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Teriyaki sauce with carvacrol or thymol effectively controls Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and indigenous flora in marinated beef and marinade
Meat Science 129 (2017) 147–152
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Teriyaki sauce with carvacrol or thymol effectively controls Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and indigenous flora in marinated beef and marinade Hyeree Moon a, Nam Hee Kim a, Soon Han Kim b, Younghoon Kim c, Jee Hoon Ryu a, Min Suk Rhee a,⁎ a b c
Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, North Chungcheong Province 363-951, Republic of Korea BK 21 Plus Graduate Program, Department of Animal Science and Institute of Rare Earth for Biological Application, Chonbuk National University, Jeonju 561-756, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 October 2016 Received in revised form 2 March 2017 Accepted 2 March 2017 Available online 06 March 2017 Chemical compounds: Carvacrol (PubChem CID: 10364) Thymol (PubChem CID: 6989) Keywords: Teriyaki sauce Marinated beef Carvacrol Thymol Antibacterial activity Synergism
a b s t r a c t An effective bactericidal cold-marinating method for beef products is described, exploiting the synergism between soy sauce and natural compounds (carvacrol, CV or thymol, TM) to reduce microbiological risks. Beef slices inoculated with Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium (3.1–3.5 log CFU/g) were marinated in a teriyaki sauce with or without CV and TM (0.3 and 0.5%). After 1, 3, and 7 days at 4 °C, indigenous microflora population, color, lipid oxidation, marinade uptake, and pH of marinated beef and leftover marinade samples were examined. Teriyaki sauce alone did not reduce or inhibit any of the target pathogens or indigenous bacteria, while 0.5% CV- or TM-containing teriyaki sauce inactivated all inocula without recovery within 7 days (p b 0.05). The pathogens relocated from the beef into the leftover marinade (3.0–3.4 log CFU/mL) were also completely inactivated. The treatment inhibited growth of indigenous aerobic bacteria (p b 0.05) and inactivated coliform bacteria. Physicochemical parameters were not significantly affected (p N 0.05). © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Undercooked meat products have been identified as a leading cause of several outbreaks of foodborne illnesses worldwide (Scanga et al., 2000; Sofos, 2008). In particular, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium are frequently associated with outbreaks caused by processed meat products (CDC, 2006, 2014; Choi, Bae, Kim, Kim, & Rhee, 2009; Choi, Kim, Kim, Kim, & Rhee, 2009; Rhoades, Kargiotou, Katsanidis, & Koutsoumanis, 2013). Rangel, Sparling, Crowe, Griffin, and Swerdlow (2005) reported that ~ 47% E. coli O157:H7 outbreaks have been associated with beef products. Since consumers generally prefer medium rare or less-well cooked beef to over-cooked beef (Behrends et al., 2005; Neely et al., 1999; Røssvoll et al., 2014), it is imperative to devise an effective technique for eliminating or controlling those pathogens in commercial beef products at cold temperatures.
⁎ Corresponding author at: Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 5–1 Anam–dong, Sungbuk–gu, Seoul 02841, Republic of Korea. E-mail address: [email protected] (M.S. Rhee).
http://dx.doi.org/10.1016/j.meatsci.2017.03.001 0309-1740/© 2017 Elsevier Ltd. All rights reserved.
Marination, the soaking of food in a seasoned sauce before cooking, is one of many effective flavoring methods, not only enhancing flavor but also improving tenderness and juiciness of meat products (Björkroth, 2005). A number of marinated meat products are currently commercially manufactured and on sale, including barbecue ribs and steaks. Commercially marinated meat products, as ready-to-cook foods, are gaining popularity due to their convenience, whereas their hygiene and safety have not been extensively demonstrated (Jo, Lee, Kang, Shin, & Byun, 2004; Kanatt, Rao, Chawla, & Sharma, 2013). Indeed, coliform bacteria, typical indicators of food hygiene and food safety potential indicators (Brown et al., 2000; Lues & Van Tonder, 2007), were occasionally found in significant amounts (0.3–5.5 log CFU/g, 61.0% detection rate) in sold packaged marinated meat products (Rhee, Ryu, Kang, Kim, & Jang, 2014). Marination in fermented soy sauce is a typical beef marinating procedure and several manufacturers apply this method in their products. Since international consumption of ethnic foods has been growing because of food consumer globalization and tourism (Mak, Lumbers, & Eves, 2012), beef products marinated in soy sauce, including ‘bulgogi’, have become popular worldwide (Jo et al., 2004; Kim, Cha, Chung, Kim, & Chung, 2009). Although the marinade itself is generally regarded as a bacterial growth inhibitor, because of its acidic pH and high
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concentration of salt, preservatives, and spices (Björkroth, 2005; Kargiotou, Katsanidis, Rhoades, Kontominas, & Koutsoumanis, 2011), several researchers report that the marinade alone did not completely control pathogen growth under real processing conditions (e.g., refrigeration) (Masuda, Hara-Kudo, & Kumagai, 1998; Pathania, McKee, Bilgili, & Singh, 2010). Cured meat products, including marinated beef, traditionally contain synthetic chemical preservatives, such as nitrate and nitrite (Sebranek & Bacus, 2007). However, because of the growing popularity of natural and organic foods, there has been a consumer shift away from chemical preservatives in foods. Antibacterial activity of various plant-derived essential oils (EOs) and their components has long been studied, and they have become popular as natural food additives (Burt, 2004). In our previous study, we discovered a significant synergistic antibacterial interaction between soy sauce and six different EOs [carvacrol (CV), thymol (TM), eugenol, trans-cinnamaldehyde, β-resorcyclic acid, and vanillin]. CV and TM showed significantly higher bactericidal synergism with soy sauce than other EOs against E. coli O157:H7, L. monocytogenes, and S. Typhimurium within 10 min exposure at refrigeration temperatures, even during refrigeration (Moon & Rhee, 2016). Therefore, it is expected that the inclusion of CV and TM in soy sauce beef marination may effectively control potentially hazardous pathogens in meat products and could result in a microbiologically safe and high-quality final product. In this study, therefore, we aimed to develop an effective antimicrobial marinating method using soy sauce-based marinade and EO components (CV and TM) to control (1) artificial inoculum of foodborne pathogenic bacteria (E. coli O157:H7, L. monocytogenes, and S. Typhimurium), and (2) indigenous microorganisms (aerobic plate counts and total coliform counts) in both marinated beef and leftover marinades. In addition, physicochemical parameters (color, lipid oxidation, marinade uptake, and pH) were also measured and compared between EO-untreated and EO-treated groups to validate industrial applicability of the developed marinating method. 2. Materials and methods 2.1. Bacterial cell suspensions Three strains of each species were obtained from American Type Culture Collection. These were: E. coli O157:H7 strains ATCC35150, 43889, 43895; L. monocytogenes strains ATCC 19111, 19115, 19117; and S. Typhimurium strains ATCC 14028, 19585, and DT104 Killer Cow. They were used as test microorganisms. Each strain was stored at − 20 °C in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) with 20% glycerol and sub-cultured on monthly basis. The strains were separately cultured in 10 mL fresh TSB at 37 °C for 18 h, and three strains of the same species were mixed in a plastic 50-mL centrifuge tube (Difco). After centrifugation at 1800 × g for 15 min (Centra-CL2; International Equipment Company, Needham Heights, MA, USA), bacterial pellets were washed twice with 0.85% sterile saline solution. The final pellet was re-suspended in 10 mL 0.85% sterile saline (density ~8–9 log CFU/mL).
(see Section 2.1.), as follows. Bacterial suspensions were diluted 10-fold with 0.85% saline to obtain 4–5 log CFU/mL cell densities. The suspensions were dispensed over beef piece surface (final concentration 3–4 log CFU/g inoculated meat). Inoculated beef pieces were incubated at 4 °C overnight to allow the inoculum to attach to the sliced beef surface. After refrigeration, two 10 g sample pieces were randomly selected and used for further experiments. To assess the changes in indigenous bacterium concentration and physicochemical parameters associated with marination (with or without EO addition), uninoculated beef slices were used as experimental samples.
