- Email: [email protected]
Combined effects of slightly acidic electrolyzed water and fumaric acid on the reduction of foodborne pathogens and shelf life extension of fresh pork
Food Control 47 (2015) 277e284
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Combined effects of slightly acidic electrolyzed water and fumaric acid on the reduction of foodborne pathogens and shelf life extension of fresh pork Ahmad Rois Mansur, Charles Nkufi Tango, Gwang-Hee Kim, Deog-Hwan Oh* Department of Food Science and Biotechnology, School of Bioconvergence Science and Technology, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2014 Received in revised form 3 July 2014 Accepted 8 July 2014 Available online 15 July 2014
This study evaluated the efficacy of the individual treatments (slightly acidic electrolyzed water [SAcEW] or fumaric acid [FA]) and their combination to reduce Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella Typhimurium in fresh pork as well as to study the shelf life and sensory quality (color, odor, and texture) of pork during storage at 4 and 10 C. The inoculated pork samples (10 g) were dipped for 3 min in each treatment (tap water [TW], SAcEW, strong acidic electrolyzed water [StAEW], 0.5% FA, or SAcEW þ 0.5% FA) with or without mild heat (40 C). Decontamination of fresh pork with SAcEW þ0.5% FA at 40 C for 3 min showed greater bactericidal effect compared to other treatments, which significantly (P < 0.05) reduced E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium by 2.59, 2.69, 2.38, and 2.99 log CFU/g, respectively. This combined treatment significantly (P < 0.05) yielded in a longer lag time of naturally occurring bacteria (TBC) on pork stored at 4 C. This combined treatment also prolonged the shelf life of pork up to 6 days and 4e5 days when stored at 4 C and 10 C, respectively, compared to those of the untreated pork. The results suggest that the combined treatment of SAcEW þ 0.5% FA has potential as a novel method to enhance the microbial safety and quality of fresh pork. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Pork Slightly acidic electrolyzed water Fumaric acid Pathogens reduction Shelf life
1. Introduction Pork is the most consumed meat in the world, but pathogen contaminations such as Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella Typhimurium have impacted its safety and quality (Baer, Miller, & Dilger, 2013; Mataragas, Skandamis, & Drosinos, 2008). Salmonella spp., L. monocytogenes, and S. aureus are among the top pathogens associated with pork and pork products, which cause foodborne illnesses and deaths annually. The Centers for Disease Control and Prevention (CDC) reported that these pathogens have caused 47.8 million illnesses and 3000 deaths each year (CDC, 2011). Moreover, product recalls, decreased sales from damaged organizational reputation, and spoilage of meat can lead to food waste and economic losses as well as the loss of consumer confidence (Nychas, Marshall, & Sofos, 2007; Scharff, 2010). Therefore, to improve the microbial safety and
* Corresponding author. Tel./fax: þ82 33 250 6457. E-mail address: [email protected] (D.-H. Oh). http://dx.doi.org/10.1016/j.foodcont.2014.07.019 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
quality of pork during processing and storage, various chemical sanitizers have been investigated to reduce its microbial contaminations and extend shelf life. In recent years, antimicrobial agents from chemicals such as chlorinated solutions, electrolyzed water (EW), organic acids, and salts, alone or in combination have been investigated and shown to reduce microbial contaminations of pork (Chen et al., 2012; Smulders & Greer, 1998). Slightly acidic electrolyzed water (SAcEW) is a type of EW and promising sanitizer for food products (Huang, Hung, Hsu, Huang, & Hwang, 2008). However, the use of SAcEW as an individual treatment has not effectively reduced bacterial contaminations in fresh meat (Ding, Rahman, Purev, & Oh, 2010; Rahman, Wang, & Oh, 2013). Therefore, it has frequently been combined with other chemical sanitizers to enhance its bactericidal efficacy. Organic acids are GRAS (Generally Recognized as Safe) compounds, which have ability to inactivate foodborne pathogens (Chen et al., 2012). Among the organic acids used for antimicrobial agent on meat, fumaric acid (FA) has shown stronger bactericidal effect compared to acetic and lactic acid (Podolak, Zayas, Kastner, & Fung, 1995, 1996). Even though the SAcEW and FA seem to be promising sanitizers for meat
278
A.R. Mansur et al. / Food Control 47 (2015) 277e284
and meat products, their application on meat and meat products remains limited. Moreover, there is no research on the application of the combined treatment (SAcEW and FA) for decontamination and extending the shelf life of meat and meat products published to date. Therefore, the current study was conducted to evaluate the efficacy of the individual treatments (SAcEW or FA) and their combination on reducing E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium in fresh pork. The shelf life and sensory quality of pork during storage at 4 and 10 C were also studied. 2. Materials and methods 2.1. Preparation of bacterial cultures The two strains of E. coli O157:H7 (ATCC 43894 and ATCC 43895), L. monocytogenes (ATCC 19114 and ATCC 19115), S. aureus (ATCC 12598 and ATCC 14458), and S. Typhimurium (ATCC 13311 and ATCC 14028) were obtained from the Department of Food Science, University of Georgia (Griffin, GA, USA), the Korean National Institute of Health (Seoul, Korea), and the Health Research Department (Gyeonggi-do, Republic of Korea), respectively. Prior to use, each strain of pathogens was separately grown in tryptic soy broth (TSB; Difco, Becton, Dickinson and Company Sparks, MD, USA) at 37 C with two consecutive transfers after a 24 h period for a total 48 h of incubation. All working cultures grown in TSB were separately centrifuged at 4000 g for 10 min at 4 C, and the supernatants were discarded. The cell pellets were washed twice with 0.1% sterile buffered peptone water (BPW; Difco, Becton, Dickinson and Company Sparks, MD, USA) pH 7.1, and resuspended in 10 mL of the same solution to obtain a final cell concentration of approximately 8 log CFU/mL. The two strains each of E. coli O157:H7, L. monocytogenes, S. aureus, or S. Typhimurium was combined in a cocktail with approximately equal numbers in the final population (8 log CFU/ mL). The bacterial population in each culture cocktail was confirmed by plating 0.1 mL portions of appropriately diluted culture on tryptic soy agar (TSA; Difco, Becton, Dickinson and Company Sparks, MD, USA) plates, and then incubating the plates at 37 C for 24 h. The prepared culture cocktails were then used in subsequent experiments. 2.2. Preparation of pork samples Boneless pork loin was transported from a local slaughterhouse (Kangwon LPC, Wonju, Kangwon, Korea) to the laboratory under temperature-controlled conditions in a sterile plastic container. All pork samples were then separately cut into pieces using a sterile knife. Aliquots with a weight of 10 ± 0.3 g were used for the decontamination and storage tests, and those of 25 ± 0.3 g were used for the sensory analysis and measurement of the pH value. 2.3. Inoculation of pathogens to pork samples Pork samples were placed on sterile petri dishes in a laminar flow hood, then spot inoculated by pipetting 0.1 mL of each culture cocktail (approximately 8 log CFU/mL) onto the surface to obtain an initial level of approximately 6 log CFU/g. The inoculated pork samples were air-dried in a laminar flow hood for 1 h at room temperature to allow attachment of bacteria, and then immediately exposed to the treatments. 2.4. Preparation of sanitizing solutions The commercially available slightly acidic electrolyzed water (SAcEW; 30 mg/L) and strong acidic electrolyzed water (StAEW;
30 mg/L) used in this study were obtained from the Korea Advanced Institute of Science and Technology (KAIST, Daejeon, Korea). SAcEW and StAEW were produced by electrolysis of 6% and 9% HCl solutions, respectively, using an electrolysis device. The crystalline fumaric acid (FA) (Daejung Chemicals and Metals Co., Siheung, Gyeonggi, Korea) was dissolved in 1 L of deionized water (DW) to give a final concentration of 0.5% FA solutions (w/v). The pH, oxidation reduction potential (ORP), and available chlorine concentration (ACC) of the sanitizers were measured with a dualscale pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ, USA) bearing pH and ORP electrodes. The ACC was determined by a colorimetric method using a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan). The detection limit was 1e300 mg/L. The physicochemical properties of the tested sanitizing solutions are summarized in Table 1. 2.5. Decontamination treatment and microbiological analysis The preliminary in vitro experiments were carried out to determine the optimal condition for each individual treatment of SAcEW, StAEW, and organic acids (lactic acid [LA], citric acid [CA], acetic acid [AA], and fumaric acid [FA]). The experiments were conducted at different temperatures (25, 30, 40, 50, and 60 C), different ACCs for EW (5, 10, 20, and 30 mg/L), different concentrations for organic acids (0.125, 0.25, and 0.5%), and different exposure times (1, 3, and 5 min). The results suggested that dipping with EW (30 mg/L) or 0.5% FA at 40 C for 3 min was the most satisfactory method (data not shown). These methods were then employed in the in vivo study. For the in vivo study, inoculated pork samples (10 g) were placed in a sterile container and dipped for 3 min in each treatment (tap water [TW], SAcEW, StAEW, 0.5% FA, SAcEW þ 0.5% FA) with or without mild heat (40 C). Unwashed pork samples were used as control. The excess solution was removed from pork samples with sterile paper towels. Each 10 g pork sample was then mixed with 90 mL of 0.1% sterile BPW and homogenized for 2 min in a Seward stomacher (400 Circulator, Seward, London, UK). After homogenization, 1 mL aliquot of each sample was serially diluted in 9 mL of 0.1% sterile BPW, and 0.1 mL of diluents was spread-plated onto sorbitol MacConkey's agar (Difco, Becton, Dickinson and Company Sparks, MD, USA), modified Oxford agar base with the addition of the Oxford antimicrobic supplement (Oxoid, Basingstoke, UK), Baird Parker agar (Difco, Becton, Dickinson and Company Sparks, MD, USA) supplemented with 50 mL of the egg yolk Tellurite, and Brilliant Green agar (Oxoid, Basingstoke, UK) to enumerate E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium,
Table 1 Physicochemical properties of the tested sanitizing solutions.a Tested solutions TW SAcEWd StAEWe 0.5% FAf SAcEW þ 0.5% FAg
ORP (mV)b
pH 7.17 6.29 2.31 2.34 2.95
± ± ± ± ±
A
0.12 0.17B 0.08C 0.11C 0.13C
358 826 1159 570 1091
± ± ± ± ±
A
10 16C 12D 9B 19D
ACC (mg/L)c NDh 30 ± 0.2 30 ± 0.6 ND 15 ± 0.09
AD Numbers within each column followed by different capital letters are significantly different (P < 0.05). a Values are mean ± standard deviation, n ¼ 6. b Oxidation reduction potential. c Available chlorine concentration. d Slightly acidic electrolyzed water. e Strong acidic electrolyzed water. f 0.5% fumaric acid. g Slightly acidic electrolyzed water (30 mg/L) þ 0.5% fumaric acid. h Not detected.
