retain moisture within the containers, and placed at refrigerated storage temperatures, approximately 4 °C. After 0, 2, 24, 48 h, nine frankfurters were chosen randomly from treatment or control containers. Three frankfurters per treatment were placed into a sterile plastic bag and 9 ml of PW was added to each bag for a 10 1 dilution of the inoculum, serial 10-fold dilutions were made and plated onto MOX agar.
The weigh dishes containing the samples were carefully placed into new vacuum pouches and sealed at 100 kPa with a Koch Ultravac sealer (Ultravac Solutions, Kansas City, MO). Pouches were either analyzed immediately (0 h) or stored at 4 °C until the 8 or 12 h sampling time. Samples were processed as described above, using only MOX agar for plating. 3. Results 3.1. Antimicrobial effect on frankfurters contaminated post application Fig. 3 is a graphical representation of the effect of liquid smoke applied to frankfurters after their casings (used during cooking) have been removed. After the liquid smoke application the frankfurters were subsequently surface inoculated with Lm. This simulated the post process scenario where an antimicrobial spray would immediately follow the casing peeler and then contamination of the frankfurters could take place in the processing plant before packaging. The control samples remained at approximately 2 log cfu/cm2 during the 48 h of storage while the Lm levels on the liquid smoke treated frankfurters continued to decline until they were below detection level (1 cfu/100 cm2) at 48 h. 3.2. Shelf life of post treatment contaminated frankfurters The 140 day shelf-life study indicated that liquid smoke suppressed the growth of Lm for up to 130 days (Fig. 4). Aerobic plate counts (data not shown) on liquid smoke treated samples exhibited counts similar to those on MOX, and characterization of several colonies from the aerobic plate counts indicated these colonies were primarily Lm. Aerobic counts of samples not treated with liquid smoke were also too numerous to count at the dilutions
2.6. Shelf life of post application surface contaminated frankfurters Frankfurters were hand-sprayed with approximately 1.8 ml per frankfurter with either liquid smoke or sterile deionized water. Frankfurters were allowed to drip momentarily and then were placed in vacuum pouches (three frankfurters per pouch). Pouches were placed in a Biosafety cabinet where 0.5 ml of the approximately 106 CFU/ml Lm ‘‘cocktail” inoculum was added to each pouch, manually massaged for 2 min and vacuum sealed. Samples were stored at approximately 4 °C for shelf life testing. After 0, 30, 60, 90, 100, 110, 120, 130, and 140 days, pouches were opened aseptically, 18 ml of PW was added and the pouches were hand massaged to release attached Lm. Serial 10-fold dilutions were made with PW and plated onto MOX agar for Lm counts, Petrifilm™ for aerobic plate counts and MRS for lactic acid bacteria counts.
Fig. 3. Effect of liquid smoke on survival of Lm surface inoculated on frankfurters.
2.7. Simulated two-stage application of liquid smoke For this portion of the study, eight frankfurters were transferred into vacuum pouches (20.3 30.5 cm, 3 mil) and 1 ml Lm ‘‘cocktail” inoculum (approximately 105 CFU/ml) was added to each pouch. Inoculated product was massaged, by hand, for 2 min. Polystyrene weigh dishes (14.6 cm square) were sprayed with ½ of the total application of liquid smoke and inoculated frankfurters were carefully transferred to the dish. The second ½ aliquot of liquid smoke was sprayed on top of the frankfurters. The total amount of liquid smoke added was 1, 2, or 3 ml. Positive control samples were placed in weigh dishes without application of liquid smoke.
Fig. 4. Survival of Lm on frankfurters surface inoculated with Lm after treatment with liquid smoke during 140 day shelf life.