2.3. Marination Teriyaki sauce (Preservative-Free Kikkoman Teriyaki Sauce, Kikkoman Co., Japan) was purchased from a local market and stored at 4 °C until use. Ingredients in teriyaki sauce were soy sauce, wine, high fructose corn syrup, water, vinegar, salt, spices, onion powder, and garlic powder. CV and TM (Sigma-Aldrich, St. Louis, MO, USA) stock solutions (50 ×) were prepared by dissolving the appropriate amount of each compound in 98% ethanol. Stock solutions (200 μL) were added to 9.8 mL teriyaki sauce in a sterile plastic 50-mL tube (Difco), to adjust the final EO concentration to 0.3% and 0.5% (v/v). Raw sliced beef samples and samples treated with teriyaki sauce +2% ethanol were used as negative (unmarinated) and positive controls (teriyaki sauce-marinated), respectively. Beef samples (10 g) were then immersed in the prepared CV- or TM-supplemented marinades and gently agitated with sterile forceps. After fastening the lid of the plastic 50-mL tube, the marinated beef was incubated at 4 °C for 7 days. The experiment was performed in triplicate using different beef sample produced in a different batch. 2.4. Microbiological analysis Following 1, 3, and 7-day refrigeration, marinated beef and leftover marinades were collected separately. Each marinated beef piece and leftover marinade was placed in a sterile stomacher bag (JS-010, Jin Sung Uni-Tech, Seoul, Korea) and homogenized in 10 volumes of sterile 0.85% saline (230 rpm for 2 min) (Circulator 400, Seward, Worthing, UK). Aliquots (0.1 mL) of the homogenate were serially diluted in 9 mL 0.85% saline 104 ×. Each dilution (0.1 mL) was then spread onto the following selective agar plates: MacConkey sorbitol agar (Difco) for E. coli O157:H7 detection; Oxford Agar supplemented with Listeria selective supplement (Difco) for L. monocytogenes detection; and xylose-lysine-desoxycholate agar (Difco) for S. Typhimurium detection. Plate count agar (Difco) and violet red bile agar (Difco) were used for aerobic plate count (APC) analysis and total coliform counts (TCC), respectively. To lower the detection limit to 10 CFU/g beef and 1 CFU/mL marinade, undiluted homogenate (0.2 mL) was spread onto five plates of each agar. All inoculated plates were incubated at 37 °C for 24 h except for PCA (48 h at 37 ± 2 °C), and typical colonies were counted.
2.2. Beef preparation and inoculation
2.5. Validation of antimicrobial marination
Beef sirloins, sliced to 5 mm thickness, were purchased from a local supermarket and stored at − 20 °C. The day before use, beef sirloins were defrosted at 4 °C overnight. Samples were cut into 10 g pieces (30 × 25 × 5 mm) with a flame-sterilized kitchen knife. Presence of E. coli O157:H7, L. monocytogenes, and S. Typhimurium in sliced beef was tested by examining 25 g samples according to method described in the Bacteriological Analytical Manual by the United States Food and Drug Administration. Detection of target microorganisms led to rejection of the entire beef sirloin from which the sample was obtained. Thus prepared samples were inoculated with bacterial cell suspensions
If none of the inoculum was detected by the plate counting method, recovery of injured bacterial cells was assessed to identify treatment conditions that resulted in a complete inactivation of the inoculum. Beef samples subjected to each condition were separately transferred into sterile stomacher bags and homogenized at 230 rpm for 2 min in 10 volumes of fresh TSB. After 24 h incubation at 37 °C, enriched samples were streaked on selective agar plates, as mentioned in Section 2.4, and further incubated at 37 °C for 24 h. The absence or presence of typical colonies was recorded as a positive or negative result, respectively. The experiments were performed in triplicate.
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2.6. Measurements of physicochemical parameters Beef color and lipid oxidation, marinade uptake by the marinated beef, and pH of the marinated beef and leftover marinades were determined after 1, 3, and 7 days of marination, as physicochemical parameters indicative of the quality of marinated beef products. Marinated beef color was examined with Chroma meter (DP–400, Minolta Camera Co., Osaka, Japan) and expressed as L* (lightness), a* (redness), and b* (yellowness) values. The extent of lipid oxidation was evaluated by measuring thiobarbituric acid reactive substance (TBARS) levels, according to method by Buege and Aust (1978). TBARS values were expressed as (mg malondialdehyde)/(kg beef). Marinated beef marinade uptake was estimated according to the method of Yusop, O'Sullivan, Kerry, and Kerry (2010). The pH values of treated marinated beef samples and leftover marinades were measured at room temperature with pH-meter (Testo 206, Testo, Lenzkirch, Germany).
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uninoculated teriyaki sauce marinade was microorganisms-free. During 7-day refrigeration of beef without marination, APC increased to 5.0 log CFU/g (Fig. 2a), whereas TCC remained stable and within 0.9– 1.3 log CFU/g (Fig. 2c). When the beef was marinated only in teriyaki sauce, APC maintained the initial level (3.2–3.4 log CFU/g) for 3 days and then slightly increased, to 3.9 log CFU/g, after 7 days. Over 7-day period, TCC was slightly reduced, by 0.3–0.5 log CFU/g. By contrast, teriyaki sauce containing CV or TM resulted in a complete TCC elimination from marinated beef samples without recovery within 1 day. In the leftover marinade (Fig. 2b and d), sliced beef microorganisms may have relocated to the marinade. Without CV or TM, APC in the leftover marinade increased to 3.7 log CFU/mL. teriyaki sauce with CV or TM inhibited the increase of APC by 1.2–2.8 log CFU/mL or 1.5–3.3 log CFU/mL, respectively (Fig. 2b). There are no significant differences in APC levels of beef samples between all of the CV or TM treated groups. However, TCC data trends were different from APC. Leftover marinade TCC remained undetectable until the end of the experiment (Fig. 2d).
2.7. Statistical analysis Triplicate experimental data on bacterial populations in marinated beef (CFU/g) and in leftover marinade (CFU/mL) were expressed as Log10 CFU/g values. Bacterial population log values and physicochemical parameter values were expressed as mean ± standard error. The data were analyzed using SAS software version 9.4 (SAS Institute, Cary, NC, USA) with general linear mixed model. The fixed effects in the model were storage time (0, 1, 3, and 7 days), and the random effects were treatment (non-marinated, teriyaki sauce, teriyaki sauce + CV or TM). If each effect was significant, mean values were subjected to post-hoc Tukey's multiple range test for mean separation. 3. Results 3.1. Bactericidal activity of cold-marination with carvacrol or thymol against foodborne pathogens Fig. 1 shows changes in bacterial populations of E. coli O157:H7, L. monocytogenes, and S. Typhimurium in marinated beef and leftover marinades during cold-marination with teriyaki sauce containing CV or TM (0.3 and 0.5%). The overall data showed similar tendencies for all the bacterial species tested. More bacteria were inactivated with increasing EO concentrations and marinating time. Bactericidal effects of CV and TM were not significantly different at the same treatment condition (p N 0.05). In the non-marinated beef sample (Fig. 1a–c), the initial inoculum (3.1–3.5 log CFU/g) did not significantly change (p N 0.05) during 7day refrigeration. When sliced beef was marinated only in teriyaki sauce, bacterial numbers initially decreased by 0.4–0.5 log CFU/g and remained at that level until the end of the experiment. Whereas teriyaki sauce alone did not significantly reduce bacterial numbers (b1 log CFU/g reduction), teriyaki sauce with CV or TM effectively inactivated the inoculated foodborne pathogens. In particular, all inocula were completely inactivated without recovery by 7-day cold-marination with 0.5% CV or TM, except S. Typhimurium by teriyaki sauce + CV 0.5%. In the leftover marinade without CV or TM (Fig. 1d–f), the inoculum relocated into the marinade from sliced beef surface, increasing marinade bacterial levels up to 3.0–3.4 log CFU/mL. By comparison, supplementation of the marinade with CV or TM inhibited the survival of the translocated inoculum to undetectable levels. 3.2. Inhibitory activity of cold-marination with carvacrol or thymol against beef sample indigenous bacteria Fig. 2 shows bacterial growth inhibition (APC) or indigenous bacterium inactivation (TCC) by cold-marination with 0.3% and 0.5% concentrations of CV and TM. Before marination, 3.5 log CFU/g APC and 1.1 log CFU/g TCC were detected in the sliced beef samples, while