A.R. Mansur et al. / Food Control 47 (2015) 277e284
respectively. All plates were then incubated at 37 C for 24e48 h. Each independent trial was in triplicates.
279
Sciences, Chicago, IL, USA). The analysis included treatment, storage temperature, and bacteria as fixed effects. Differences between effects were assessed by the Tukey test (P < 0.05).
2.6. Storage tests 3. Results and discussion The inoculated (E. coli O157:H7 and S. Typhimurium) and uninoculated pork samples were divided into four different groups and a control (untreated pork samples). Samples in group 1 to 3 were only dipped for 3 min in 100 mL SAcEW, StAEW, or 0.5% FA at 40 C, while samples in group 4 were dipped for 3 min in a combined treatment (SAcEW þ 0.5% FA) at 40 C. Each treated sample was then mixed with 100 mL of neutralizing solution (0.85% NaCl containing 0.5% Na2S2O3) for 3 min to remove residual solutions and stop their bactericidal effects. The excess solution was then removed from pork samples with sterile paper towels. All pork samples (treated and untreated) were then airpackaged using a new stomacher bag (Nasco Whirl-Pak, Janesville, WI, USA) and marked carefully before storage at 4 and 10 C. Each sample was analyzed on days 0, 2, 4, 6, 8, and 10 at 4 C, while on days 0, 1, 2, 3, 5, and 7 at 10 C. At each sampling interval, 90 mL of 0.1% sterile BPW was poured into the stomacher bag containing the samples. After 2 min of homogenization, 0.1 mL aliquots of the appropriate dilution were plated onto plate count agar (Difco, Becton, Dickinson and Company Sparks, MD, USA) to enumerate total bacterial counts (TBC). All plates were incubated at 37 C for 24 h. The end of shelf life was considered to occur when TBC was 7 log CFU/g (Nychas, Skandamis, Tassou, & Koutsoumanis, 2008) or when the sensory score of the pork samples reached an unacceptable level. Each storage test consisted of two trials with triplicates of samples per trial and three plates per replicate at each time interval. 2.7. Sensory analysis and measurement of the pH value Sensory evaluation of pork samples (25 g each) were carried out by a four member sensory panel (each panel consisted of eight panelists) (Latha, Sherikar, Waskar, Dubal, & Ahmed, 2009; Rahman et al., 2013). Each sample was evaluated on days 0, 2, 4, 6, 8, and 10 at 4 C and on days 0, 1, 2, 3, 5, and 7 at 10 C for sensorial items (color, odor, and texture) using a simple four-point scoring system (1e4); 1 is the highest score and 4 is the lowest score. A sensory index (SI) was calculated using Eq. (1) (Kreyenschmidt et al., 2010).
SI ¼
2*C þ 1*ðO þ TÞ 4
3.1. Decontamination of fresh pork Initial populations of E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium on pork used for evaluating the efficacy of sanitizing treatments either alone or in combination were approximately 6.27, 5.95, 6.16, and 6.12 log CFU/g, respectively. The individual treatments (SAcEW [30 mg/L], StAEW [30 mg/L], and 0.5% FA) and a combined treatment (SAcEW þ 0.5% FA) with mild heat (40 C) for 3 min significantly (P < 0.05) reduced pathogens compared to the treatments applied at 25 C and control. After 3 min dipping at 40 C, the individual treatments yielded pathogens reductions ranging from 0.23e0.33, 1.19e1.55, 1.20e1.43, and 1.17e1.44 log CFU/g for the TW, SAcEW, StAEW, and 0.5% FA treatment, respectively (Figs. 1e4). The use of SAcEW treatment showed no significant (P > 0.05) difference in bactericidal effects compared to StAEW treatment, even though the pH and ORP values of SAcEW were significantly (P < 0.05) different compared to those of StAEW (Table 1). The results revealed that the bactericidal effects of EW appeared to be due more to the available chlorine concentration (ACC) than to other factors such as low pH and high ORP value. Huang et al. (2008) stated that the chlorine compounds can kill the microbial cell through inhibiting glucose oxidation by chlorine-oxidizing sulfhydryl groups of certain enzymes used in carbohydrate metabolism, disrupting protein synthesis, inhibiting oxygen uptake and oxidative phosphorylation coupled with leakage of some macromolecules, and forming toxic N-chlorine derivatives of cytosine. The bactericidal effects of 0.5% FA were also not significantly (P > 0.05) different compared to those of SAcEW and StAEW. According to Baird Parker (1980), the undissociated molecules of organic acids are likely responsible for their antimicrobial properties. Booth (1985) reported that the accumulation of undissociated molecules of weak acids in the cytoplasm of the cell leads to the dissociation of protonated acid, the release of a proton,
(1)
where C is color, O is odor, and T is texture. The cut-off score was fixed at an SI of 2.2. The pork samples (in triplicates in each sample) were then homogenized in a Seward stomacher (400 Circulator, Seward, London, UK) with 125 mL of DW, and filtered. The pH of the filtrate was measured with a dual-scale pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ, USA). 2.8. Curve fitting and statistical analyses The values of all experimental data (in triplicates) represent the mean of triplicate determinations for each sample. The modified Gompertz model (Zwietering, Jongenburger, Rombouts, & van’t Riet, 1990) was used to describe the bacterial growth curves at 4 and 10 C, and growth parameters (specific growth rate [SGR]: log CFU/day; lag time [LT]: day) were analyzed by the GraphPad prism software (version 5.01, GraphPad Software, Inc., San Diego, CA). The data (means ± standard deviation) were analyzed using One-way ANOVA in the SPSS ver. 13.0 (Statistical Package for the Social
Fig. 1. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on E. coli O157:H7 in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
280
A.R. Mansur et al. / Food Control 47 (2015) 277e284
Fig. 2. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on L. monocytogenes in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
and the acidification of the cytoplasm of the microorganism, if intracellular pH is higher than the pKa of the acid. Several previous studies have examined the effectiveness of using individual treatments of SAcEW (10 mg/L, dipping at room temperature for 5 min), StAEW (50 mg/L, dipping at room temperature for 5 min or spraying for 15 s), and FA (1e1.5%, dipping at 55 C for 15e30 s) for reducing E. coli, E. coli O157:H7, L. monocytogenes, and S. Typhimurium on fresh pork surfaces. The reductions of these pathogens were also less than 2 log CFU/g (Fabrizio & Cutter, 2004; Podolak et al., 1995, 1996; Rahman et al., 2013). The application of combined treatment with mild heat (SAcEW þ 0.5% FA 40 C) yielded in greater reductions of pathogens compared to all individual treatments and control (P < 0.05), which reduced E. coli O157:H7, L. monocytogenes, S. aureus, and S.