E.M. Martin et al. / Meat Science 85 (2010) 640–644
plated, and characterization of several of these colonies also revealed them to be predominantly Lm. Lactic acid bacteria plate counts (data not shown) of liquid smoke samples showed no noticeable growth up to 110 days. After 110 days there were similar plate counts to those of MOX. Only a small number (2–3 logs) of colonies resembling lactic acid bacteria were seen at 130 days, post-inoculation of the liquid smoke samples. 3.3. Simulated two-stage application of liquid smoke Although shelf life results indicated that an ‘‘in-process” application of liquid smoke could be capable of achieving an initial 1 log reduction of Lm and would suppress growth of Lm throughout shelf life we considered a second alternative, application of liquid smoke at packaging. The two-stage application of 2 or 3 ml liquid smoke at packaging resulted in at least a 1 log reduction of Lm at 8 and 12 h post packaging (Fig. 5). To verify that the liquid smoke would sufficiently cover the frankfurter surfaces, a food dye was added to the liquid smoke and 3 ml of this dyed liquid smoke was added to the packaged product and vacuum sealed. From careful visual observation of the sample it was noted that the dyed solution uniformly covered all areas of the frankfurters including the crimped ends of the frankfurters (data not shown). 4. Discussion Both the control and the liquid smoke treatments showed a 2log decrease from inoculation levels at 2 h, possibly due to loss in purge dilution effects. In agreement with Gedela et al. (2007) liquid smoke administered as an antimicrobial for control of Lm also exhibited an initial 1 log reduction and a suppression of Lm growth when used post contamination. This demonstrated that there was a positive impact of using liquid smoke as an antimicrobial just after removing the casing and before packaging. Potentially, if there were random, low levels of Lm contamination the liquid smoke would provide an extra margin of safety. The approximate shelf life of frankfurters is 90 days, therefore the 140 days (20 weeks) went well beyond the 10 week shelf-life study Gedela et al. (2007) reported, indicating that the liquid smoke suppression from the initial lethality continued past the stated shelf life. As suggested by Gedela, the liquid smoke product has a possible application for Alternative 1 as a post-lethality treatment that reduces or eliminates Lm and can also suppress growth of Lm on surface inoculated frankfurters through and beyond shelf life. A two-stage application of liquid smoke would position the frankfurters to be sprayed with the liquid smoke at the time of packaging after removal from the casing and transportation through the plant environment. In addition, the two-stage application seems to be a reasonable method for administering the liquid smoke in a
Fig. 5. Effects of two-stage application of liquid smoke on Lm surface inoculated onto frankfurters.
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fashion that would fit into existing plant equipment. This two-stage application is similar to the Sprayed Lethality in Container (SLIC™) method described by Luchansky et al. (2005) in their research on hams. However, the SLIC™ method is a one-stage spray administered within the intact package, while this two-stage application is administered with a two section package system, where the bottom ply and top ply are two separate units that are vacuum/heat sealed after the frankfurters are placed into the bottom section. In addition to the microbial testing, it is essential that the antimicrobial agent does not change the product’s quality in an adverse way. To address sensory concerns of liquid smoke, consumer triangle sensory tests were performed in the Food Science Sensory Laboratory, Department of Food Science, University of Arkansas. Results indicated no significant difference between liquid smoke treated frankfurters and the control frankfurters (personal communication, Food Science Sensory Laboratory, Department of Food Science, University of Arkansas). 