3.3. Quality parameters of the marinated beef and leftover marinade during cold-marination with carvacrol or thymol Beef color and lipid oxidation, marinade uptake by the marinated beef, and marinated beef and leftover marinade pH values are presented in Table 1. As indicated by L*, a*, and b* values, the addition of CV or TM to teriyaki sauce marinade did not significantly affect marinated beef color (p N 0.05). TBARS values ranged between 3.2 and 3.7 mg malondialdehyde per kg marinated beef, and no statistically significant differences were observed comparing teriyaki sauce-only marination (3.2–3.7 mg/kg) with teriyaki sauce + CV (3.2–3.7 mg/kg) or teriyaki sauce + TM (3.3–3.6 mg/kg) (p N 0.05) during 7-day refrigeration. The sliced beef absorbed on average 21.2–24.9% teriyaki sauce marinade in the absence or presence of CV or TM, no significant differences in marinade uptake (%) were observed between the experimental groups (p N 0.05). Marinated beef and leftover marinade pH values ranged between 5.0 and 5.2 and 4.8–5.0, respectively, and were not significantly affected in any of the experimental groups (p N 0.05). 4. Discussion This study suggests an effective intervention, using teriyaki sauce supplemented with a small amount of EOs, that can completely inactivate major foodborne pathogens (E. coli O157:H7, L. monocytogenes, and S. Typhimurium) and coliform bacteria in marinated beef and leftover marinade during cold-marination. Results of this study indicated that soy sauce-based teriyaki sauce alone did not significantly reduce foodborne pathogen levels in a marinated beef product during refrigeration. The commercially available teriyaki sauce marinade used in this study contains soy sauce, wine, high fructose corn syrup, water, vinegar, salt, spices, onion powder, and garlic powder as major ingredients, and was “preservative-free”. This is in agreement with a previous publication by Masuda et al. (1998), who reported that E. coli O157:H7 levels were not affected by dipping in soy sauce (main component of teriyaki sauce) at 4 °C for 9 days. Thus the refrigeration temperature (4 °C), at which marinated beef products were tested in this study, might not have been optimal for the antibacterial action of teriyaki sauce. Several groups previously reported the various antibacterial parameters of teriyaki sauce (e.g., high sodium content, low pH, ethanol, and preservatives) (Kataoka, 2005; Rhoades et al., 2013). Indeed, Pathania et al. (2010) found that teriyaki sauce inactivated Salmonella spp. to below the detection limit at 4 °C within 32 h. However, the antibacterial activity of teriyaki sauce marinade might vary depending on brand of the basic marinade used in the experiments, e.g., They applied teriyaki sauce with pH 3.7–3.8, which was significantly lower than pH of teriyaki sauce used in this study (pH 4.9–5.0) (Pathania et al., 2010). High sodium chloride levels in the soy sauce included in teriyaki sauce marinade may damage bacterial cell membranes (Hajmeer, Ceylan, Marsden, &
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Fig. 1. Bacterial populations in marinated beef and marinade following 1, 3, and 7-day marination at refrigeration temperature (4 °C). Escherichia coli O157:H7 population in the marinated beef (a) and leftover marinade (d); Salmonella Typhimurium population in the marinated beef (b) and leftover marinade (e); and Listeria monocytogenes population in the marinated beef (c) and leftover marinade (f). Data points represent population means and error bars indicate standard error from triplicate experiments.
Fung, 2006), but the final sodium chloride concentration in the teriyaki sauce marinade (610 mg per 15 mL, i.e., 4.1%) was not sufficient to exert an antibacterial effect. In addition, meat and meat products might constitute a pathogen survival-favoring environment (Veldhuizen, Creutzberg, Burt, & Haagsman, 2007). The antimicrobial activity of teriyaki sauce was likely reduced because of interaction with beef components (Quintavalla & Vicini, 2002; Veldhuizen et al., 2007). To enhance the antibacterial efficacy of soy sauce-based marinade that would reflect consumer avoidance of synthetic chemical preservatives, some attempts have been made to find the appropriate natural antimicrobial agents. Although the target microorganisms differed (i.e., spoilage bacteria instead of foodborne pathogens), Schirmer and Langsrud (2010) tested the inhibitory effects of several natural antimicrobials, including allyl isothiocyanate, ascorbic acid, cinnamaldehyde,
citric acid, grapefruit seed extract, rosemary extract, etc. They concluded that the use of these antimicrobials in meat products was limited because of insufficient antimicrobial activity, and strong odor and flavor which consumers may find unacceptable. Rhoades et al. (2013) reported the antibacterial activity of soy sauce marinade with lactic acid against S. enterica and L. monocytogenes in beef, with bacterial levels declining from 6 to 3 log CFU/g during 10-day marination at 5 °C. However, the pH of that marinade was 3.8 potentially affecting the quality of meat, possibly affecting the quality of meat (Alvarado & McKee, 2007). On the other hand, previous studies reported inhibitory effects of various EOs toward E. coli O157:H7, L. monocytogenes, and S. Typhimurium in a variety of meat products. In particular, antibacterial effects of oregano and thyme oil, whose predominant components are CV and TM, have been studied in meat products (Govaris, Solomakos,
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Fig. 2. Aerobic plate counts from marinated beef (a) and leftover marinade (b), and total coliform counts in marinated beef (c) and leftover marinade (d), after 1, 3, and 7-day marination at refrigeration temperature (4 °C). Data points represent population means and error bars indicate standard error from triplicate experiments.
Pexara, & Chatzopoulou, 2010; Solomakos, Govaris, Koidis, & Botsoglou, 2008a, 2008b). For instance, when Solomakos et al. (2008b) treated L. monocytogenes in minced beef with 0.6% thyme oil at 4 °C for 12 days, the bacterial levels were reduced from 4 to 3 log CFU/g. Similarly, Govaris et al. (2010) reported that 2-day refrigeration (4 °C) of minced shrimp with 0.6 and 0.9% oregano oil reduced Salmonella Enteritidis numbers, from ~ 4 log CFU/g to ~ 2.5 and 0.5 log CFU/g, respectively, maintained until the end of the experiment (12 days). However, several publications reported that EOs alone did not exert a significant bactericidal effect against foodborne pathogens. Indeed, when Lin, Labbe, and Shetty (2004) applied oregano extract to beef and fish slices, the numbers of inoculated L. monocytogenes cells remained largely at the initial level (8 log CFU/g) during 8-day incubation at 4 °C. Also, Veldhuizen et al. (2007) reported that 2.5 mM CV (0.04%) did not inactivate L. monocytogenes in steak tartare at 10 °C and 20 °C during 100-h storage. Solomakos et al. (2008a) similarly concluded that thyme oil did not reduce E. coli O157:H7 numbers in minced beef samples. By comparison, the effective cold bactericidal marinating method suggested in this study resulted in a complete inactivation without recovery of the major foodborne pathogens, E. coli O157:H7, L. monocytogenes, and S. Typhimurium, and also food hygiene indicator bacteria (coliform bacteria), over 7-day refrigeration (4 °C). Individual
teriyaki sauce (Fig. 1a–c) or EOs (CV and TM) (data not shown) treatments did not exert a significant antibacterial effect against beef sample inocula. Thus, the significantly higher bactericidal effect of teriyaki sauce with EOs might be caused by their synergistic interaction, as we previously reported (Moon & Rhee, 2016). Moreover, no significant differences in physicochemical sample parameters (i.e., color, lipid oxidation, marinade uptake, and pH) were observed between samples marinated with teriyaki sauce and EOs-supplemented teriyaki sauce. Solomakos et al. (2008a) evaluated the odor of thyme oil-treated minced beef, and found that 0.3% and 0.6% thyme oil treatment was acceptable to beef consumers, whereas the odor of 0.9% thyme oil was described as unpleasant. Thus, the method developed in this study can be successfully used in the marinated beef industry, because EO amounts used in this study (0.3% and 0.5%) were lower than 0.6% cut-off. In addition, teriyaki sauce marinade, with its unique odor and flavor, is expected to mask the characteristic CV and TM odors. The marked synergism between teriyaki sauce and EOs (CV and TM) will also contribute to reducing several possible microbiological risk factors affecting the microbiological safety and quality of marinated beef product manufacture: cross-contamination and growth of harmful bacteria. Cross-contamination has been proposed as one of the major risk factors affecting microbiological safety and quality of marinated meat products (Björkroth, 2005; Choi, Bae, et al., 2009). As indicated in
Table 1 Physicochemical parameters of marinated beef and leftover marinade. pH
Color Day
Essential oil
%
L*
1
– Carvacrol
– 0.3 0.5 0.3 0.5 – 0.3 0.5 0.3 0.5 – 0.3 0.5 0.3 0.5
31.7 30.6 31.4 30.3 31.0 31.3 32.4 31.8 31.3 31.7 31.5 31.6 31.1 31.7 32.1
Thymol 3
– Carvacrol Thymol
7
– Carvacrol Thymol
a* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.3 0.4 1.6 1.6 0.4 0.5 0.7 0.6 0.3 0.2 0.4 0.5 0.5 0.7
9.6 9.7 9.9 9.9 10.7 10.7 10.0 10.0 10.5 10.2 10.1 10.3 9.8 10.4 10.0
b* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.2 0.0 0.4 0.4 0.3 0.3 0.5 0.5 0.1 0.5 0.5 0.6 0.6 0.4
17.6 16.0 17.1 16.0 17.6 15.5 15.7 14.8 16.7 15.9 15.8 16.2 15.5 15.8 16.0
Lipid oxidation (mg/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.1 0.1 1.6 1.4 0.1 0.3 0.6 0.8 0.4 0.3 0.4 0.5 0.8 0.3
3.2 3.2 3.7 3.5 3.6 3.7 3.4 3.5 3.6 3.5 3.6 3.4 3.4 3.4 3.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0d 0.1bcd 0.1bcd 0.1bcd 0.1bc 0.2b 0.1bcd 0.2bcd 0.0bcd 0.1bcd 0.1bcd 0.0bcd 0.0bcd 0.1bcd 0.1bcd
a
Marinade uptake (%)
Marinated beef
Leftover marinade
24.8 24.5 23.7 22.7 22.1 23.7 24.1 23.2 24.9 24.6 24.0 22.2 25.0 21.2 21.8
5.1 5.0 5.0 5.0 5.1 5.0 5.0 5.1 5.1 5.1 5.1 5.1 5.2 5.1 5.0
4.8 4.9 4.9 4.9 4.8 4.8 4.9 4.8 4.9 4.9 4.9 4.9 5.0 4.8 4.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Data represent means ± standard error (n = 3). b–d Values designated by different superscripts in the same column are significantly different (p b 0.05). a Lipid oxidation was determined as thiobarbituric acid reactive substances (TBARS), mg malondialdehyde per kg beef.