Fig. 3. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on S. aureus in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
Fig. 4. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on S. Typhimurium in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
Typhimurium by 2.59, 2.69, 2.38, and 2.99 log CFU/g, respectively (Figs. 1e4). The enhanced bactericidal effect of SAcEW þ 0.5% FA might occur from the synergistic physicochemical properties of both sanitizers. When 0.5% FA was added into SAcEW, the pH and
Fig. 5. Effect of various treatments with mild heat (40 C) for 3 min on E. coli O157:H7 counts in pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
A.R. Mansur et al. / Food Control 47 (2015) 277e284
281
Fig. 6. Effect of various treatments with mild heat (40 C) for 3 min on S. Typhimurium counts in pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
Fig. 7. Effect of various treatments with mild heat (40 C) for 3 min on total bacterial counts in pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
ACC values of SAcEW were decreased, while its ORP value was increased. Thus, SAcEW þ 0.5% FA treatment contained similar values of pH and ORP with those of StAEW treatment, higher value of ORP compared to those of its individual treatments, and lower ACC value compared to those of EW treatments (Table 1). The synergistic effects of chlorine compound, pH, ACC, and undissociated molecules of SAcEW þ 0.5% FA were likely responsible for its enhanced bactericidal efficacy on the reduction of pathogens on fresh pork. However, the effectiveness of using SAcEW þ 0.5% FA was decreased significantly (P < 0.05) when it was applied at 25 C (Figs. 1e4). It means that the mild heat treatment also enhanced the efficacy of the combined treatment as it did to each individual treatment. In addition, the reduced chlorine concentration of the combined treatment might benefit to its applicability to meat industries in terms of safety. In the United States, the use of chlorine at the concentration of 20 mg/L has been approved in poultry washes/sprays (Byelashov & Sofos, 2009). The reductions of pathogens obtained from the application of SAcEW þ 0.5% FA 40 C were similar to those of applying 5% potassium sorbate or combined salts (5% sodium chloride, sodium acetate, sodium citrate, sodium lactate, and potassium sorbate each at 2.5%) for 2e4 min, which reduced E. coli, L. monocytogenes, S. aureus, and S. Typhimurium on fresh pork by 3.29, 2.88, 3.29, and 2.95 log CFU/g, respectively (Latha et al., 2009). Moreover, Rahman et al. (2013) also reported the reductions of E. coli O157:H7 and L. monocytogenes around 3 log CFU/g on fresh pork treated with
SAcEW (10 mg/L) þ 3% calcium lactate (CaL) at room temperature for 5 min. In addition, the effectiveness of using EW in combination with other antimicrobial treatments for reducing Campylobacter coli, Campylobacter jejuni, E. coli, L. monocytogenes, and S. Typhimurium on pork and poultry surfaces have been reported. The reductions of these pathogens were also ranging from 2 to 3 log CFU/g (Fabrizio & Cutter, 2004, 2005; Fabrizio, Sharma, Demirci, & Cutter, 2002; Kim, Hung, & Russell, 2005; Park, Hung, & Brackett, 2002).
3.2. Effects of decontamination with mild heat (40 C) on bacterial growth during storage There has been concern that decontamination with organic acids might result in the emergence of pathogens that have become acid tolerant. Generally, mesophilic enterobacteria (E. coli O157:H7 and S. Typhimurium) are among the most resistant to organic acids in comparison to other pathogens, which able to grow on treated meat during storage (Smulders & Greer, 1998). Therefore, we employed these pathogens in the storage test. To investigate the bacterial growth and shelf life of pork during storage test, the untreated and treated (SAcEW 40 C, StAEW 40 C, 0.5% FA 40 C, and SAcEW þ 0.5% FA 40 C) pork samples were stored at 4 and 10 C. The untreated and treated pork samples at each interval were then independently analyzed for bacterial growth (E. coli O157:H7, S. Typhimurium, and TBC), pH, and sensory quality.
282
A.R. Mansur et al. / Food Control 47 (2015) 277e284
Table 2 Specific growth rate (SGR) and lag time (LT) of pathogens and naturally occurring bacteria on treated pork. Treatments
Controlf SAcEW 40 C StAEW 40 C 0.5% FA 40 C SAcEW þ0.5% FA 40 C
Storage temperature ( C)
Specific growth rate (log CFU/day)a,b
Lag time (day)a,b
E. coli O157:H7c
S. Typhimuriumd
TBCe
E. coli O157:H7c
S. Typhimuriumd
TBCe
4 10 4 10 4 10 4 10 4 10
0.491Aa 0.860Abc 0.509Aab 1.021Ac 0.419Aa 0.977Ac 0.436Aa 0.921Ac 0.524Aab 1.132Ac
0.544Aa 1.263Ab 0.553Aa 1.171Ab 0.619Aa 1.167Ab 0.621Aa 1.141Ab 0.565Aa 1.160Ab
0.320Aa 1.009Abcd 0.693Aabcd 1.267Ad 0.639Aabc 1.180Acd 0.731Aabcd 1.122Abcd 0.554Aab 1.101Abcd
3.746Cf 1.489Bc 2.381Bd 0.563Aab 2.505Bde 0.666Aab 2.249Bd 0.446Aa 2.976Ae 1.069Abc
3.116Bd 1.699Babc 2.761Bd 1.979Cbc 2.789Bd 1.499Bab 2.150Bc 1.616Bab 2.840Ad 1.448ABa
1.993Ac 0.919Aa 1.786Abc 1.402Bab 1.613Abc 1.389Bab 1.560Abc 1.208Bab 2.692Ad 1.590Bbc
a
Within the same column, values not followed by the same small letter are significantly different (P < 0.05). Within the same row, values not followed by the same capital letter are significantly different (P < 0.05). c 2 R > 0.96. d 2 R > 0.95. e 2 R > 0.88. f Untreated sample. b
The growth curves of E. coli O157:H7, S. Typhimurium, and TB counts on each pork sample during storage at 4 and 10 C are shown in Figs. 5e7, respectively, while the bacterial growth parameters (SGR and LT) in each pork sample are described in Table 2. The results showed that the lower storage temperature (4 C) yielded in lower SGRs and longer LTs of pathogens. However, the SGRs and LTs of naturally occurring bacteria (TBC) were likely not affected by storage temperature; in other words, the naturally occurring bacteria could grow well on pork stored either at 4 or 10 C. Nychas et al. (2008) stated that fresh meat such as pork can harbor high numbers of aerobic mesophilic and psychrotrophic bacteria, which are the major spoilage agents especially during cold storage. Most of which is Pseudomonas spp. and Enterobacteriaceae. Most of the SGRs and LTs of all bacteria in treated pork samples were not significantly different compared to those in control samples. However, the SAcEW þ 0.5% FA 40 C treatment significantly (P < 0.05) yielded in a longer lag time of naturally occurring bacteria (TBC) on pork stored at 4 C compared to other pork samples. Thus, the longer lag time of TBC on treated (SAcEW þ 0.5% FA 40 C) pork might affect to its shelf life extension during storage.
the shelf life of pork from 5 to 6 days compared to those of untreated pork. This finding was similar with a previous study that reported the application of combined treatment (SAcEW þ 3% CaL) for 5 min prolongs the shelf life of fresh pork up to 6 days at 4 C (Rahman et al., 2013). Latha et al. (2009) also observed the
3.3. Shelf life based on microbial and sensory quality The initial populations of TBC on pork samples treated with the individual treatments (SAcEW, StAEW, and 0.5% FA) and a combined treatment (SAcEW þ 0.5%) at 40 C for 3 min decreased approximately by 2.28, 2.35, 2.41, and 2.75 log CFU/g, respectively (Fig. 7). The results were in agreement with the findings of Latha et al. (2009) and Rahman et al. (2013), which are approximately 2.2e2.3 log CFU/g. The end of shelf life was considered to occur when the TBC reached 7 log CFU/g (Nychas et al., 2008). The TBC in untreated pork reached unacceptable levels within 4 (7.07 ± 0.33 log CFU/g) and 2 days (7.62 ± 0.13 log CFU/g) of storage at 4 and 10 C, respectively. The spoilage of treated (SAcEW, StAEW, 0.5% FA 40 C) pork stored at 4 and 10 C were evidenced within 6 and 4 days, respectively, with corresponding increases ranging from 6.99 to 7.34 log CFU/g. The combined treatment (SAcEW þ 0.5% FA 40 C) more effectively controlled TBC during storage at 4 and 10 C for 10 and 7 days, respectively, when compared to those of the untreated and the individually treated samples. The TBC on treated (SAcEW þ 0.5% FA 40 C) pork stored at 4 and 10 C increased up to 6.77 ± 0.23 and 6.98 ± 0.19 log CFU/g after 10 and 7 days, respectively. Thus, the SAcEW þ 0.5% FA 40 C treatment could prolong
Fig. 8. Effect of various treatments with mild heat (40 C) for 3 min on sensory quality of pork stored at 4 and 10 C. Values shown are means ± standard error.