5. Conclusions This study indicated that liquid smoke was an effective antimicrobial for frankfurters in that it provided at least a one log initial reduction of surface inoculated Lm and suppressed growth during the extended shelf life of this product. A two-stage application at the time of packaging rather than at the time of peeling also provided the initial one log reduction of Lm. No sensory changes were noted with the use of liquid smoke on frankfurters. Acknowledgements This study was supported by Bar-S Foods, Inc. and Mastertaste, Inc. Arun Ramadadran and Rick Grat from AutoJet Technologies Division of Spraying Systems Co. were helpful with technical assistance on the spray system. We would also like to thank Brandon Beard, Carol Boger, Erik Friedly and Jim Smith for their technical assistance. References Anonymous (2003). Control of L. monocytogenes in ready-to-eat meat and poultry products: Final rule. Federal Register, 68, 34207–34254. Bakal, G., & Diaz, A. (2005). The lowdown on lauric arginate. Food Quality, 12, 54–61. Barmpalia, I. M., Geornaras, I., Belk, K. E., Scanga, J. A., Kendall, P. A., Smith, G. C., et al. (2004). Control of Listeria monocytogenes on frankfurters with antimicrobials in the formulation and by dipping in organic acid solutions. Journal of Food Protection, 67, 2456–2464. Gedela, S., Escoubas, J. R., & Muriana, P. M. (2007). Effect of inhibitory liquid smoke fractions on Listeria monocytogenes during long-term storage of frankfurters. Journal of Food Protection, 70, 386–391. Geornaras, I., Skandamis, P. N., Belk, K. E., Scanga, J. A., Kendall, P. A., Smith, G. C., et al. (2006). Postprocess control of Listeria monocytogenes on commercial frankfurters formulated with and without antimicrobials and stored at 10 °C. Journal of Food Protection, 69, 53–61. Lu, Z., Sebranek, J. G., Dickson, J. S., Mendonca, A. F., & Bailey, T. B. (2005). Inhibitory effects of organic acid salts for control of Listeria monocytogenes on frankfurters. Journal of Food Protection, 68, 499–506. Luchansky, J. B., Call, J. E., Hristova, B., Rumery, L., Yoder, L., & Oser, A. (2005). Viability of Listeria monocytogenes on commercially-prepared hams surface treated with acidic calcium sulfate and lauric arginate and stored at 4 °C. Meat Science, 71, 92–99. Maga, J. A. (1988). Smoke in food processing. Boca Raton, FL: CRC Press. Martin, E., Lary, Y., Griffis, C., Vaughn, K., Boger C., & Marcy, J. 2008. Antimicrobial effects of A&B Ingredients CytoGuardÒ on the survival of Listeria monocytogenes surface-inoculated onto Bar-S Foods Co. hot dogs, vacuum-packed and stored at 4 °C. In: Proceeding of the 95th Annual meeting, IAFP 2008, Columbus, Ohio, USA, 3–6 August, 2008. Abstract P2-25. Mead, P. S., Slutsker, L., Dietz, V., McCaig, F., Bresee, J. S., Shapiro, C., et al. (1999). Food-related illness and death in the United States. Emerging Infectious Diseases, 5, 607–625. Murphy, R. Y., Hanson, R. E., Johnson, N. R., Scott, L. L., Feze, N., & Chappa, K. (2005). Combining antimicrobial and steam treatments in a vacuum-packaging system to control L. monocytogenes on ready-to-eat franks. Journal of Food Science, 70, 138–140.
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Norton, D. M., & Braden, C. R. (2007). Foodborne listeriosis. In E. T. Ryser & E. H. Marth (Eds.), Listeria, listeriosis and food safety (pp. 305–356). New York: CRC Press. Painter, J., & Slutsker, L. (2007). Listeriosis in humans. In E. T. Ryser & E. H. Marth (Eds.), Listeria, listeriosis and food safety (pp. 85–109). New York: CRC Press. Porto, A. C. S., Franco, B. D. G. M., Sant’Anna, E. S., Call, J. E., Piva, A., & Luchansky, J. B. (2002). Viability of a five-strain mixture of Listeria monocytogenes in vacuumsealed packages of frankfurters, commercially prepared with and without 2.0 or 3.0% added potassium lactate, during extended storage at 4 and 10 °C. Journal of Food Protection, 65, 308–315. Samelis, J., & Metaxopoulos, J. (1999). Incidence and principal sources of Listeria spp. and Listeria monocytogenes contamination in processed meats and a meat processing plant. Food Microbiology, 16, 465–477.
Schlech, W. F. I., Lavigne, R. A., Bortolussi, A. C., Allen, A. C., Haldane, E. V., Wort, A. J., et al. (1983). Epidemic listeriosis – Evidence for transmission by food. New England Journal of Medicine, 308, 203–206. Schuchat, A., Swaminathan, B., & Broome, C. V. (1991). Epidemiology of human listeriosis. Clinical Microbiological Reviews, 4, 169–183. United States Department of Agriculture/Food Safety Inspection Service, USDA/FSIS. 2003. Quantitative assessment of relative risk to public health from foodborne Listeria monocytogenes among selected categories of ready-to-eat foods. Available at: Accessed 11.07.08. Wenger, J. D., Swaminathan, B., Hayes, P. S., Green, S. S., Pratt, M., Pinner, R. W., et al. (1990). Listeria monocytogenes contamination of turkey franks: evaluation of a production facility. Journal of Food Protection, 53, 1015–1019.