0.8 1.0 2.3 1.8 2.1 1.3 3.7 3.8 0.9 0.6 1.9 5.2 0.3 1.6 2.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.0
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Fig. 1d–f, the addition of small amounts of CV or TM resulted in reduced (undetectable, b1 CFU/mL) foodborne pathogen and coliform levels in the leftover marinade. Considering that consumers may reuse spent marinade, causing a potential contamination risk to products during subsequent marination (Muras, 2009), the developed method will be helpful in controlling this problem. Moreover, as this approach inhibited bacterial growth in both marinated beef and leftover marinade (Fig. 2a), it is also expected that it will control rapid growth of harmful bacteria that adversely affect the quality of marinated beef, such as typical spoilage bacteria. In conclusion, we developed an effective antibacterial beef coldmarinating method against E. coli O157:H7, L. monocytogenes, and S. Typhimurium, and indigenous coliform bacteria, exploiting the remarkable synergism of soy sauce in teriyaki sauce with common natural compounds, EOs (CV and TM). Practical application of this method in marinated beef processing has several advantages, as follows: (1) use of only natural compounds (preventing consumer avoidance), (2) simplicity and convenience (a simple addition of the appropriate CV or TM amounts results in significant bacterial inactivation), and (3) continuity of the antibacterial effect under refrigeration (i.e., the bactericidal effect is preserved during distribution). The method will also contribute to preventing bacterial contamination via re-use of contaminated marinade and, ultimately, to delivery of microbiologically safe marinated meat products. We demonstrated that the synergistic antibacterial effect of soy sauce with EOs (CV or TM) is applicable in a real food scenario. This technique will contribute to various marinades and marinated foods industry. Acknowledgements This study was supported by a grant (13162MFDS045) from the Korean Ministry of Food and Drug Safety (2013–2014) and, partially, by a Korea University Grant. Authors also thank the School of Life Sciences and Biotechnology of Korea University for BK 21 PLUS and the Institute of Biomedical Science and Food Safety, CJ-Korea University Food Safety Hall, for providing equipment and facilities. References Alvarado, C., & McKee, S. (2007). Marination to improve functional properties and safety of poultry meat. The Journal of Applied Poultry Research, 16(1), 113–120. Behrends, J. M., Goodson, K. J., Koohmaraie, M., Shackelford, S. D., Wheeler, T. L., Morgan, W. W., ... Savell, J. W. (2005). Beef customer satisfaction: USDA quality grade and marination effects on consumer evaluations of top round steaks. Journal of Animal Science, 83(3), 662–670. Björkroth, J. (2005). Microbiological ecology of marinated meat products. Meat Science, 70(3), 477–480. Brown, M. H., Gill, C. O., Hollingsworth, J., Nickelson, R., Seward, S., Sheridan, J. J., ... Usborne, W. R. (2000). The role of microbiological testing in systems for assuring the safety of beef. International Journal of Food Microbiology, 62(1), 7–16. Buege, J. A., & Aust, S. D. (1978). [30] Microsomal lipid peroxidation. Methods in Enzymology, 52, 302–310. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods—A review. International Journal of Food Microbiology, 94(3), 223–253. CDC (2006). Multistate outbreak of Salmonella Typhimurium infections associated with eating ground beef-United States, 2004. Morbidity and Mortality Weekly Report, 55, 180–182. CDC (2014). Multistats outbreak of Shiga toxin-producing Escherichia coli O157:H7 infections linked to ground beef (final update). Retrieved from http://www.cdc.gov/ecoli/ 2014/O157H7-05-14/index.html (Accessed on January 13, 2016) Choi, Y. M., Bae, Y. Y., Kim, K. H., Kim, B. C., & Rhee, M. S. (2009a). Effects of supercritical carbon dioxide treatment against generic Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, and E. coli O157: H7 in marinades and marinated pork. Meat Science, 82(4), 419–424. Choi, Y. M., Kim, O. Y., Kim, K. H., Kim, B. C., & Rhee, M. S. (2009b). Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium and E. coli O157: H7 in fresh pork. Letters in Applied Microbiology, 49(4), 510–515.
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(2011). Efficacies of soy sauce and wine base marinades for controlling spoilage of raw beef. Food Microbiology, 28(1), 158–163. Kataoka, S. (2005). Functional effects of Japanese style fermented soy sauce (shoyu) and its components. Journal of Bioscience and Bioengineering, 100(3), 227–234. Kim, J. Y., Cha, S. M., Chung, L. N., Kim, K. O., & Chung, S. J. (2009). Developing an attitude scale for Korean style flavors. Journal of the Korean Society of Food Culture, 24(6), 805–812. Lin, Y. T., Labbe, R. G., & Shetty, K. (2004). Inhibition of Listeria monocytogenes in fish and meat systems by use of oregano and cranberry phytochemical synergies. Applied and Environmental Microbiology, 70(9), 5672–5678. Lues, J. F. R., & Van Tonder, I. (2007). The occurrence of indicator bacteria on hands and aprons of food handlers in the delicatessen sections of a retail group. Food Control, 18(4), 326–332. Mak, A. H. N., Lumbers, M., & Eves, A. (2012). Globalisation and food consumption in tourism. Annals of Tourism Research, 39(1), 171–196. Masuda, S., Hara-Kudo, Y., & Kumagai, S. (1998). Reduction of Escherichia coli O157: H7 populations in soy sauce, a fermented seasoning. Journal of Food Protection, 61(6), 657–661. Moon, H., & Rhee, M. S. (2016). Synergism between carvacrol or thymol increases the antimicrobial efficacy of soy sauce with no sensory impact. International Journal of Food Microbiology, 217, 35–41. Muras, T. M. (2009). Escherichia coli O157: H7 and Salmonella Typhimurium risk assessment during the production of marinated beef inside skirts and tri-tip roasts. Mater of Science. College Station, TX, USA: Texas A&M University. Neely, T. R., Lorenzen, C. L., Miller, R. K., Tatum, J. D., Wise, J. W., Taylor, J. F., ... Savell, J. W. (1999). Beef customer satisfaction: Cooking method and degree of doneness effects on the top round steak. Journal of Animal Science, 77(3), 653–660. Pathania, A., McKee, S. R., Bilgili, S. F., & Singh, M. (2010). Antimicrobial activity of commercial marinades against multiple strains of Salmonella spp. International Journal of Food Microbiology, 139(3), 214–217. Quintavalla, S., & Vicini, L. (2002). Antimicrobial food packaging in meat industry. Meat Science, 62(3), 373–380. Rangel, J. M., Sparling, P. H., Crowe, C., Griffin, P. M., & Swerdlow, D. L. (2005). Epidemiology of Escherichia coli O157: H7 outbreaks, United States, 1982–2002. Emerging Infectious Diseases, 11(4), 603–609. Rhee, M. S., Ryu, J. H., Kang, S. H., Kim, Y. H., & Jang, S. H. (2014). Control of pathogenic Escherichia coli (research project number: 13162MFDS045). Seoul, Korea: Ministry of Food and Drug Safety. Rhoades, J., Kargiotou, C., Katsanidis, E., & Koutsoumanis, K. P. (2013). Use of marination for controlling Salmonella enterica and Listeria monocytogenes in raw beef. Food Microbiology, 36(2), 248–253. Røssvoll, E., Sørheim, O., Heir, E., Møretrø, T., Olsen, N. V., & Langsrud, S. (2014). Consumer preferences, internal color and reduction of shigatoxigenic Escherichia coli in cooked hamburgers. Meat Science, 96(2), 695–703. Scanga, J. A., Grona, A. D., Belk, K. E., Sofos, J. N., Bellinger, G. R., & Smith, G. C. (2000). Microbiological contamination of raw beef trimmings and ground beef. Meat Science, 56(2), 145–152. Schirmer, B. C., & Langsrud, S. (2010). Evaluation of natural antimicrobials on typical meat spoilage bacteria in vitro and in vacuum-packed pork meat. Journal of Food Science, 75(2), M98–M102. Sebranek, J. G., & Bacus, J. N. (2007). Cured meat products without direct addition of nitrate or nitrite: What are the issues? Meat Science, 77(1), 136–147. Sofos, J. N. (2008). Challenges to meat safety in the 21st century. Meat Science, 78(1), 3–13. Solomakos, N., Govaris, A., Koidis, P., & Botsoglou, N. (2008a). The antimicrobial effect of thyme essential oil, nisin and their combination against Escherichia coli O157: H7 in minced beef during refrigerated storage. Meat Science, 80(2), 159–166. Solomakos, N., Govaris, A., Koidis, P., & Botsoglou, N. (2008b). The antimicrobial effect of thyme essential oil, nisin, and their combination against Listeria monocytogenes in minced beef during refrigerated storage. Food Microbiology, 25(1), 120–127. Veldhuizen, E. J. A., Creutzberg, T. O., Burt, S. A., & Haagsman, H. P. (2007). Low temperature and binding to food components inhibit the antibacterial activity of carvacrol against Listeria monocytogenes in steak tartare. Journal of Food Protection, 70(9), 2127–2132. Yusop, S. M., O'Sullivan, M. G., Kerry, J. F., & Kerry, J. P. (2010). Effect of marinating time and low pH on marinade performance and sensory acceptability of poultry meat. Meat Science, 85(4), 657–663.