A.R. Mansur et al. / Food Control 47 (2015) 277e284
enhanced shelf life of 4e5 days for fresh pork treated with salt or salt combinations and stored at refrigeration temperature (5e7 C). The SI of sensory properties (color, odor, and texture) of all pork samples decreased as the storage time increased, and the higher storage temperature resulted in a faster decrease of sensory acceptance (Fig. 8). The spoilage of the control pork at 4 and 10 C was evident on days 2 and 1, respectively, whereas the treated (SAcEW, StAEW, and 0.5% FA 40 C) pork stored at 4 and 10 C were spoiled on days 4 and 2, respectively. The more prolonged sensory quality was observed on days 8 and 5 in treated (SAcEW and 0.5% FA 40 C) pork stored at 4 and 10 C, respectively. Similar extended shelf life also has been reported by Latha et al. (2009) and Rahman et al. (2013) in their previous studies. 3.4. Changes in pH during storage The initial pH values of all pork samples were slightly acidic (5.20e6.16), except that of treated (StAEW) pork, which decreased into 4.88 ± 0.18. However, the pH of all pork samples increased gradually over time and then became slightly acidic or close to neutral. The values of final pH of all pork samples after storage at 4 and 10 C were 5.89e6.48 and 6.20e6.73, respectively (Fig. 9). The results were similar to those reported by Tan and Shelef (2002) and Rahman et al. (2013). Gill (1983) stated that an augmented pH is typical for fresh meat during storage, caused by Gram-negative bacteria. These bacteria break down the protein compounds of meat and then release basic compounds such as amines. The
Fig. 9. Changes in pH of untreated and treated pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
283
current study also revealed that the pH value increased as TBC increased. Rousset and Renerre (1991) and Rahman et al. (2013) reported that TBC were 10e100-fold greater on high pH meat than those on normal pH meat at various aging durations. Moreover, Holmer et al. (2009) also reported that higher pH and a longer aging period enhance the microbial proliferation and decrease the shelf life. Based on the results of the present study, the decontamination of fresh pork by SAcEW þ 0.5% FA at 40 C for 3 min was capable to reduce bacterial contaminations and prolong the shelf life of pork during storage at 4 and 10 C. However, the microscopic tests and mathematical modeling might be needed in the future study to provide more fundamental understanding of the interaction of the treatments with bacterial cells and the mode of action of the treatment. Also, a large number of experiments are much needed to ascertain the effects of SAcEW þ 0.5% FA in a commercial slaughter plant or other meat industries. Acknowledgments This work was supported by grant no. 13072-957 from the Ministry of Food and Drug Safety (MFDS) of Republic of Korea. References Baer, A. A., Miller, M. J., & Dilger, A. C. (2013). Pathogens of interest to the pork Industry: a review of research on interventions to assure food safety. Comprehensive Reviews in Food Science and Food Safety, 12, 183e217. Baird Parker, A. C. (1980). Organic acids. In J. H. Silliker, R. P. Elliott, A. C. Baird Parker, S. L. Bryan, J. H. B. Christian, D. S. Clarke, et al. (Eds.), International Commission on Microbiological Specifications for Foods: Vol. I. Microbial ecology of foods (pp. 126e135). New York: Academic Press. Booth, I. R. (1985). Regulation of cytoplasmic pH in bacteria. Microbiological Reviews, 49, 359e378. Byelashov, O. A., & Sofos, J. N. (2009). Strategies for on-line decontamination of carcasses. In F. Toldr0 a (Ed.), Safety of meat and processed meat (pp. 164e165). New York: Springer. CDC. (2011). CDC estimates of foodborne illness in the United States. Center for Disease Control. Available from http://www.cdc.gov/foodborneburden/2011-foodborneestimates.html Accessed 15.03.14. Chen, J. H., Ren, Y., Seow, J., Liu, T., Bang, W. S., & Yuk, H. G. (2012). Intervention technologies for ensuring microbiological safety of meat: current and future trends. Comprehensive Reviews in Food Science and Food Safety, 11, 119e132. Ding, T., Rahman, S. M. E., Purev, U., & Oh, D. H. (2010). Modelling of Escherichia coli O157:H7 growth at various storage temperatures on beef treated with electrolyzed oxidizing water. Journal of Food Engineering, 97, 497e503. Fabrizio, K. A., & Cutter, C. N. (2004). Comparison of electrolyzed oxidizing water with other antimicrobial interventions to reduce pathogens on fresh pork. Meat Science, 68, 463e468. Fabrizio, K. A., & Cutter, C. N. (2005). Application of electrolyzed oxidizing water to reduce Listeria monocytogenes on ready-to-eat meats. Meat Science, 71, 327e333. Fabrizio, K. A., Sharma, R. R., Demirci, A., & Cutter, C. N. (2002). Comparison of electrolyzed oxidizing water with various antimicrobial interventions to reduce Salmonella on poultry. Poultry Science, 66(10), 1379e1384. Gill, C. O. (1983). Meat spoilage and evaluation of the potential storage life of fresh meat. Journal of Food Protection, 46, 444e448. Holmer, S. F., McKeith, R. O., Boler, D. D., Dilger, A. C., Eggert, J. M., Petry, D. B., et al. (2009). The effect of pH on shelf-life of pork during aging and simulated retail display. Meat Science, 82, 86e93. Huang, Y. R., Hung, Y. C., Hsu, S. Y., Huang, Y. W., & Hwang, D. F. (2008). Application of electrolyzed water in the food industry. Food Control, 19, 329e345. Kim, C., Hung, Y. C., & Russell, S. M. (2005). Efficacy of electrolyzed water in the prevention and removal of fecal material attachment and its microbicidal effectiveness during simulated industrial poultry processing. Poultry Science, 84, 1778e1784. Kreyenschmidt, J., Hubner, A., Beierle, E., Chonsch, L., Scherer, A., & Petersen, B. (2010). Determination of the shelf life of sliced cooked ham based on the growth of lactic acid bacteria in different steps of the chain. Journal of Applied Microbiology, 108, 510e520. Latha, C., Sherikar, A. T., Waskar, V. S., Dubal, Z. B., & Ahmed, S. N. (2009). Sanitizing effect of salts on experimentally inoculated organisms on pork carcasses. Meat Science, 83, 796e799. Mataragas, M., Skandamis, P. N., & Drosinos, E. H. (2008). Risk profiles of pork and poultry meat and risk ratings of various pathogen/product combinations e a review. International Journal of Food Microbiology, 126, 1e12.
284
A.R. Mansur et al. / Food Control 47 (2015) 277e284
Nychas, G. J. E., Marshall, D. L., & Sofos, J. N. (2007). Meat, poultry and seafood. In M. P. Doyle, L. R. Beuchat, & T. J. Montville (Eds.), Food microbiology: Fundamentals and frontiers (3rd ed.) (pp. 105e140). Washington, USA: ASM Press. Nychas, G. J. E., Skandamis, P. N., Tassou, C. C., & Koutsoumanis, K. P. (2008). Meat spoilage during distribution. Meat Science, 78, 77e89. Park, H., Hung, Y. C., & Brackett, R. E. (2002). Antimicrobial effect of electrolyzed water for inactivating Campylobacter jejuni during poultry washing. International Journal of Food Microbiology, 72, 77e83. Podolak, R. K., Zayas, J. F., Kastner, C. L., & Fung, D. Y. C. (1995). Reduction of Listeria monocytogenes, Escherichia coli 0157:H7 and Salmonella typhimurium during storage on beef sanitized with fumaric, acetic and lactic acids. Journal of Food Safety, 15, 283e290. Podolak, R. K., Zayas, J. F., Kastner, C. L., & Fung, D. Y. C. (1996). Reduction of bacterial populations on vaccum-packaged ground beef patties with fumaric and lactic acids. Journal of Food Protection, 59, 1037e1040. Rahman, S. M. E., Wang, J., & Oh, D. H. (2013). Synergistic effect of low concentration electrolyzed water and calcium lactate to ensure microbial safety, shelf life and sensory quality of fresh pork. Food Control, 30, 176e183.
Rousset, S., & Renerre, M. (1991). Effect of CO2 or vacuum packaging on normal and high pH meat shelf-life. International Journal of Food Science and Technology, 26(6), 641e652. Scharff, R. L. (2010). Health-related costs from foodborne illness in the United States. Produce Safety Project at Georgetown Univ. Available from http://www. producesafetyproject.org/admin/assets/files/Health-Related-Foodborne-IllnessCosts-Report.pdf-1.pdf. Georgetown Univ. Accessed 17.03.14. Smulders, F. J. M., & Greer, G. G. (1998). Integrating microbial decontamination with organic acids in HACCP programmes for muscle foods: prospects and controversies. International Journal of Food Microbiology, 44, 149e169. Tan, W., & Shelef, L. A. (2002). Effects of sodium chloride and lactates on chemical and microbiological changes in refrigerated and frozen fresh ground pork. Meat Science, 62, 27e32. Zwietering, M. H., Jongenburger, I., Rombouts, F. M., & van't Riet, K. (1990). Modeling of the bacterial growth curve. Applied and Environmental Microbiology, 56, 1875e1881.