Contents lists available at ScienceDirect
Meat Science journal homepage: www.elsevier.com/locate/meatsci
Spray application of liquid smoke to reduce or eliminate Listeria monocytogenes surface inoculated on frankfurters Elizabeth M. Martin a, Corliss A. O’Bryan b,*, Robert Y. Lary Jr. c, Carl L. Griffis a, Katherine L.S. Vaughn a, John A. Marcy d, Steven C. Ricke b,d, Philip G. Crandall b a
Department of Biological and Agricultural Engineering, University of Arkansas, Fayetteville, AR 72704, USA Department of Food Science and Center for Food Safety, University of Arkansas, 2650 Young Ave., Fayetteville, AR 72704, USA Sysco Corporation, 1390 Enclave Parkway, Houston, TX 77077, USA d Department of Poultry Science, Division of Agriculture, University of Arkansas, Fayetteville, AR 72704, USA b c
a r t i c l e
i n f o
Article history: Received 30 April 2009 Received in revised form 6 November 2009 Accepted 11 March 2010
Keywords: Liquid smoke Listeria monocytogenes Frankfurters
a b s t r a c t In a simulated post process contamination scenario liquid smoke was sprayed on the frankfurters after peeling, and then inoculated with Listeria monocytogenes (Lm). Samples that did not receive a liquid smoke spray remained at approximately 2 log cfu/cm2 during the 48 h of storage while the levels on the liquid smoke treated frankfurters continued to decline until they were below detection level (1 cfu/100 cm2). A shelf-life study lasting 140 days indicated that liquid smoke suppressed the growth of Lm for up to 130 days. An application of 2 or 3 ml liquid smoke at packaging resulted in at least a 1 log reduction of Lm within 12 h post packaging. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Listeria monocytogenes (Lm) is a Gram-positive, non spore forming rod that can cause a disease in humans known as listeriosis. Although listeriosis can occur in healthy adults, most (80%) of the cases occur in immunocompromised persons, pregnant women, newborns, or the elderly (Schuchat, Swaminathan, & Broome, 1991). A pregnant woman is likely to experience only mild discomfort, but the Listeria infection can cause a miscarriage, premature birth or stillbirth; in addition newborns infected during pregnancy are at increased risk of dying (Painter & Slutsker, 2007). Although there is considerable data obtained from outbreaks of listeriosis, the vast majority of the cases are isolated and sporadic (Painter & Slutsker, 2007). Mead et al. (1999) estimated that twice as many cases of listeriosis actually occurred as were reported and projected that 2518 cases of listeriosis occur each year, with 504 cases resulting in death. The realization that Listeria infections could be considered as a foodborne illness stems from the 1981 investigation of a large number of cases of listeriosis in Canada which was subsequently traced to coleslaw (Schlech et al., 1983). Since this recognition, investigations of Listeria outbreaks as well as sporadic cases of listeriosis have implicated a variety of foods, but a common characteristic of the foods is that the consumer eats them without * Corresponding author. Tel.: +1 479 575 5328; fax: +1 479 575 6936. E-mail address: [email protected] (C.A. O’Bryan). 0309-1740/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.meatsci.2010.03.017
cooking (Norton & Braden, 2007). Many of these ‘‘ready to eat” foods are cooked, cured or pasteurized during manufacturing but are susceptible to post processing contamination before packaging (Wenger et al., 1990). The United States Department of Agriculture’s Food Safety and Inspection Service (USDA/FSIS) has designated frankfurters and hot dogs to be at the top of their ‘‘high risk” foods list (USDA/FSIS, 2003) because of their long shelf life, being marketed as ready to eat, and their high levels of consumption by children or immunocompromised persons such as the elderly. USDA issued a ruling in 2003 that affects foods that are ready to eat and that might be exposed to Lm in the food processing plant after cooking, but before or during packaging (Anonymous, 2003). These USDA guidelines give processors three alternative methods of Lm control. Alternative 1 dictates the use of a ‘‘post-lethality treatment” that reduces or eliminates Listeria on the product and an antimicrobial agent that suppresses or limits the growth of Listeria throughout the shelf life of the product. Alternative 2 allows either a post-lethality treatment or an agent that suppresses or limits growth of Listeria throughout shelf life. Alternative 3 allows the use of sanitation measures only. Extensive research has been done on post-lethality treatments and agents that suppress Lm growth, including the use of organic salts and acids. Sodium lactate, sodium diacetate, lactic acid, and acetic acid have been reported to have antimicrobial effects on Lm when used alone or in combination (Barmpalia et al., 2004; Geornaras et al., 2006; Lu, Sebranek, Dickson, Mendonca, & Bailey, 2005; Porto et al., 2002; Samelis &