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Teriyaki sauce with carvacrol or thymol effectively controls Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and indigenous flora in marinated beef and marinade Hyeree Moon a, Nam Hee Kim a, Soon Han Kim b, Younghoon Kim c, Jee Hoon Ryu a, Min Suk Rhee a,⁎ a b c
Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea Food Microbiology Division, National Institute of Food and Drug Safety Evaluation, North Chungcheong Province 363-951, Republic of Korea BK 21 Plus Graduate Program, Department of Animal Science and Institute of Rare Earth for Biological Application, Chonbuk National University, Jeonju 561-756, Republic of Korea
a r t i c l e
i n f o
Article history: Received 19 October 2016 Received in revised form 2 March 2017 Accepted 2 March 2017 Available online 06 March 2017 Chemical compounds: Carvacrol (PubChem CID: 10364) Thymol (PubChem CID: 6989) Keywords: Teriyaki sauce Marinated beef Carvacrol Thymol Antibacterial activity Synergism
a b s t r a c t An effective bactericidal cold-marinating method for beef products is described, exploiting the synergism between soy sauce and natural compounds (carvacrol, CV or thymol, TM) to reduce microbiological risks. Beef slices inoculated with Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium (3.1–3.5 log CFU/g) were marinated in a teriyaki sauce with or without CV and TM (0.3 and 0.5%). After 1, 3, and 7 days at 4 °C, indigenous microflora population, color, lipid oxidation, marinade uptake, and pH of marinated beef and leftover marinade samples were examined. Teriyaki sauce alone did not reduce or inhibit any of the target pathogens or indigenous bacteria, while 0.5% CV- or TM-containing teriyaki sauce inactivated all inocula without recovery within 7 days (p b 0.05). The pathogens relocated from the beef into the leftover marinade (3.0–3.4 log CFU/mL) were also completely inactivated. The treatment inhibited growth of indigenous aerobic bacteria (p b 0.05) and inactivated coliform bacteria. Physicochemical parameters were not significantly affected (p N 0.05). © 2017 Elsevier Ltd. All rights reserved.
1. Introduction Undercooked meat products have been identified as a leading cause of several outbreaks of foodborne illnesses worldwide (Scanga et al., 2000; Sofos, 2008). In particular, Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella Typhimurium are frequently associated with outbreaks caused by processed meat products (CDC, 2006, 2014; Choi, Bae, Kim, Kim, & Rhee, 2009; Choi, Kim, Kim, Kim, & Rhee, 2009; Rhoades, Kargiotou, Katsanidis, & Koutsoumanis, 2013). Rangel, Sparling, Crowe, Griffin, and Swerdlow (2005) reported that ~ 47% E. coli O157:H7 outbreaks have been associated with beef products. Since consumers generally prefer medium rare or less-well cooked beef to over-cooked beef (Behrends et al., 2005; Neely et al., 1999; Røssvoll et al., 2014), it is imperative to devise an effective technique for eliminating or controlling those pathogens in commercial beef products at cold temperatures.
⁎ Corresponding author at: Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 5–1 Anam–dong, Sungbuk–gu, Seoul 02841, Republic of Korea. E-mail address: [email protected] (M.S. Rhee).
http://dx.doi.org/10.1016/j.meatsci.2017.03.001 0309-1740/© 2017 Elsevier Ltd. All rights reserved.
Marination, the soaking of food in a seasoned sauce before cooking, is one of many effective flavoring methods, not only enhancing flavor but also improving tenderness and juiciness of meat products (Björkroth, 2005). A number of marinated meat products are currently commercially manufactured and on sale, including barbecue ribs and steaks. Commercially marinated meat products, as ready-to-cook foods, are gaining popularity due to their convenience, whereas their hygiene and safety have not been extensively demonstrated (Jo, Lee, Kang, Shin, & Byun, 2004; Kanatt, Rao, Chawla, & Sharma, 2013). Indeed, coliform bacteria, typical indicators of food hygiene and food safety potential indicators (Brown et al., 2000; Lues & Van Tonder, 2007), were occasionally found in significant amounts (0.3–5.5 log CFU/g, 61.0% detection rate) in sold packaged marinated meat products (Rhee, Ryu, Kang, Kim, & Jang, 2014). Marination in fermented soy sauce is a typical beef marinating procedure and several manufacturers apply this method in their products. Since international consumption of ethnic foods has been growing because of food consumer globalization and tourism (Mak, Lumbers, & Eves, 2012), beef products marinated in soy sauce, including ‘bulgogi’, have become popular worldwide (Jo et al., 2004; Kim, Cha, Chung, Kim, & Chung, 2009). Although the marinade itself is generally regarded as a bacterial growth inhibitor, because of its acidic pH and high
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concentration of salt, preservatives, and spices (Björkroth, 2005; Kargiotou, Katsanidis, Rhoades, Kontominas, & Koutsoumanis, 2011), several researchers report that the marinade alone did not completely control pathogen growth under real processing conditions (e.g., refrigeration) (Masuda, Hara-Kudo, & Kumagai, 1998; Pathania, McKee, Bilgili, & Singh, 2010). Cured meat products, including marinated beef, traditionally contain synthetic chemical preservatives, such as nitrate and nitrite (Sebranek & Bacus, 2007). However, because of the growing popularity of natural and organic foods, there has been a consumer shift away from chemical preservatives in foods. Antibacterial activity of various plant-derived essential oils (EOs) and their components has long been studied, and they have become popular as natural food additives (Burt, 2004). In our previous study, we discovered a significant synergistic antibacterial interaction between soy sauce and six different EOs [carvacrol (CV), thymol (TM), eugenol, trans-cinnamaldehyde, β-resorcyclic acid, and vanillin]. CV and TM showed significantly higher bactericidal synergism with soy sauce than other EOs against E. coli O157:H7, L. monocytogenes, and S. Typhimurium within 10 min exposure at refrigeration temperatures, even during refrigeration (Moon & Rhee, 2016). Therefore, it is expected that the inclusion of CV and TM in soy sauce beef marination may effectively control potentially hazardous pathogens in meat products and could result in a microbiologically safe and high-quality final product. In this study, therefore, we aimed to develop an effective antimicrobial marinating method using soy sauce-based marinade and EO components (CV and TM) to control (1) artificial inoculum of foodborne pathogenic bacteria (E. coli O157:H7, L. monocytogenes, and S. Typhimurium), and (2) indigenous microorganisms (aerobic plate counts and total coliform counts) in both marinated beef and leftover marinades. In addition, physicochemical parameters (color, lipid oxidation, marinade uptake, and pH) were also measured and compared between EO-untreated and EO-treated groups to validate industrial applicability of the developed marinating method. 2. Materials and methods 2.1. Bacterial cell suspensions Three strains of each species were obtained from American Type Culture Collection. These were: E. coli O157:H7 strains ATCC35150, 43889, 43895; L. monocytogenes strains ATCC 19111, 19115, 19117; and S. Typhimurium strains ATCC 14028, 19585, and DT104 Killer Cow. They were used as test microorganisms. Each strain was stored at − 20 °C in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) with 20% glycerol and sub-cultured on monthly basis. The strains were separately cultured in 10 mL fresh TSB at 37 °C for 18 h, and three strains of the same species were mixed in a plastic 50-mL centrifuge tube (Difco). After centrifugation at 1800 × g for 15 min (Centra-CL2; International Equipment Company, Needham Heights, MA, USA), bacterial pellets were washed twice with 0.85% sterile saline solution. The final pellet was re-suspended in 10 mL 0.85% sterile saline (density ~8–9 log CFU/mL).