Contents lists available at ScienceDirect
Food Control journal homepage: www.elsevier.com/locate/foodcont
Combined effects of slightly acidic electrolyzed water and fumaric acid on the reduction of foodborne pathogens and shelf life extension of fresh pork Ahmad Rois Mansur, Charles Nkufi Tango, Gwang-Hee Kim, Deog-Hwan Oh* Department of Food Science and Biotechnology, School of Bioconvergence Science and Technology, Kangwon National University, Chuncheon, Gangwon 200-701, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 April 2014 Received in revised form 3 July 2014 Accepted 8 July 2014 Available online 15 July 2014
This study evaluated the efficacy of the individual treatments (slightly acidic electrolyzed water [SAcEW] or fumaric acid [FA]) and their combination to reduce Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella Typhimurium in fresh pork as well as to study the shelf life and sensory quality (color, odor, and texture) of pork during storage at 4 and 10 C. The inoculated pork samples (10 g) were dipped for 3 min in each treatment (tap water [TW], SAcEW, strong acidic electrolyzed water [StAEW], 0.5% FA, or SAcEW þ 0.5% FA) with or without mild heat (40 C). Decontamination of fresh pork with SAcEW þ0.5% FA at 40 C for 3 min showed greater bactericidal effect compared to other treatments, which significantly (P < 0.05) reduced E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium by 2.59, 2.69, 2.38, and 2.99 log CFU/g, respectively. This combined treatment significantly (P < 0.05) yielded in a longer lag time of naturally occurring bacteria (TBC) on pork stored at 4 C. This combined treatment also prolonged the shelf life of pork up to 6 days and 4e5 days when stored at 4 C and 10 C, respectively, compared to those of the untreated pork. The results suggest that the combined treatment of SAcEW þ 0.5% FA has potential as a novel method to enhance the microbial safety and quality of fresh pork. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Pork Slightly acidic electrolyzed water Fumaric acid Pathogens reduction Shelf life
1. Introduction Pork is the most consumed meat in the world, but pathogen contaminations such as Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella Typhimurium have impacted its safety and quality (Baer, Miller, & Dilger, 2013; Mataragas, Skandamis, & Drosinos, 2008). Salmonella spp., L. monocytogenes, and S. aureus are among the top pathogens associated with pork and pork products, which cause foodborne illnesses and deaths annually. The Centers for Disease Control and Prevention (CDC) reported that these pathogens have caused 47.8 million illnesses and 3000 deaths each year (CDC, 2011). Moreover, product recalls, decreased sales from damaged organizational reputation, and spoilage of meat can lead to food waste and economic losses as well as the loss of consumer confidence (Nychas, Marshall, & Sofos, 2007; Scharff, 2010). Therefore, to improve the microbial safety and
* Corresponding author. Tel./fax: þ82 33 250 6457. E-mail address: [email protected] (D.-H. Oh). http://dx.doi.org/10.1016/j.foodcont.2014.07.019 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
quality of pork during processing and storage, various chemical sanitizers have been investigated to reduce its microbial contaminations and extend shelf life. In recent years, antimicrobial agents from chemicals such as chlorinated solutions, electrolyzed water (EW), organic acids, and salts, alone or in combination have been investigated and shown to reduce microbial contaminations of pork (Chen et al., 2012; Smulders & Greer, 1998). Slightly acidic electrolyzed water (SAcEW) is a type of EW and promising sanitizer for food products (Huang, Hung, Hsu, Huang, & Hwang, 2008). However, the use of SAcEW as an individual treatment has not effectively reduced bacterial contaminations in fresh meat (Ding, Rahman, Purev, & Oh, 2010; Rahman, Wang, & Oh, 2013). Therefore, it has frequently been combined with other chemical sanitizers to enhance its bactericidal efficacy. Organic acids are GRAS (Generally Recognized as Safe) compounds, which have ability to inactivate foodborne pathogens (Chen et al., 2012). Among the organic acids used for antimicrobial agent on meat, fumaric acid (FA) has shown stronger bactericidal effect compared to acetic and lactic acid (Podolak, Zayas, Kastner, & Fung, 1995, 1996). Even though the SAcEW and FA seem to be promising sanitizers for meat
278
A.R. Mansur et al. / Food Control 47 (2015) 277e284
and meat products, their application on meat and meat products remains limited. Moreover, there is no research on the application of the combined treatment (SAcEW and FA) for decontamination and extending the shelf life of meat and meat products published to date. Therefore, the current study was conducted to evaluate the efficacy of the individual treatments (SAcEW or FA) and their combination on reducing E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium in fresh pork. The shelf life and sensory quality of pork during storage at 4 and 10 C were also studied. 2. Materials and methods 2.1. Preparation of bacterial cultures The two strains of E. coli O157:H7 (ATCC 43894 and ATCC 43895), L. monocytogenes (ATCC 19114 and ATCC 19115), S. aureus (ATCC 12598 and ATCC 14458), and S. Typhimurium (ATCC 13311 and ATCC 14028) were obtained from the Department of Food Science, University of Georgia (Griffin, GA, USA), the Korean National Institute of Health (Seoul, Korea), and the Health Research Department (Gyeonggi-do, Republic of Korea), respectively. Prior to use, each strain of pathogens was separately grown in tryptic soy broth (TSB; Difco, Becton, Dickinson and Company Sparks, MD, USA) at 37 C with two consecutive transfers after a 24 h period for a total 48 h of incubation. All working cultures grown in TSB were separately centrifuged at 4000 g for 10 min at 4 C, and the supernatants were discarded. The cell pellets were washed twice with 0.1% sterile buffered peptone water (BPW; Difco, Becton, Dickinson and Company Sparks, MD, USA) pH 7.1, and resuspended in 10 mL of the same solution to obtain a final cell concentration of approximately 8 log CFU/mL. The two strains each of E. coli O157:H7, L. monocytogenes, S. aureus, or S. Typhimurium was combined in a cocktail with approximately equal numbers in the final population (8 log CFU/ mL). The bacterial population in each culture cocktail was confirmed by plating 0.1 mL portions of appropriately diluted culture on tryptic soy agar (TSA; Difco, Becton, Dickinson and Company Sparks, MD, USA) plates, and then incubating the plates at 37 C for 24 h. The prepared culture cocktails were then used in subsequent experiments. 2.2. Preparation of pork samples Boneless pork loin was transported from a local slaughterhouse (Kangwon LPC, Wonju, Kangwon, Korea) to the laboratory under temperature-controlled conditions in a sterile plastic container. All pork samples were then separately cut into pieces using a sterile knife. Aliquots with a weight of 10 ± 0.3 g were used for the decontamination and storage tests, and those of 25 ± 0.3 g were used for the sensory analysis and measurement of the pH value. 2.3. Inoculation of pathogens to pork samples Pork samples were placed on sterile petri dishes in a laminar flow hood, then spot inoculated by pipetting 0.1 mL of each culture cocktail (approximately 8 log CFU/mL) onto the surface to obtain an initial level of approximately 6 log CFU/g. The inoculated pork samples were air-dried in a laminar flow hood for 1 h at room temperature to allow attachment of bacteria, and then immediately exposed to the treatments. 2.4. Preparation of sanitizing solutions The commercially available slightly acidic electrolyzed water (SAcEW; 30 mg/L) and strong acidic electrolyzed water (StAEW;
30 mg/L) used in this study were obtained from the Korea Advanced Institute of Science and Technology (KAIST, Daejeon, Korea). SAcEW and StAEW were produced by electrolysis of 6% and 9% HCl solutions, respectively, using an electrolysis device. The crystalline fumaric acid (FA) (Daejung Chemicals and Metals Co., Siheung, Gyeonggi, Korea) was dissolved in 1 L of deionized water (DW) to give a final concentration of 0.5% FA solutions (w/v). The pH, oxidation reduction potential (ORP), and available chlorine concentration (ACC) of the sanitizers were measured with a dualscale pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ, USA) bearing pH and ORP electrodes. The ACC was determined by a colorimetric method using a digital chlorine test kit (RC-3F, Kasahara Chemical Instruments Corp., Saitama, Japan). The detection limit was 1e300 mg/L. The physicochemical properties of the tested sanitizing solutions are summarized in Table 1. 2.5. Decontamination treatment and microbiological analysis The preliminary in vitro experiments were carried out to determine the optimal condition for each individual treatment of SAcEW, StAEW, and organic acids (lactic acid [LA], citric acid [CA], acetic acid [AA], and fumaric acid [FA]). The experiments were conducted at different temperatures (25, 30, 40, 50, and 60 C), different ACCs for EW (5, 10, 20, and 30 mg/L), different concentrations for organic acids (0.125, 0.25, and 0.5%), and different exposure times (1, 3, and 5 min). The results suggested that dipping with EW (30 mg/L) or 0.5% FA at 40 C for 3 min was the most satisfactory method (data not shown). These methods were then employed in the in vivo study. For the in vivo study, inoculated pork samples (10 g) were placed in a sterile container and dipped for 3 min in each treatment (tap water [TW], SAcEW, StAEW, 0.5% FA, SAcEW þ 0.5% FA) with or without mild heat (40 C). Unwashed pork samples were used as control. The excess solution was removed from pork samples with sterile paper towels. Each 10 g pork sample was then mixed with 90 mL of 0.1% sterile BPW and homogenized for 2 min in a Seward stomacher (400 Circulator, Seward, London, UK). After homogenization, 1 mL aliquot of each sample was serially diluted in 9 mL of 0.1% sterile BPW, and 0.1 mL of diluents was spread-plated onto sorbitol MacConkey's agar (Difco, Becton, Dickinson and Company Sparks, MD, USA), modified Oxford agar base with the addition of the Oxford antimicrobic supplement (Oxoid, Basingstoke, UK), Baird Parker agar (Difco, Becton, Dickinson and Company Sparks, MD, USA) supplemented with 50 mL of the egg yolk Tellurite, and Brilliant Green agar (Oxoid, Basingstoke, UK) to enumerate E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium,
Table 1 Physicochemical properties of the tested sanitizing solutions.a Tested solutions TW SAcEWd StAEWe 0.5% FAf SAcEW þ 0.5% FAg
ORP (mV)b
pH 7.17 6.29 2.31 2.34 2.95
± ± ± ± ±
A
0.12 0.17B 0.08C 0.11C 0.13C
358 826 1159 570 1091
± ± ± ± ±
A
10 16C 12D 9B 19D
ACC (mg/L)c NDh 30 ± 0.2 30 ± 0.6 ND 15 ± 0.09
AD Numbers within each column followed by different capital letters are significantly different (P < 0.05). a Values are mean ± standard deviation, n ¼ 6. b Oxidation reduction potential. c Available chlorine concentration. d Slightly acidic electrolyzed water. e Strong acidic electrolyzed water. f 0.5% fumaric acid. g Slightly acidic electrolyzed water (30 mg/L) þ 0.5% fumaric acid. h Not detected.