E.M. Martin et al. / Meat Science 85 (2010) 640–644
Metaxopoulos, 1999). Lauric arginate, a newly introduced Generally Recognized as Safe (GRAS) antimicrobial agent has proven to be effective as a post-lethality treatment in ham and frankfurters (Bakal & Diaz, 2005; Luchansky et al., 2005; Martin et al., 2008). Smoking meats for preservation is a traditional method of preservation and smoking is still practiced (Maga, 1988). Commercially manufactured liquid smoke extracts have been approved as GRAS and are being used as antimicrobial agents by the ready to eat (RTE) industry. Recent studies have indicated reductions of Lm utilizing commercially available liquid smoke extracts (Zesti-B and AM3) as a surface application on frankfurters that were formulated with or without lactate/diacetate (Gedela, Escoubas, & Muriana, 2007). Frankfurters without lactate/diacetate allowed growth of Lm to the level of 9 log CFU in 10 weeks, while Lm levels on frankfurters treated with liquid smoke remained below 1 log CFU during this time. Murphy et al. (2005) combined the use of liquid smoke (Select 23P, Red Arrow Intl., Manitowoc, Wis., USA) with steam pasteurization on surface inoculated frankfurters and found no growth of Lm during 47 days of storage. These studies all present data for controlling Listeria in RTE foods. However, there is still a need for additional research to address an effective mechanical application of liquid smoke, the optimum time period for application, and a way devised to integrate the antimicrobial application equipment onto existing plant machinery, which were the objectives of this research.
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The specifications of this fraction were titratable acidity 1.8–2.1%, specific gravity 1.095–1.120 at 25 °C, phenol level 0.3–0.8 mg/ml, pH 4.25–4.85 and color light amber to yellow. The fraction was stored at room temperature and used as received. Liquid smoke was sprayed onto frankfurters using either a hand held spray bottle (Fig. 1) or an AutoJet PWM control panel with a Pulsajet 10000AUH-03 spray nozzle (Spraying Systems Co., Wheaton, IL) (Fig. 2). The pressure to the spray gun was maintained at 30 psig and controller was set to ‘‘one shot mode”. Spray time was calibrated with a gram scale to deliver 1.5 ml per shot. One shot was dispensed in the sterile weigh boat, frankfurters were added to the boat and one shot dispensed over the top of the frankfurters. For simulated post processing contamination frankfurters were individually sprayed with liquid smoke using a bottle sprayer, placed in a sterile stainless steel pan and inoculated (see Section 2.6). For shelf-life studies, frankfurters were individually sprayed with liquid smoke using a bottle sprayer, inoculated with Lm, placed in a sterile pouch and vacuum sealed (see Section 2.7). For the simulated two-stage application process, the AutoJet sprayer was used as described (see Section 2.7). 2.4. Microbiological analysis At set times (as described in each experimental design) vacuum pouches (3 mil Barrier Pouches, Ultravac Solutions) containing
2. Materials and methods 2.1. Frankfurters Frankfurters were formulated without lactate/diacetate and thermally processed in a commercial processing plant using the following formulation: mechanically separated chicken (55.7%), high fat pork trim (14.7%), 50% fat beef (2.0%), modified food starch (2.7%), dextrose (2.0%), corn syrup solids (1.5%), salt (2.35%), spices (0.75%), phosphate (0.32%), nitrite (0.039%), and water (19%). After stuffing, the frankfurters were thermally processed in a continuous run oven, cooked to a minimum internal temperature of 170 °F and held at that temperature for 10–15 min. Frankfurters were delivered to our lab, refrigerated and held at 4 °C prior to the study. 2.2. Bacterial cultures Six strains of Lm were used in these studies; five strains (ARS V67, ARS V72, ARS V113, ARS V125, and ARS 105) were obtained from Dr. M.E. Berrang at USDA Agricultural Research Service, Athens, GA. Lm strain LCDC 81-861 (4b) was obtained from Dr. M. Johnson at the Department of Food Science, University of Arkansas, Fayetteville, AR. Stock cultures were maintained frozen at 80 °C. One loop of the frozen stock cultures of each strain of Lm was inoculated into tryptic soy broth with 0.06% yeast extract (TSBYE; Becton Dickinson, Sparks, MD) and incubated at 37 °C for 18–20 h. One loop of each strain was streaked for isolated colonies onto Modified Oxford agar (MOX: Becton Dickinson, Sparks, MD). Individual colonies were picked and grown in TSBYE for two or three additional passages to build up inoculum levels. One ml was taken from each six cultured strains and placed in a tube and vortexed to form the inoculum ‘‘cocktail”. The cocktail was diluted with sterile 0.1% peptone water (PW) to a concentration of 105/ml. 2.3. Liquid smoke A refined liquid smoke fraction (AM-3, Mastertaste, Inc., Crossville, TN) with reduced color and flavor was used in these studies.