(see Section 2.1.), as follows. Bacterial suspensions were diluted 10-fold with 0.85% saline to obtain 4–5 log CFU/mL cell densities. The suspensions were dispensed over beef piece surface (final concentration 3–4 log CFU/g inoculated meat). Inoculated beef pieces were incubated at 4 °C overnight to allow the inoculum to attach to the sliced beef surface. After refrigeration, two 10 g sample pieces were randomly selected and used for further experiments. To assess the changes in indigenous bacterium concentration and physicochemical parameters associated with marination (with or without EO addition), uninoculated beef slices were used as experimental samples.
2.3. Marination Teriyaki sauce (Preservative-Free Kikkoman Teriyaki Sauce, Kikkoman Co., Japan) was purchased from a local market and stored at 4 °C until use. Ingredients in teriyaki sauce were soy sauce, wine, high fructose corn syrup, water, vinegar, salt, spices, onion powder, and garlic powder. CV and TM (Sigma-Aldrich, St. Louis, MO, USA) stock solutions (50 ×) were prepared by dissolving the appropriate amount of each compound in 98% ethanol. Stock solutions (200 μL) were added to 9.8 mL teriyaki sauce in a sterile plastic 50-mL tube (Difco), to adjust the final EO concentration to 0.3% and 0.5% (v/v). Raw sliced beef samples and samples treated with teriyaki sauce +2% ethanol were used as negative (unmarinated) and positive controls (teriyaki sauce-marinated), respectively. Beef samples (10 g) were then immersed in the prepared CV- or TM-supplemented marinades and gently agitated with sterile forceps. After fastening the lid of the plastic 50-mL tube, the marinated beef was incubated at 4 °C for 7 days. The experiment was performed in triplicate using different beef sample produced in a different batch. 2.4. Microbiological analysis Following 1, 3, and 7-day refrigeration, marinated beef and leftover marinades were collected separately. Each marinated beef piece and leftover marinade was placed in a sterile stomacher bag (JS-010, Jin Sung Uni-Tech, Seoul, Korea) and homogenized in 10 volumes of sterile 0.85% saline (230 rpm for 2 min) (Circulator 400, Seward, Worthing, UK). Aliquots (0.1 mL) of the homogenate were serially diluted in 9 mL 0.85% saline 104 ×. Each dilution (0.1 mL) was then spread onto the following selective agar plates: MacConkey sorbitol agar (Difco) for E. coli O157:H7 detection; Oxford Agar supplemented with Listeria selective supplement (Difco) for L. monocytogenes detection; and xylose-lysine-desoxycholate agar (Difco) for S. Typhimurium detection. Plate count agar (Difco) and violet red bile agar (Difco) were used for aerobic plate count (APC) analysis and total coliform counts (TCC), respectively. To lower the detection limit to 10 CFU/g beef and 1 CFU/mL marinade, undiluted homogenate (0.2 mL) was spread onto five plates of each agar. All inoculated plates were incubated at 37 °C for 24 h except for PCA (48 h at 37 ± 2 °C), and typical colonies were counted.
2.2. Beef preparation and inoculation
2.5. Validation of antimicrobial marination
Beef sirloins, sliced to 5 mm thickness, were purchased from a local supermarket and stored at − 20 °C. The day before use, beef sirloins were defrosted at 4 °C overnight. Samples were cut into 10 g pieces (30 × 25 × 5 mm) with a flame-sterilized kitchen knife. Presence of E. coli O157:H7, L. monocytogenes, and S. Typhimurium in sliced beef was tested by examining 25 g samples according to method described in the Bacteriological Analytical Manual by the United States Food and Drug Administration. Detection of target microorganisms led to rejection of the entire beef sirloin from which the sample was obtained. Thus prepared samples were inoculated with bacterial cell suspensions
If none of the inoculum was detected by the plate counting method, recovery of injured bacterial cells was assessed to identify treatment conditions that resulted in a complete inactivation of the inoculum. Beef samples subjected to each condition were separately transferred into sterile stomacher bags and homogenized at 230 rpm for 2 min in 10 volumes of fresh TSB. After 24 h incubation at 37 °C, enriched samples were streaked on selective agar plates, as mentioned in Section 2.4, and further incubated at 37 °C for 24 h. The absence or presence of typical colonies was recorded as a positive or negative result, respectively. The experiments were performed in triplicate.
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2.6. Measurements of physicochemical parameters Beef color and lipid oxidation, marinade uptake by the marinated beef, and pH of the marinated beef and leftover marinades were determined after 1, 3, and 7 days of marination, as physicochemical parameters indicative of the quality of marinated beef products. Marinated beef color was examined with Chroma meter (DP–400, Minolta Camera Co., Osaka, Japan) and expressed as L* (lightness), a* (redness), and b* (yellowness) values. The extent of lipid oxidation was evaluated by measuring thiobarbituric acid reactive substance (TBARS) levels, according to method by Buege and Aust (1978). TBARS values were expressed as (mg malondialdehyde)/(kg beef). Marinated beef marinade uptake was estimated according to the method of Yusop, O'Sullivan, Kerry, and Kerry (2010). The pH values of treated marinated beef samples and leftover marinades were measured at room temperature with pH-meter (Testo 206, Testo, Lenzkirch, Germany).
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uninoculated teriyaki sauce marinade was microorganisms-free. During 7-day refrigeration of beef without marination, APC increased to 5.0 log CFU/g (Fig. 2a), whereas TCC remained stable and within 0.9– 1.3 log CFU/g (Fig. 2c). When the beef was marinated only in teriyaki sauce, APC maintained the initial level (3.2–3.4 log CFU/g) for 3 days and then slightly increased, to 3.9 log CFU/g, after 7 days. Over 7-day period, TCC was slightly reduced, by 0.3–0.5 log CFU/g. By contrast, teriyaki sauce containing CV or TM resulted in a complete TCC elimination from marinated beef samples without recovery within 1 day. In the leftover marinade (Fig. 2b and d), sliced beef microorganisms may have relocated to the marinade. Without CV or TM, APC in the leftover marinade increased to 3.7 log CFU/mL. teriyaki sauce with CV or TM inhibited the increase of APC by 1.2–2.8 log CFU/mL or 1.5–3.3 log CFU/mL, respectively (Fig. 2b). There are no significant differences in APC levels of beef samples between all of the CV or TM treated groups. However, TCC data trends were different from APC. Leftover marinade TCC remained undetectable until the end of the experiment (Fig. 2d).