A.R. Mansur et al. / Food Control 47 (2015) 277e284
respectively. All plates were then incubated at 37 C for 24e48 h. Each independent trial was in triplicates.
279
Sciences, Chicago, IL, USA). The analysis included treatment, storage temperature, and bacteria as fixed effects. Differences between effects were assessed by the Tukey test (P < 0.05).
2.6. Storage tests 3. Results and discussion The inoculated (E. coli O157:H7 and S. Typhimurium) and uninoculated pork samples were divided into four different groups and a control (untreated pork samples). Samples in group 1 to 3 were only dipped for 3 min in 100 mL SAcEW, StAEW, or 0.5% FA at 40 C, while samples in group 4 were dipped for 3 min in a combined treatment (SAcEW þ 0.5% FA) at 40 C. Each treated sample was then mixed with 100 mL of neutralizing solution (0.85% NaCl containing 0.5% Na2S2O3) for 3 min to remove residual solutions and stop their bactericidal effects. The excess solution was then removed from pork samples with sterile paper towels. All pork samples (treated and untreated) were then airpackaged using a new stomacher bag (Nasco Whirl-Pak, Janesville, WI, USA) and marked carefully before storage at 4 and 10 C. Each sample was analyzed on days 0, 2, 4, 6, 8, and 10 at 4 C, while on days 0, 1, 2, 3, 5, and 7 at 10 C. At each sampling interval, 90 mL of 0.1% sterile BPW was poured into the stomacher bag containing the samples. After 2 min of homogenization, 0.1 mL aliquots of the appropriate dilution were plated onto plate count agar (Difco, Becton, Dickinson and Company Sparks, MD, USA) to enumerate total bacterial counts (TBC). All plates were incubated at 37 C for 24 h. The end of shelf life was considered to occur when TBC was 7 log CFU/g (Nychas, Skandamis, Tassou, & Koutsoumanis, 2008) or when the sensory score of the pork samples reached an unacceptable level. Each storage test consisted of two trials with triplicates of samples per trial and three plates per replicate at each time interval. 2.7. Sensory analysis and measurement of the pH value Sensory evaluation of pork samples (25 g each) were carried out by a four member sensory panel (each panel consisted of eight panelists) (Latha, Sherikar, Waskar, Dubal, & Ahmed, 2009; Rahman et al., 2013). Each sample was evaluated on days 0, 2, 4, 6, 8, and 10 at 4 C and on days 0, 1, 2, 3, 5, and 7 at 10 C for sensorial items (color, odor, and texture) using a simple four-point scoring system (1e4); 1 is the highest score and 4 is the lowest score. A sensory index (SI) was calculated using Eq. (1) (Kreyenschmidt et al., 2010).
SI ¼
2*C þ 1*ðO þ TÞ 4
3.1. Decontamination of fresh pork Initial populations of E. coli O157:H7, L. monocytogenes, S. aureus, and S. Typhimurium on pork used for evaluating the efficacy of sanitizing treatments either alone or in combination were approximately 6.27, 5.95, 6.16, and 6.12 log CFU/g, respectively. The individual treatments (SAcEW [30 mg/L], StAEW [30 mg/L], and 0.5% FA) and a combined treatment (SAcEW þ 0.5% FA) with mild heat (40 C) for 3 min significantly (P < 0.05) reduced pathogens compared to the treatments applied at 25 C and control. After 3 min dipping at 40 C, the individual treatments yielded pathogens reductions ranging from 0.23e0.33, 1.19e1.55, 1.20e1.43, and 1.17e1.44 log CFU/g for the TW, SAcEW, StAEW, and 0.5% FA treatment, respectively (Figs. 1e4). The use of SAcEW treatment showed no significant (P > 0.05) difference in bactericidal effects compared to StAEW treatment, even though the pH and ORP values of SAcEW were significantly (P < 0.05) different compared to those of StAEW (Table 1). The results revealed that the bactericidal effects of EW appeared to be due more to the available chlorine concentration (ACC) than to other factors such as low pH and high ORP value. Huang et al. (2008) stated that the chlorine compounds can kill the microbial cell through inhibiting glucose oxidation by chlorine-oxidizing sulfhydryl groups of certain enzymes used in carbohydrate metabolism, disrupting protein synthesis, inhibiting oxygen uptake and oxidative phosphorylation coupled with leakage of some macromolecules, and forming toxic N-chlorine derivatives of cytosine. The bactericidal effects of 0.5% FA were also not significantly (P > 0.05) different compared to those of SAcEW and StAEW. According to Baird Parker (1980), the undissociated molecules of organic acids are likely responsible for their antimicrobial properties. Booth (1985) reported that the accumulation of undissociated molecules of weak acids in the cytoplasm of the cell leads to the dissociation of protonated acid, the release of a proton,
(1)
where C is color, O is odor, and T is texture. The cut-off score was fixed at an SI of 2.2. The pork samples (in triplicates in each sample) were then homogenized in a Seward stomacher (400 Circulator, Seward, London, UK) with 125 mL of DW, and filtered. The pH of the filtrate was measured with a dual-scale pH meter (Accumet model 15, Fisher Scientific Co., Fair Lawn, NJ, USA). 2.8. Curve fitting and statistical analyses The values of all experimental data (in triplicates) represent the mean of triplicate determinations for each sample. The modified Gompertz model (Zwietering, Jongenburger, Rombouts, & van’t Riet, 1990) was used to describe the bacterial growth curves at 4 and 10 C, and growth parameters (specific growth rate [SGR]: log CFU/day; lag time [LT]: day) were analyzed by the GraphPad prism software (version 5.01, GraphPad Software, Inc., San Diego, CA). The data (means ± standard deviation) were analyzed using One-way ANOVA in the SPSS ver. 13.0 (Statistical Package for the Social
Fig. 1. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on E. coli O157:H7 in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
280
A.R. Mansur et al. / Food Control 47 (2015) 277e284
Fig. 2. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on L. monocytogenes in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
and the acidification of the cytoplasm of the microorganism, if intracellular pH is higher than the pKa of the acid. Several previous studies have examined the effectiveness of using individual treatments of SAcEW (10 mg/L, dipping at room temperature for 5 min), StAEW (50 mg/L, dipping at room temperature for 5 min or spraying for 15 s), and FA (1e1.5%, dipping at 55 C for 15e30 s) for reducing E. coli, E. coli O157:H7, L. monocytogenes, and S. Typhimurium on fresh pork surfaces. The reductions of these pathogens were also less than 2 log CFU/g (Fabrizio & Cutter, 2004; Podolak et al., 1995, 1996; Rahman et al., 2013). The application of combined treatment with mild heat (SAcEW þ 0.5% FA 40 C) yielded in greater reductions of pathogens compared to all individual treatments and control (P < 0.05), which reduced E. coli O157:H7, L. monocytogenes, S. aureus, and S.
Fig. 3. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on S. aureus in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
Fig. 4. Inactivation effect of various treatments with or without mild heat (40 C) for 3 min on S. Typhimurium in fresh pork. Vertical bars represent means ± standard deviation of triplicate assays. Bars labeled with different letters indicate a significant difference (P < 0.05). (TW: Tap water; SAcEW: slightly acidic electrolyzed water (30 mg/L); StAEW: strong acidic electrolyzed water (30 mg/L); 0.5% FA: 0.5% fumaric acid; SAcEW þ 0.5% FA: A mixed solution of SAcEW (30 mg/L) plus 0.5% fumaric acid).