Fig. 1. Spray bottles for hand delivery of antimicrobials, control sterile water on the left, liquid smoke (AM3) on the right.
Fig. 2. Spray technique for frankfurters, using an AutoJet PWM control panel with a Pulsajet 10000AUH-03 spray nozzle (Spraying Systems Co., Wheaton, IL). Half of the liquid smoke was dispensed in the weigh boat, frankfurters added, and other half of liquid smoke sprayed on top of frankfurters.
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control or treated frankfurters were removed from refrigeration (4 °C) and aseptically opened. Sterile 0.1% PW was added to the pouch at the rate of 9 ml per frankfurter and the pouches were hand massaged to release attached Lm. Serial 10-fold dilutions were made with 0.1% sterile PW and plated onto MOX for Lm counts. Plates were incubated at 37 °C for 72 h and enumerated. Sampling was done in triplicate. Total aerobic plate counts were done on Petrifilm™ aerobic plate count which was incubated at 37 °C for 48–72 h. Lactic acid bacteria were enumerated on Man– Rogosa–Sharp agar plates (MRS: Becton Dickinson and Co., Sparks, MD) incubated at 30 °C for 5 days, using the MGC AnaeroPack System (Mitsubishi Gas Chemical Company of America, Inc., New York, NY 10017). 2.5. Antimicrobial activity on post application contaminated frankfurters All manipulations were carried out in a Labconco Class II Biosafety Cabinet (Labconco Corporation, 8811 Prospect Avenue, Kansas City, Missouri 64132-2696). To simulate application just after peeling the frankfurters from the casings the liquid smoke was applied to the frankfurters prior to inoculation. Frankfurters were individually hand-sprayed to simulate in process plant application of the liquid smoke with either 3 ml of liquid smoke or 3.0 ml sterile deionized water. Frankfurters were then placed in sterile stainless steel pans, and surface inoculated with 105 CFU/ml of the six strain Lm ‘‘cocktail” to provide 1 ml of inoculum per three frankfurters, and the inoculum was dispersed over the frankfurters by gently shaking the pans. Containers were sealed with sterile lids, covered with aluminum foil to retain moisture within the containers, and placed at refrigerated storage temperatures, approximately 4 °C. After 0, 2, 24, 48 h, nine frankfurters were chosen randomly from treatment or control containers. Three frankfurters per treatment were placed into a sterile plastic bag and 9 ml of PW was added to each bag for a 10 1 dilution of the inoculum, serial 10-fold dilutions were made and plated onto MOX agar.
The weigh dishes containing the samples were carefully placed into new vacuum pouches and sealed at 100 kPa with a Koch Ultravac sealer (Ultravac Solutions, Kansas City, MO). Pouches were either analyzed immediately (0 h) or stored at 4 °C until the 8 or 12 h sampling time. Samples were processed as described above, using only MOX agar for plating. 3. Results 3.1. Antimicrobial effect on frankfurters contaminated post application Fig. 3 is a graphical representation of the effect of liquid smoke applied to frankfurters after their casings (used during cooking) have been removed. After the liquid smoke application the frankfurters were subsequently surface inoculated with Lm. This simulated the post process scenario where an antimicrobial spray would immediately follow the casing peeler and then contamination of the frankfurters could take place in the processing plant before packaging. The control samples remained at approximately 2 log cfu/cm2 during the 48 h of storage while the Lm levels on the liquid smoke treated frankfurters continued to decline until they were below detection level (1 cfu/100 cm2) at 48 h. 3.2. Shelf life of post treatment contaminated frankfurters The 140 day shelf-life study indicated that liquid smoke suppressed the growth of Lm for up to 130 days (Fig. 4). Aerobic plate counts (data not shown) on liquid smoke treated samples exhibited counts similar to those on MOX, and characterization of several colonies from the aerobic plate counts indicated these colonies were primarily Lm. Aerobic counts of samples not treated with liquid smoke were also too numerous to count at the dilutions
2.6. Shelf life of post application surface contaminated frankfurters Frankfurters were hand-sprayed with approximately 1.8 ml per frankfurter with either liquid smoke or sterile deionized water. Frankfurters were allowed to drip momentarily and then were placed in vacuum pouches (three frankfurters per pouch). Pouches were placed in a Biosafety cabinet where 0.5 ml of the approximately 106 CFU/ml Lm ‘‘cocktail” inoculum was added to each pouch, manually massaged for 2 min and vacuum sealed. Samples were stored at approximately 4 °C for shelf life testing. After 0, 30, 60, 90, 100, 110, 120, 130, and 140 days, pouches were opened aseptically, 18 ml of PW was added and the pouches were hand massaged to release attached Lm. Serial 10-fold dilutions were made with PW and plated onto MOX agar for Lm counts, Petrifilm™ for aerobic plate counts and MRS for lactic acid bacteria counts.