2.7. Statistical analysis Triplicate experimental data on bacterial populations in marinated beef (CFU/g) and in leftover marinade (CFU/mL) were expressed as Log10 CFU/g values. Bacterial population log values and physicochemical parameter values were expressed as mean ± standard error. The data were analyzed using SAS software version 9.4 (SAS Institute, Cary, NC, USA) with general linear mixed model. The fixed effects in the model were storage time (0, 1, 3, and 7 days), and the random effects were treatment (non-marinated, teriyaki sauce, teriyaki sauce + CV or TM). If each effect was significant, mean values were subjected to post-hoc Tukey's multiple range test for mean separation. 3. Results 3.1. Bactericidal activity of cold-marination with carvacrol or thymol against foodborne pathogens Fig. 1 shows changes in bacterial populations of E. coli O157:H7, L. monocytogenes, and S. Typhimurium in marinated beef and leftover marinades during cold-marination with teriyaki sauce containing CV or TM (0.3 and 0.5%). The overall data showed similar tendencies for all the bacterial species tested. More bacteria were inactivated with increasing EO concentrations and marinating time. Bactericidal effects of CV and TM were not significantly different at the same treatment condition (p N 0.05). In the non-marinated beef sample (Fig. 1a–c), the initial inoculum (3.1–3.5 log CFU/g) did not significantly change (p N 0.05) during 7day refrigeration. When sliced beef was marinated only in teriyaki sauce, bacterial numbers initially decreased by 0.4–0.5 log CFU/g and remained at that level until the end of the experiment. Whereas teriyaki sauce alone did not significantly reduce bacterial numbers (b1 log CFU/g reduction), teriyaki sauce with CV or TM effectively inactivated the inoculated foodborne pathogens. In particular, all inocula were completely inactivated without recovery by 7-day cold-marination with 0.5% CV or TM, except S. Typhimurium by teriyaki sauce + CV 0.5%. In the leftover marinade without CV or TM (Fig. 1d–f), the inoculum relocated into the marinade from sliced beef surface, increasing marinade bacterial levels up to 3.0–3.4 log CFU/mL. By comparison, supplementation of the marinade with CV or TM inhibited the survival of the translocated inoculum to undetectable levels. 3.2. Inhibitory activity of cold-marination with carvacrol or thymol against beef sample indigenous bacteria Fig. 2 shows bacterial growth inhibition (APC) or indigenous bacterium inactivation (TCC) by cold-marination with 0.3% and 0.5% concentrations of CV and TM. Before marination, 3.5 log CFU/g APC and 1.1 log CFU/g TCC were detected in the sliced beef samples, while
3.3. Quality parameters of the marinated beef and leftover marinade during cold-marination with carvacrol or thymol Beef color and lipid oxidation, marinade uptake by the marinated beef, and marinated beef and leftover marinade pH values are presented in Table 1. As indicated by L*, a*, and b* values, the addition of CV or TM to teriyaki sauce marinade did not significantly affect marinated beef color (p N 0.05). TBARS values ranged between 3.2 and 3.7 mg malondialdehyde per kg marinated beef, and no statistically significant differences were observed comparing teriyaki sauce-only marination (3.2–3.7 mg/kg) with teriyaki sauce + CV (3.2–3.7 mg/kg) or teriyaki sauce + TM (3.3–3.6 mg/kg) (p N 0.05) during 7-day refrigeration. The sliced beef absorbed on average 21.2–24.9% teriyaki sauce marinade in the absence or presence of CV or TM, no significant differences in marinade uptake (%) were observed between the experimental groups (p N 0.05). Marinated beef and leftover marinade pH values ranged between 5.0 and 5.2 and 4.8–5.0, respectively, and were not significantly affected in any of the experimental groups (p N 0.05). 4. Discussion This study suggests an effective intervention, using teriyaki sauce supplemented with a small amount of EOs, that can completely inactivate major foodborne pathogens (E. coli O157:H7, L. monocytogenes, and S. Typhimurium) and coliform bacteria in marinated beef and leftover marinade during cold-marination. Results of this study indicated that soy sauce-based teriyaki sauce alone did not significantly reduce foodborne pathogen levels in a marinated beef product during refrigeration. The commercially available teriyaki sauce marinade used in this study contains soy sauce, wine, high fructose corn syrup, water, vinegar, salt, spices, onion powder, and garlic powder as major ingredients, and was “preservative-free”. This is in agreement with a previous publication by Masuda et al. (1998), who reported that E. coli O157:H7 levels were not affected by dipping in soy sauce (main component of teriyaki sauce) at 4 °C for 9 days. Thus the refrigeration temperature (4 °C), at which marinated beef products were tested in this study, might not have been optimal for the antibacterial action of teriyaki sauce. Several groups previously reported the various antibacterial parameters of teriyaki sauce (e.g., high sodium content, low pH, ethanol, and preservatives) (Kataoka, 2005; Rhoades et al., 2013). Indeed, Pathania et al. (2010) found that teriyaki sauce inactivated Salmonella spp. to below the detection limit at 4 °C within 32 h. However, the antibacterial activity of teriyaki sauce marinade might vary depending on brand of the basic marinade used in the experiments, e.g., They applied teriyaki sauce with pH 3.7–3.8, which was significantly lower than pH of teriyaki sauce used in this study (pH 4.9–5.0) (Pathania et al., 2010). High sodium chloride levels in the soy sauce included in teriyaki sauce marinade may damage bacterial cell membranes (Hajmeer, Ceylan, Marsden, &
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Fig. 1. Bacterial populations in marinated beef and marinade following 1, 3, and 7-day marination at refrigeration temperature (4 °C). Escherichia coli O157:H7 population in the marinated beef (a) and leftover marinade (d); Salmonella Typhimurium population in the marinated beef (b) and leftover marinade (e); and Listeria monocytogenes population in the marinated beef (c) and leftover marinade (f). Data points represent population means and error bars indicate standard error from triplicate experiments.
Fung, 2006), but the final sodium chloride concentration in the teriyaki sauce marinade (610 mg per 15 mL, i.e., 4.1%) was not sufficient to exert an antibacterial effect. In addition, meat and meat products might constitute a pathogen survival-favoring environment (Veldhuizen, Creutzberg, Burt, & Haagsman, 2007). The antimicrobial activity of teriyaki sauce was likely reduced because of interaction with beef components (Quintavalla & Vicini, 2002; Veldhuizen et al., 2007). To enhance the antibacterial efficacy of soy sauce-based marinade that would reflect consumer avoidance of synthetic chemical preservatives, some attempts have been made to find the appropriate natural antimicrobial agents. Although the target microorganisms differed (i.e., spoilage bacteria instead of foodborne pathogens), Schirmer and Langsrud (2010) tested the inhibitory effects of several natural antimicrobials, including allyl isothiocyanate, ascorbic acid, cinnamaldehyde,
citric acid, grapefruit seed extract, rosemary extract, etc. They concluded that the use of these antimicrobials in meat products was limited because of insufficient antimicrobial activity, and strong odor and flavor which consumers may find unacceptable. Rhoades et al. (2013) reported the antibacterial activity of soy sauce marinade with lactic acid against S. enterica and L. monocytogenes in beef, with bacterial levels declining from 6 to 3 log CFU/g during 10-day marination at 5 °C. However, the pH of that marinade was 3.8 potentially affecting the quality of meat, possibly affecting the quality of meat (Alvarado & McKee, 2007). On the other hand, previous studies reported inhibitory effects of various EOs toward E. coli O157:H7, L. monocytogenes, and S. Typhimurium in a variety of meat products. In particular, antibacterial effects of oregano and thyme oil, whose predominant components are CV and TM, have been studied in meat products (Govaris, Solomakos,
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Fig. 2. Aerobic plate counts from marinated beef (a) and leftover marinade (b), and total coliform counts in marinated beef (c) and leftover marinade (d), after 1, 3, and 7-day marination at refrigeration temperature (4 °C). Data points represent population means and error bars indicate standard error from triplicate experiments.