Typhimurium by 2.59, 2.69, 2.38, and 2.99 log CFU/g, respectively (Figs. 1e4). The enhanced bactericidal effect of SAcEW þ 0.5% FA might occur from the synergistic physicochemical properties of both sanitizers. When 0.5% FA was added into SAcEW, the pH and
Fig. 5. Effect of various treatments with mild heat (40 C) for 3 min on E. coli O157:H7 counts in pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
A.R. Mansur et al. / Food Control 47 (2015) 277e284
281
Fig. 6. Effect of various treatments with mild heat (40 C) for 3 min on S. Typhimurium counts in pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
Fig. 7. Effect of various treatments with mild heat (40 C) for 3 min on total bacterial counts in pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
ACC values of SAcEW were decreased, while its ORP value was increased. Thus, SAcEW þ 0.5% FA treatment contained similar values of pH and ORP with those of StAEW treatment, higher value of ORP compared to those of its individual treatments, and lower ACC value compared to those of EW treatments (Table 1). The synergistic effects of chlorine compound, pH, ACC, and undissociated molecules of SAcEW þ 0.5% FA were likely responsible for its enhanced bactericidal efficacy on the reduction of pathogens on fresh pork. However, the effectiveness of using SAcEW þ 0.5% FA was decreased significantly (P < 0.05) when it was applied at 25 C (Figs. 1e4). It means that the mild heat treatment also enhanced the efficacy of the combined treatment as it did to each individual treatment. In addition, the reduced chlorine concentration of the combined treatment might benefit to its applicability to meat industries in terms of safety. In the United States, the use of chlorine at the concentration of 20 mg/L has been approved in poultry washes/sprays (Byelashov & Sofos, 2009). The reductions of pathogens obtained from the application of SAcEW þ 0.5% FA 40 C were similar to those of applying 5% potassium sorbate or combined salts (5% sodium chloride, sodium acetate, sodium citrate, sodium lactate, and potassium sorbate each at 2.5%) for 2e4 min, which reduced E. coli, L. monocytogenes, S. aureus, and S. Typhimurium on fresh pork by 3.29, 2.88, 3.29, and 2.95 log CFU/g, respectively (Latha et al., 2009). Moreover, Rahman et al. (2013) also reported the reductions of E. coli O157:H7 and L. monocytogenes around 3 log CFU/g on fresh pork treated with
SAcEW (10 mg/L) þ 3% calcium lactate (CaL) at room temperature for 5 min. In addition, the effectiveness of using EW in combination with other antimicrobial treatments for reducing Campylobacter coli, Campylobacter jejuni, E. coli, L. monocytogenes, and S. Typhimurium on pork and poultry surfaces have been reported. The reductions of these pathogens were also ranging from 2 to 3 log CFU/g (Fabrizio & Cutter, 2004, 2005; Fabrizio, Sharma, Demirci, & Cutter, 2002; Kim, Hung, & Russell, 2005; Park, Hung, & Brackett, 2002).
3.2. Effects of decontamination with mild heat (40 C) on bacterial growth during storage There has been concern that decontamination with organic acids might result in the emergence of pathogens that have become acid tolerant. Generally, mesophilic enterobacteria (E. coli O157:H7 and S. Typhimurium) are among the most resistant to organic acids in comparison to other pathogens, which able to grow on treated meat during storage (Smulders & Greer, 1998). Therefore, we employed these pathogens in the storage test. To investigate the bacterial growth and shelf life of pork during storage test, the untreated and treated (SAcEW 40 C, StAEW 40 C, 0.5% FA 40 C, and SAcEW þ 0.5% FA 40 C) pork samples were stored at 4 and 10 C. The untreated and treated pork samples at each interval were then independently analyzed for bacterial growth (E. coli O157:H7, S. Typhimurium, and TBC), pH, and sensory quality.
282
A.R. Mansur et al. / Food Control 47 (2015) 277e284
Table 2 Specific growth rate (SGR) and lag time (LT) of pathogens and naturally occurring bacteria on treated pork. Treatments
Controlf SAcEW 40 C StAEW 40 C 0.5% FA 40 C SAcEW þ0.5% FA 40 C
Storage temperature ( C)
Specific growth rate (log CFU/day)a,b
Lag time (day)a,b
E. coli O157:H7c
S. Typhimuriumd
TBCe
E. coli O157:H7c
S. Typhimuriumd
TBCe
4 10 4 10 4 10 4 10 4 10
0.491Aa 0.860Abc 0.509Aab 1.021Ac 0.419Aa 0.977Ac 0.436Aa 0.921Ac 0.524Aab 1.132Ac
0.544Aa 1.263Ab 0.553Aa 1.171Ab 0.619Aa 1.167Ab 0.621Aa 1.141Ab 0.565Aa 1.160Ab
0.320Aa 1.009Abcd 0.693Aabcd 1.267Ad 0.639Aabc 1.180Acd 0.731Aabcd 1.122Abcd 0.554Aab 1.101Abcd
3.746Cf 1.489Bc 2.381Bd 0.563Aab 2.505Bde 0.666Aab 2.249Bd 0.446Aa 2.976Ae 1.069Abc
3.116Bd 1.699Babc 2.761Bd 1.979Cbc 2.789Bd 1.499Bab 2.150Bc 1.616Bab 2.840Ad 1.448ABa
1.993Ac 0.919Aa 1.786Abc 1.402Bab 1.613Abc 1.389Bab 1.560Abc 1.208Bab 2.692Ad 1.590Bbc
a
Within the same column, values not followed by the same small letter are significantly different (P < 0.05). Within the same row, values not followed by the same capital letter are significantly different (P < 0.05). c 2 R > 0.96. d 2 R > 0.95. e 2 R > 0.88. f Untreated sample. b
The growth curves of E. coli O157:H7, S. Typhimurium, and TB counts on each pork sample during storage at 4 and 10 C are shown in Figs. 5e7, respectively, while the bacterial growth parameters (SGR and LT) in each pork sample are described in Table 2. The results showed that the lower storage temperature (4 C) yielded in lower SGRs and longer LTs of pathogens. However, the SGRs and LTs of naturally occurring bacteria (TBC) were likely not affected by storage temperature; in other words, the naturally occurring bacteria could grow well on pork stored either at 4 or 10 C. Nychas et al. (2008) stated that fresh meat such as pork can harbor high numbers of aerobic mesophilic and psychrotrophic bacteria, which are the major spoilage agents especially during cold storage. Most of which is Pseudomonas spp. and Enterobacteriaceae. Most of the SGRs and LTs of all bacteria in treated pork samples were not significantly different compared to those in control samples. However, the SAcEW þ 0.5% FA 40 C treatment significantly (P < 0.05) yielded in a longer lag time of naturally occurring bacteria (TBC) on pork stored at 4 C compared to other pork samples. Thus, the longer lag time of TBC on treated (SAcEW þ 0.5% FA 40 C) pork might affect to its shelf life extension during storage.
the shelf life of pork from 5 to 6 days compared to those of untreated pork. This finding was similar with a previous study that reported the application of combined treatment (SAcEW þ 3% CaL) for 5 min prolongs the shelf life of fresh pork up to 6 days at 4 C (Rahman et al., 2013). Latha et al. (2009) also observed the
3.3. Shelf life based on microbial and sensory quality The initial populations of TBC on pork samples treated with the individual treatments (SAcEW, StAEW, and 0.5% FA) and a combined treatment (SAcEW þ 0.5%) at 40 C for 3 min decreased approximately by 2.28, 2.35, 2.41, and 2.75 log CFU/g, respectively (Fig. 7). The results were in agreement with the findings of Latha et al. (2009) and Rahman et al. (2013), which are approximately 2.2e2.3 log CFU/g. The end of shelf life was considered to occur when the TBC reached 7 log CFU/g (Nychas et al., 2008). The TBC in untreated pork reached unacceptable levels within 4 (7.07 ± 0.33 log CFU/g) and 2 days (7.62 ± 0.13 log CFU/g) of storage at 4 and 10 C, respectively. The spoilage of treated (SAcEW, StAEW, 0.5% FA 40 C) pork stored at 4 and 10 C were evidenced within 6 and 4 days, respectively, with corresponding increases ranging from 6.99 to 7.34 log CFU/g. The combined treatment (SAcEW þ 0.5% FA 40 C) more effectively controlled TBC during storage at 4 and 10 C for 10 and 7 days, respectively, when compared to those of the untreated and the individually treated samples. The TBC on treated (SAcEW þ 0.5% FA 40 C) pork stored at 4 and 10 C increased up to 6.77 ± 0.23 and 6.98 ± 0.19 log CFU/g after 10 and 7 days, respectively. Thus, the SAcEW þ 0.5% FA 40 C treatment could prolong
Fig. 8. Effect of various treatments with mild heat (40 C) for 3 min on sensory quality of pork stored at 4 and 10 C. Values shown are means ± standard error.
