Synergism between carvacrol or thymol increases the antimicrobial efficacy of soy sauce with no sensory impact

International Journal of Food Microbiology 217 (2016) 35–41

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International Journal of Food Microbiology journal homepage: www.elsevier.com/locate/ijfoodmicro

Synergism between carvacrol or thymol increases the antimicrobial efficacy of soy sauce with no sensory impact Hyeree Moon, Min Suk Rhee ⁎ Department of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul 02841, Republic of Korea

a r t i c l e

i n f o

Article history: Received 21 May 2015 Received in revised form 7 October 2015 Accepted 10 October 2015 Available online xxxx Chemical compounds studied in this article: Carvacrol (PubChem CID: 10364) Thymol (PubChem CID: 6989) Eugenol (PubChem CID: 3314) Trans-cinnamaldehyde (PubChem CID: 637511) β-resorcylic acid (PubChem CID: 1491) Vanillin (PubChem CID: 1183) Keywords: Soy sauce Carvacrol Thymol E. coli O157:H7 S. Typhimurium L. monocytogenes

a b s t r a c t Here, we examined the antimicrobial effects of soy sauce containing essential oils (EOs) against Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes at 22 °C and 4 °C. To screen a variety of combined effects, soy sauce was mixed with six different EOs (carvacrol, thymol, eugenol, trans-cinnamaldehyde, β-resorcylic acid, and vanillin), each at a concentration of 1 mM for 10 min. None of the oils showed bactericidal activity when used alone. Soy sauce combined with carvacrol and thymol induced the greatest antibacterial activity against all tested bacteria; therefore, these oils were further tested at 0.25, 0.5, and 1 mM (0.0039%, 0.0078%, and 0.0157%) for 1, 5, and 10 min at 4 °C and 22 °C. In addition, sensory evaluation of soy sauce containing each EO at 0.25, 0.5, 1, and 2 mM was performed using the nine point hedonic test. Carvacrol or thymol (1 mM) eliminated all the test bacteria (initial population, 7.0–7.5 log CFU/ml) in 1–5 min at 22 °C and within 10 min at 4 °C. L. monocytogenes was slightly more tolerant at 4 °C, which may be attributable to the ability of the cell membrane to adapt to low temperatures. The sensory scores for soy sauce containing EOs were not significantly different from that of soy sauce without EOs (P N 0.05). The stability of EO efficacy in soy sauce was also verified. These results suggest that carvacrol and thymol act synergistically with other factors present in soy sauce to increase antimicrobial activity against major foodborne pathogens at both 4 °C and 22 °C. The synergism may be attributable to the combination of factors (mainly high salt concentration and low pH imparted by organic acids) present in soy sauce and the membrane attacking properties of carvacrol and thymol. This method will facilitate the production of microbiologically safe soy sauce, soy sauce-based marinades, and various marinated foods. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Soy sauce, made by fermenting soy bean and wheat flour with molds or yeasts, is one of the popular condiments worldwide. Indeed, it is the third popular condiment in the USA (after mayonnaise and ketchup), with a market share worth 725 million dollars in 2014 (Ferdman and King, 2014). The soy sauce market in the USA has grown 40% since 2000 (Silva, 2014). In addition, because soy sauce contains much less sodium (307 mg per teaspoon) than salt (2325 mg per teaspoon), it is a potential salt replacement for those on a low sodium diet; this will only serve to increase market share even further (USDA, 2014a,b). Soy sauce is often used to complement salads, meat products, sea foods, and fried rice (amongst other things). Because of its antimicrobial factors such as high concentration of salts (17%), low pH (4.6 by lactic acid), ethanol (2%), and preservatives (mainly sorbates or benzoates), soy sauce has been utilized to control food deterioration, and that is ⁎ Corresponding author at: Department 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.ijfoodmicro.2015.10.009 0168-1605/© 2015 Elsevier B.V. All rights reserved.

one of the reasons why soy sauce is used to preserve and season foods (Kataoka, 2005; Masuda et al., 1998). Unfortunately, contrary to expectation, foods containing soy sauce have caused several outbreaks of food poisoning (CDC, 1995, 1998; Mizuta et al., 1956). Furthermore, previous studies show that long exposure (6 h–7 days) to soy sauce merely inhibits the growth of foodborne pathogens such as Escherichia coli, E. coli O157:H7, Salmonella spp., Shigella flexneri, Shigella sonnei, and Staphylococcus aureus, rather than killing them or reducing their numbers to a safe limit, particularly at temperatures used for refrigeration (Kataoka, 2005; Masuda et al., 1998). The US Department of Agriculture (USDA) and the US Food and Drug Administration (USFDA) recommend that seasoned or marinated foods be stored at refrigeration temperatures; however, once soy sauce is contaminated with foodborne pathogens, it cannot inactivate the pathogens at these temperatures, and can even cross-contaminate foods seasoned with it (USDA, 2013; USFDA, 2014). However, combining soy sauce with other antimicrobial factors may improve the safety of various foods containing soy sauce. There is a growing interest in “natural” food additives to guarantee food safety and to satisfy health conscious people (Kim and Rhee, 2013). Essential oils (EOs) are natural plant extracts that have antibacterial properties (Bakkali et al., 2008; Burt, 2004; Janssen et al., 1987;

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H. Moon, M.S. Rhee / International Journal of Food Microbiology 217 (2016) 35–41