Fig. 3. Effect of liquid smoke on survival of Lm surface inoculated on frankfurters.
2.7. Simulated two-stage application of liquid smoke For this portion of the study, eight frankfurters were transferred into vacuum pouches (20.3 30.5 cm, 3 mil) and 1 ml Lm ‘‘cocktail” inoculum (approximately 105 CFU/ml) was added to each pouch. Inoculated product was massaged, by hand, for 2 min. Polystyrene weigh dishes (14.6 cm square) were sprayed with ½ of the total application of liquid smoke and inoculated frankfurters were carefully transferred to the dish. The second ½ aliquot of liquid smoke was sprayed on top of the frankfurters. The total amount of liquid smoke added was 1, 2, or 3 ml. Positive control samples were placed in weigh dishes without application of liquid smoke.
Fig. 4. Survival of Lm on frankfurters surface inoculated with Lm after treatment with liquid smoke during 140 day shelf life.
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plated, and characterization of several of these colonies also revealed them to be predominantly Lm. Lactic acid bacteria plate counts (data not shown) of liquid smoke samples showed no noticeable growth up to 110 days. After 110 days there were similar plate counts to those of MOX. Only a small number (2–3 logs) of colonies resembling lactic acid bacteria were seen at 130 days, post-inoculation of the liquid smoke samples. 3.3. Simulated two-stage application of liquid smoke Although shelf life results indicated that an ‘‘in-process” application of liquid smoke could be capable of achieving an initial 1 log reduction of Lm and would suppress growth of Lm throughout shelf life we considered a second alternative, application of liquid smoke at packaging. The two-stage application of 2 or 3 ml liquid smoke at packaging resulted in at least a 1 log reduction of Lm at 8 and 12 h post packaging (Fig. 5). To verify that the liquid smoke would sufficiently cover the frankfurter surfaces, a food dye was added to the liquid smoke and 3 ml of this dyed liquid smoke was added to the packaged product and vacuum sealed. From careful visual observation of the sample it was noted that the dyed solution uniformly covered all areas of the frankfurters including the crimped ends of the frankfurters (data not shown). 4. Discussion Both the control and the liquid smoke treatments showed a 2log decrease from inoculation levels at 2 h, possibly due to loss in purge dilution effects. In agreement with Gedela et al. (2007) liquid smoke administered as an antimicrobial for control of Lm also exhibited an initial 1 log reduction and a suppression of Lm growth when used post contamination. This demonstrated that there was a positive impact of using liquid smoke as an antimicrobial just after removing the casing and before packaging. Potentially, if there were random, low levels of Lm contamination the liquid smoke would provide an extra margin of safety. The approximate shelf life of frankfurters is 90 days, therefore the 140 days (20 weeks) went well beyond the 10 week shelf-life study Gedela et al. (2007) reported, indicating that the liquid smoke suppression from the initial lethality continued past the stated shelf life. As suggested by Gedela, the liquid smoke product has a possible application for Alternative 1 as a post-lethality treatment that reduces or eliminates Lm and can also suppress growth of Lm on surface inoculated frankfurters through and beyond shelf life. A two-stage application of liquid smoke would position the frankfurters to be sprayed with the liquid smoke at the time of packaging after removal from the casing and transportation through the plant environment. In addition, the two-stage application seems to be a reasonable method for administering the liquid smoke in a
Fig. 5. Effects of two-stage application of liquid smoke on Lm surface inoculated onto frankfurters.