A.R. Mansur et al. / Food Control 47 (2015) 277e284
enhanced shelf life of 4e5 days for fresh pork treated with salt or salt combinations and stored at refrigeration temperature (5e7 C). The SI of sensory properties (color, odor, and texture) of all pork samples decreased as the storage time increased, and the higher storage temperature resulted in a faster decrease of sensory acceptance (Fig. 8). The spoilage of the control pork at 4 and 10 C was evident on days 2 and 1, respectively, whereas the treated (SAcEW, StAEW, and 0.5% FA 40 C) pork stored at 4 and 10 C were spoiled on days 4 and 2, respectively. The more prolonged sensory quality was observed on days 8 and 5 in treated (SAcEW and 0.5% FA 40 C) pork stored at 4 and 10 C, respectively. Similar extended shelf life also has been reported by Latha et al. (2009) and Rahman et al. (2013) in their previous studies. 3.4. Changes in pH during storage The initial pH values of all pork samples were slightly acidic (5.20e6.16), except that of treated (StAEW) pork, which decreased into 4.88 ± 0.18. However, the pH of all pork samples increased gradually over time and then became slightly acidic or close to neutral. The values of final pH of all pork samples after storage at 4 and 10 C were 5.89e6.48 and 6.20e6.73, respectively (Fig. 9). The results were similar to those reported by Tan and Shelef (2002) and Rahman et al. (2013). Gill (1983) stated that an augmented pH is typical for fresh meat during storage, caused by Gram-negative bacteria. These bacteria break down the protein compounds of meat and then release basic compounds such as amines. The
Fig. 9. Changes in pH of untreated and treated pork stored at 4 and 10 C. Values shown are means ± standard deviation of triplicate assays.
283
current study also revealed that the pH value increased as TBC increased. Rousset and Renerre (1991) and Rahman et al. (2013) reported that TBC were 10e100-fold greater on high pH meat than those on normal pH meat at various aging durations. Moreover, Holmer et al. (2009) also reported that higher pH and a longer aging period enhance the microbial proliferation and decrease the shelf life. Based on the results of the present study, the decontamination of fresh pork by SAcEW þ 0.5% FA at 40 C for 3 min was capable to reduce bacterial contaminations and prolong the shelf life of pork during storage at 4 and 10 C. However, the microscopic tests and mathematical modeling might be needed in the future study to provide more fundamental understanding of the interaction of the treatments with bacterial cells and the mode of action of the treatment. Also, a large number of experiments are much needed to ascertain the effects of SAcEW þ 0.5% FA in a commercial slaughter plant or other meat industries. Acknowledgments This work was supported by grant no. 13072-957 from the Ministry of Food and Drug Safety (MFDS) of Republic of Korea. References Baer, A. A., Miller, M. J., & Dilger, A. C. (2013). Pathogens of interest to the pork Industry: a review of research on interventions to assure food safety. Comprehensive Reviews in Food Science and Food Safety, 12, 183e217. Baird Parker, A. C. (1980). Organic acids. In J. H. Silliker, R. P. Elliott, A. C. Baird Parker, S. L. Bryan, J. H. B. Christian, D. S. Clarke, et al. (Eds.), International Commission on Microbiological Specifications for Foods: Vol. I. Microbial ecology of foods (pp. 126e135). New York: Academic Press. Booth, I. R. (1985). Regulation of cytoplasmic pH in bacteria. Microbiological Reviews, 49, 359e378. Byelashov, O. A., & Sofos, J. N. (2009). Strategies for on-line decontamination of carcasses. In F. Toldr0 a (Ed.), Safety of meat and processed meat (pp. 164e165). New York: Springer. CDC. (2011). CDC estimates of foodborne illness in the United States. Center for Disease Control. Available from http://www.cdc.gov/foodborneburden/2011-foodborneestimates.html Accessed 15.03.14. Chen, J. H., Ren, Y., Seow, J., Liu, T., Bang, W. S., & Yuk, H. G. (2012). Intervention technologies for ensuring microbiological safety of meat: current and future trends. Comprehensive Reviews in Food Science and Food Safety, 11, 119e132. Ding, T., Rahman, S. M. E., Purev, U., & Oh, D. H. (2010). Modelling of Escherichia coli O157:H7 growth at various storage temperatures on beef treated with electrolyzed oxidizing water. Journal of Food Engineering, 97, 497e503. Fabrizio, K. A., & Cutter, C. N. (2004). Comparison of electrolyzed oxidizing water with other antimicrobial interventions to reduce pathogens on fresh pork. Meat Science, 68, 463e468. Fabrizio, K. A., & Cutter, C. N. (2005). Application of electrolyzed oxidizing water to reduce Listeria monocytogenes on ready-to-eat meats. Meat Science, 71, 327e333. Fabrizio, K. A., Sharma, R. R., Demirci, A., & Cutter, C. N. (2002). Comparison of electrolyzed oxidizing water with various antimicrobial interventions to reduce Salmonella on poultry. Poultry Science, 66(10), 1379e1384. Gill, C. O. (1983). Meat spoilage and evaluation of the potential storage life of fresh meat. Journal of Food Protection, 46, 444e448. Holmer, S. F., McKeith, R. O., Boler, D. D., Dilger, A. C., Eggert, J. M., Petry, D. B., et al. (2009). The effect of pH on shelf-life of pork during aging and simulated retail display. Meat Science, 82, 86e93. Huang, Y. R., Hung, Y. C., Hsu, S. Y., Huang, Y. W., & Hwang, D. F. (2008). Application of electrolyzed water in the food industry. Food Control, 19, 329e345. Kim, C., Hung, Y. C., & Russell, S. M. (2005). Efficacy of electrolyzed water in the prevention and removal of fecal material attachment and its microbicidal effectiveness during simulated industrial poultry processing. Poultry Science, 84, 1778e1784. Kreyenschmidt, J., Hubner, A., Beierle, E., Chonsch, L., Scherer, A., & Petersen, B. (2010). Determination of the shelf life of sliced cooked ham based on the growth of lactic acid bacteria in different steps of the chain. Journal of Applied Microbiology, 108, 510e520. Latha, C., Sherikar, A. T., Waskar, V. S., Dubal, Z. B., & Ahmed, S. N. (2009). Sanitizing effect of salts on experimentally inoculated organisms on pork carcasses. Meat Science, 83, 796e799. Mataragas, M., Skandamis, P. N., & Drosinos, E. H. (2008). Risk profiles of pork and poultry meat and risk ratings of various pathogen/product combinations e a review. International Journal of Food Microbiology, 126, 1e12.
284
A.R. Mansur et al. / Food Control 47 (2015) 277e284
Nychas, G. J. E., Marshall, D. L., & Sofos, J. N. (2007). Meat, poultry and seafood. In M. P. Doyle, L. R. Beuchat, & T. J. Montville (Eds.), Food microbiology: Fundamentals and frontiers (3rd ed.) (pp. 105e140). Washington, USA: ASM Press. Nychas, G. J. E., Skandamis, P. N., Tassou, C. C., & Koutsoumanis, K. P. (2008). Meat spoilage during distribution. Meat Science, 78, 77e89. Park, H., Hung, Y. C., & Brackett, R. E. (2002). Antimicrobial effect of electrolyzed water for inactivating Campylobacter jejuni during poultry washing. International Journal of Food Microbiology, 72, 77e83. Podolak, R. K., Zayas, J. F., Kastner, C. L., & Fung, D. Y. C. (1995). Reduction of Listeria monocytogenes, Escherichia coli 0157:H7 and Salmonella typhimurium during storage on beef sanitized with fumaric, acetic and lactic acids. Journal of Food Safety, 15, 283e290. Podolak, R. K., Zayas, J. F., Kastner, C. L., & Fung, D. Y. C. (1996). Reduction of bacterial populations on vaccum-packaged ground beef patties with fumaric and lactic acids. Journal of Food Protection, 59, 1037e1040. Rahman, S. M. E., Wang, J., & Oh, D. H. (2013). Synergistic effect of low concentration electrolyzed water and calcium lactate to ensure microbial safety, shelf life and sensory quality of fresh pork. Food Control, 30, 176e183.
Rousset, S., & Renerre, M. (1991). Effect of CO2 or vacuum packaging on normal and high pH meat shelf-life. International Journal of Food Science and Technology, 26(6), 641e652. Scharff, R. L. (2010). Health-related costs from foodborne illness in the United States. Produce Safety Project at Georgetown Univ. Available from http://www. producesafetyproject.org/admin/assets/files/Health-Related-Foodborne-IllnessCosts-Report.pdf-1.pdf. Georgetown Univ. Accessed 17.03.14. Smulders, F. J. M., & Greer, G. G. (1998). Integrating microbial decontamination with organic acids in HACCP programmes for muscle foods: prospects and controversies. International Journal of Food Microbiology, 44, 149e169. Tan, W., & Shelef, L. A. (2002). Effects of sodium chloride and lactates on chemical and microbiological changes in refrigerated and frozen fresh ground pork. Meat Science, 62, 27e32. Zwietering, M. H., Jongenburger, I., Rombouts, F. M., & van't Riet, K. (1990). Modeling of the bacterial growth curve. Applied and Environmental Microbiology, 56, 1875e1881.