Pexara, & Chatzopoulou, 2010; Solomakos, Govaris, Koidis, & Botsoglou, 2008a, 2008b). For instance, when Solomakos et al. (2008b) treated L. monocytogenes in minced beef with 0.6% thyme oil at 4 °C for 12 days, the bacterial levels were reduced from 4 to 3 log CFU/g. Similarly, Govaris et al. (2010) reported that 2-day refrigeration (4 °C) of minced shrimp with 0.6 and 0.9% oregano oil reduced Salmonella Enteritidis numbers, from ~ 4 log CFU/g to ~ 2.5 and 0.5 log CFU/g, respectively, maintained until the end of the experiment (12 days). However, several publications reported that EOs alone did not exert a significant bactericidal effect against foodborne pathogens. Indeed, when Lin, Labbe, and Shetty (2004) applied oregano extract to beef and fish slices, the numbers of inoculated L. monocytogenes cells remained largely at the initial level (8 log CFU/g) during 8-day incubation at 4 °C. Also, Veldhuizen et al. (2007) reported that 2.5 mM CV (0.04%) did not inactivate L. monocytogenes in steak tartare at 10 °C and 20 °C during 100-h storage. Solomakos et al. (2008a) similarly concluded that thyme oil did not reduce E. coli O157:H7 numbers in minced beef samples. By comparison, the effective cold bactericidal marinating method suggested in this study resulted in a complete inactivation without recovery of the major foodborne pathogens, E. coli O157:H7, L. monocytogenes, and S. Typhimurium, and also food hygiene indicator bacteria (coliform bacteria), over 7-day refrigeration (4 °C). Individual
teriyaki sauce (Fig. 1a–c) or EOs (CV and TM) (data not shown) treatments did not exert a significant antibacterial effect against beef sample inocula. Thus, the significantly higher bactericidal effect of teriyaki sauce with EOs might be caused by their synergistic interaction, as we previously reported (Moon & Rhee, 2016). Moreover, no significant differences in physicochemical sample parameters (i.e., color, lipid oxidation, marinade uptake, and pH) were observed between samples marinated with teriyaki sauce and EOs-supplemented teriyaki sauce. Solomakos et al. (2008a) evaluated the odor of thyme oil-treated minced beef, and found that 0.3% and 0.6% thyme oil treatment was acceptable to beef consumers, whereas the odor of 0.9% thyme oil was described as unpleasant. Thus, the method developed in this study can be successfully used in the marinated beef industry, because EO amounts used in this study (0.3% and 0.5%) were lower than 0.6% cut-off. In addition, teriyaki sauce marinade, with its unique odor and flavor, is expected to mask the characteristic CV and TM odors. The marked synergism between teriyaki sauce and EOs (CV and TM) will also contribute to reducing several possible microbiological risk factors affecting the microbiological safety and quality of marinated beef product manufacture: cross-contamination and growth of harmful bacteria. Cross-contamination has been proposed as one of the major risk factors affecting microbiological safety and quality of marinated meat products (Björkroth, 2005; Choi, Bae, et al., 2009). As indicated in
Table 1 Physicochemical parameters of marinated beef and leftover marinade. pH
Color Day
Essential oil
%
L*
1
– Carvacrol
– 0.3 0.5 0.3 0.5 – 0.3 0.5 0.3 0.5 – 0.3 0.5 0.3 0.5
31.7 30.6 31.4 30.3 31.0 31.3 32.4 31.8 31.3 31.7 31.5 31.6 31.1 31.7 32.1
Thymol 3
– Carvacrol Thymol
7
– Carvacrol Thymol
a* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.3 0.4 1.6 1.6 0.4 0.5 0.7 0.6 0.3 0.2 0.4 0.5 0.5 0.7
9.6 9.7 9.9 9.9 10.7 10.7 10.0 10.0 10.5 10.2 10.1 10.3 9.8 10.4 10.0
b* ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.2 0.0 0.4 0.4 0.3 0.3 0.5 0.5 0.1 0.5 0.5 0.6 0.6 0.4
17.6 16.0 17.1 16.0 17.6 15.5 15.7 14.8 16.7 15.9 15.8 16.2 15.5 15.8 16.0
Lipid oxidation (mg/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.3 0.1 0.1 1.6 1.4 0.1 0.3 0.6 0.8 0.4 0.3 0.4 0.5 0.8 0.3
3.2 3.2 3.7 3.5 3.6 3.7 3.4 3.5 3.6 3.5 3.6 3.4 3.4 3.4 3.3
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0d 0.1bcd 0.1bcd 0.1bcd 0.1bc 0.2b 0.1bcd 0.2bcd 0.0bcd 0.1bcd 0.1bcd 0.0bcd 0.0bcd 0.1bcd 0.1bcd
a
Marinade uptake (%)
Marinated beef
Leftover marinade
24.8 24.5 23.7 22.7 22.1 23.7 24.1 23.2 24.9 24.6 24.0 22.2 25.0 21.2 21.8
5.1 5.0 5.0 5.0 5.1 5.0 5.0 5.1 5.1 5.1 5.1 5.1 5.2 5.1 5.0
4.8 4.9 4.9 4.9 4.8 4.8 4.9 4.8 4.9 4.9 4.9 4.9 5.0 4.8 4.9
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
Data represent means ± standard error (n = 3). b–d Values designated by different superscripts in the same column are significantly different (p b 0.05). a Lipid oxidation was determined as thiobarbituric acid reactive substances (TBARS), mg malondialdehyde per kg beef.
0.8 1.0 2.3 1.8 2.1 1.3 3.7 3.8 0.9 0.6 1.9 5.2 0.3 1.6 2.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.0 0.0 0.1 0.1 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.0 0.0
152
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Fig. 1d–f, the addition of small amounts of CV or TM resulted in reduced (undetectable, b1 CFU/mL) foodborne pathogen and coliform levels in the leftover marinade. Considering that consumers may reuse spent marinade, causing a potential contamination risk to products during subsequent marination (Muras, 2009), the developed method will be helpful in controlling this problem. Moreover, as this approach inhibited bacterial growth in both marinated beef and leftover marinade (Fig. 2a), it is also expected that it will control rapid growth of harmful bacteria that adversely affect the quality of marinated beef, such as typical spoilage bacteria. In conclusion, we developed an effective antibacterial beef coldmarinating method against E. coli O157:H7, L. monocytogenes, and S. Typhimurium, and indigenous coliform bacteria, exploiting the remarkable synergism of soy sauce in teriyaki sauce with common natural compounds, EOs (CV and TM). Practical application of this method in marinated beef processing has several advantages, as follows: (1) use of only natural compounds (preventing consumer avoidance), (2) simplicity and convenience (a simple addition of the appropriate CV or TM amounts results in significant bacterial inactivation), and (3) continuity of the antibacterial effect under refrigeration (i.e., the bactericidal effect is preserved during distribution). The method will also contribute to preventing bacterial contamination via re-use of contaminated marinade and, ultimately, to delivery of microbiologically safe marinated meat products. We demonstrated that the synergistic antibacterial effect of soy sauce with EOs (CV or TM) is applicable in a real food scenario. This technique will contribute to various marinades and marinated foods industry. Acknowledgements This study was supported by a grant (13162MFDS045) from the Korean Ministry of Food and Drug Safety (2013–2014) and, partially, by a Korea University Grant. Authors also thank the School of Life Sciences and Biotechnology of Korea University for BK 21 PLUS and the Institute of Biomedical Science and Food Safety, CJ-Korea University Food Safety Hall, for providing equipment and facilities. References Alvarado, C., & McKee, S. (2007). Marination to improve functional properties and safety of poultry meat. The Journal of Applied Poultry Research, 16(1), 113–120. Behrends, J. M., Goodson, K. J., Koohmaraie, M., Shackelford, S. D., Wheeler, T. L., Morgan, W. W., ... Savell, J. W. (2005). Beef customer satisfaction: USDA quality grade and marination effects on consumer evaluations of top round steaks. Journal of Animal Science, 83(3), 662–670. Björkroth, J. (2005). Microbiological ecology of marinated meat products. Meat Science, 70(3), 477–480. Brown, M. H., Gill, C. O., Hollingsworth, J., Nickelson, R., Seward, S., Sheridan, J. J., ... Usborne, W. R. (2000). The role of microbiological testing in systems for assuring the safety of beef. International Journal of Food Microbiology, 62(1), 7–16. Buege, J. A., & Aust, S. D. (1978). [30] Microsomal lipid peroxidation. Methods in Enzymology, 52, 302–310. Burt, S. (2004). Essential oils: Their antibacterial properties and potential applications in foods—A review. International Journal of Food Microbiology, 94(3), 223–253. CDC (2006). Multistate outbreak of Salmonella Typhimurium infections associated with eating ground beef-United States, 2004. Morbidity and Mortality Weekly Report, 55, 180–182. CDC (2014). Multistats outbreak of Shiga toxin-producing Escherichia coli O157:H7 infections linked to ground beef (final update). Retrieved from http://www.cdc.gov/ecoli/ 2014/O157H7-05-14/index.html (Accessed on January 13, 2016) Choi, Y. M., Bae, Y. Y., Kim, K. H., Kim, B. C., & Rhee, M. S. (2009a). Effects of supercritical carbon dioxide treatment against generic Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium, and E. coli O157: H7 in marinades and marinated pork. Meat Science, 82(4), 419–424. Choi, Y. M., Kim, O. Y., Kim, K. H., Kim, B. C., & Rhee, M. S. (2009b). Combined effect of organic acids and supercritical carbon dioxide treatments against nonpathogenic Escherichia coli, Listeria monocytogenes, Salmonella Typhimurium and E. coli O157: H7 in fresh pork. Letters in Applied Microbiology, 49(4), 510–515.
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