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fashion that would fit into existing plant equipment. This two-stage application is similar to the Sprayed Lethality in Container (SLIC™) method described by Luchansky et al. (2005) in their research on hams. However, the SLIC™ method is a one-stage spray administered within the intact package, while this two-stage application is administered with a two section package system, where the bottom ply and top ply are two separate units that are vacuum/heat sealed after the frankfurters are placed into the bottom section. In addition to the microbial testing, it is essential that the antimicrobial agent does not change the product’s quality in an adverse way. To address sensory concerns of liquid smoke, consumer triangle sensory tests were performed in the Food Science Sensory Laboratory, Department of Food Science, University of Arkansas. Results indicated no significant difference between liquid smoke treated frankfurters and the control frankfurters (personal communication, Food Science Sensory Laboratory, Department of Food Science, University of Arkansas). 5. Conclusions This study indicated that liquid smoke was an effective antimicrobial for frankfurters in that it provided at least a one log initial reduction of surface inoculated Lm and suppressed growth during the extended shelf life of this product. A two-stage application at the time of packaging rather than at the time of peeling also provided the initial one log reduction of Lm. No sensory changes were noted with the use of liquid smoke on frankfurters. Acknowledgements This study was supported by Bar-S Foods, Inc. and Mastertaste, Inc. Arun Ramadadran and Rick Grat from AutoJet Technologies Division of Spraying Systems Co. were helpful with technical assistance on the spray system. We would also like to thank Brandon Beard, Carol Boger, Erik Friedly and Jim Smith for their technical assistance. References Anonymous (2003). Control of L. monocytogenes in ready-to-eat meat and poultry products: Final rule. Federal Register, 68, 34207–34254. Bakal, G., & Diaz, A. (2005). The lowdown on lauric arginate. Food Quality, 12, 54–61. Barmpalia, I. M., Geornaras, I., Belk, K. E., Scanga, J. A., Kendall, P. A., Smith, G. C., et al. (2004). Control of Listeria monocytogenes on frankfurters with antimicrobials in the formulation and by dipping in organic acid solutions. Journal of Food Protection, 67, 2456–2464. Gedela, S., Escoubas, J. R., & Muriana, P. M. (2007). Effect of inhibitory liquid smoke fractions on Listeria monocytogenes during long-term storage of frankfurters. Journal of Food Protection, 70, 386–391. Geornaras, I., Skandamis, P. N., Belk, K. E., Scanga, J. A., Kendall, P. A., Smith, G. C., et al. (2006). Postprocess control of Listeria monocytogenes on commercial frankfurters formulated with and without antimicrobials and stored at 10 °C. Journal of Food Protection, 69, 53–61. Lu, Z., Sebranek, J. G., Dickson, J. S., Mendonca, A. F., & Bailey, T. B. (2005). Inhibitory effects of organic acid salts for control of Listeria monocytogenes on frankfurters. Journal of Food Protection, 68, 499–506. Luchansky, J. B., Call, J. E., Hristova, B., Rumery, L., Yoder, L., & Oser, A. (2005). Viability of Listeria monocytogenes on commercially-prepared hams surface treated with acidic calcium sulfate and lauric arginate and stored at 4 °C. Meat Science, 71, 92–99. Maga, J. A. (1988). Smoke in food processing. Boca Raton, FL: CRC Press. Martin, E., Lary, Y., Griffis, C., Vaughn, K., Boger C., & Marcy, J. 2008. Antimicrobial effects of A&B Ingredients CytoGuardÒ on the survival of Listeria monocytogenes surface-inoculated onto Bar-S Foods Co. hot dogs, vacuum-packed and stored at 4 °C. In: Proceeding of the 95th Annual meeting, IAFP 2008, Columbus, Ohio, USA, 3–6 August, 2008. Abstract P2-25. Mead, P. S., Slutsker, L., Dietz, V., McCaig, F., Bresee, J. S., Shapiro, C., et al. (1999). Food-related illness and death in the United States. Emerging Infectious Diseases, 5, 607–625. Murphy, R. Y., Hanson, R. E., Johnson, N. R., Scott, L. L., Feze, N., & Chappa, K. (2005). Combining antimicrobial and steam treatments in a vacuum-packaging system to control L. monocytogenes on ready-to-eat franks. Journal of Food Science, 70, 138–140.
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