Kivanc and Akgäul, 1988). Although naturally occurring EOs are becoming popular with the food industry, there are several barriers to their use in foods; 1) they must be present at high concentrations to exhibit effective bactericidal activity (Burt, 2004; Gutierrez et al., 2008); 2) they are much more expensive than synthetic additives; 3) the strong smell of EOs is unacceptable from an organoleptic perspective (Angienda and Hill, 2011; Lambert et al., 2001). To overcome these barriers, hurdle technologies based on EOs have been developed, and several studies report the combined antibacterial effects of various EOs (Bevilacqua et al., 2010; Delaquis et al., 2002; Didry et al., 1993; Marino et al., 2001; Pei et al., 2009). Other studies report the combination of EOs with other food preservation methods, e.g., nisin, sodium chloride, sodium nitrite, chelators, organic acids, physical treatments, and fatty acids (Ait-Ouazzou et al., 2013; Choi et al., 2013; de Oliveira et al., 2010; Pol and Smid, 1999; Zhou et al., 2007). As mentioned above, studies show that the efficacy of EOs is increased when they are combined with other preservation methods (Angienda and Hill, 2011). Marination is such a method of food preservation; thus EOs may increase the antibacterial activity of marinades such as soy sauce. The objectives of the present study were to improve the antimicrobial activity of soy sauce, thereby increasing the safety of soy saucebased foods stored at both refrigeration and room temperatures. First, we screened six different EOs (carvacrol, thymol, eugenol, transcinnamaldehyde, β-resorcylic acid, and vanillin) and selected the most effective for further study in experiments with E. coli O157:H7, S. Typhimurium, and Listeria monocytogenes, which together cause the majority of foodborne diseases (CDC, 2013). Sensory changes (i.e., smell) were also evaluated. 2. Materials and methods 2.1. Bacterial strains E. coli O157:H7 (ATCC 35150, 43890, and 43895), S. Typhimurium (ATCC 14028, 19585, and DT104 Killercow), and L. monocytogenes (ATCC 19111, 19115, and 19117) were obtained from the Korea University Food Microbiology Culture Collection. Stock cultures were stored at −20 °C in tryptic soy broth (TSB; Difco, Becton Dickinson, Sparks, MD, USA) supplemented with 20% glycerol and sub-cultured every month. 2.2. Cell suspensions Each strain was separately resuscitated in 10 ml fresh TSB (Difco) for 18 h at 37 °C. To obtain the three-strain cocktail, the culture suspensions of each bacterial strain were combined in a plastic 50-ml centrifuge tube (Difco), which was then centrifuged at 3000 rpm for 15 min (CentraCL2; International Equipment Company, Needham Heights, MA, USA). After discarding the supernatant, the pellet was washed twice in 0.85% sterile saline solution. The final bacterial pellet was suspended in 10 ml of 0.85% sterile saline (approximately 9 log CFU/ml).

suspension. The microbial population in each soy sauce sample was then examined 10 min later. Soy sauce containing 1% ethanol was used as control. All experiments were repeated six times. 2.4. Antimicrobial efficacy of soy sauce containing carvacrol and thymol Carvacrol and thymol were tested under different conditions: concentration, 0.25, 0.5, or 1 mM; time, 1, 5, or 10 min; temperature, 22 °C or 4 °C. To obtain the final concentrations of carvacrol and thymol in soy sauce, stock solutions of 25, 50, and 100 mM were prepared in 98% ethanol. An aliquot (0.1 ml) of each was then pipetted into 9.8 ml of soy sauce. The sauce samples were prepared in a 22 °C incubator or 4 °C refrigerator. Immediately after preparation, 0.1 ml of bacterial suspension was added. Soy sauce containing 1% ethanol was used as control. All the experiments were repeated six times. 2.5. Microbiological analysis One ml of sample was serially diluted 10-fold in 9 ml of 0.85% saline until 10−4. Next, an aliquot (0.1 ml) of each was plated in duplicate onto MacConkey Agar containing Sorbitol (SMAC; Difco (E. coli O157:H7)), Xylose-Lysine-Desoxycholate Agar (XLD; Difco (S. Typhimurium)), or Oxford Agar supplemented with Listeria selective supplement (Oxford; Difco (L. monocytogenes)). For samples containing low levels of bacteria, 0.2 ml of undiluted sample was spread onto five plates of each selective agar (detection limit, 1 CFU/ml). Colonies were counted after the plates were incubated at 37 °C for 18 h. 2.6. Validation of antimicrobial efficacy The extent of recovery of injured cells (E. coli O157:H7, S. Typhimurium, and L. monocytogenes) was examined at the treatments where bacterial growth was not detected by plating method (Jang and Rhee, 2009; Rhee et al., 2003). One ml of treated sample was inoculated into 20 ml of TSB (Difco). Following incubating at 37 °C for 18 h, the enriched samples were streaked onto Tryptic Soy Agar (TSA; Difco). Whether the growth occurred was recorded as positive or negative, after the plates were incubated at 37 °C for 18 h. The experiments were performed in triplicate. 2.7. Stability of effects of carvacrol and thymol in soy sauce To evaluate the stability of effects of carvacrol and thymol in soy sauce, the long time exposure test and the long time storage test were performed. (1) Long time exposure test. The experiment was performed as same as 2.4, but the test time was 14 days (not 1, 5, 10 min). (2) Long time storage test. Soy sauce samples containing carvacrol and thymol made 14 days ago were used. The bacterial suspension was added in the stored samples, and the microbiological analysis was performed after 1, 5, 10 min.

2.3. Antimicrobial efficacy of soy sauce containing essential oils 2.8. Evaluation of pH The antibacterial effects of soy sauce containing six different EOs were investigated. Soy sauce (Preservative-Free Kikkoman Soy Sauce, Kikkoman Corporation, Japan), was purchased from a local market and stored at room temperature. EO solutions (100 mM) were prepared immediately prior to use by dissolving the oil (carvacrol, thymol, eugenol, trans-cinnamaldehyde, β-resorcylic acid, or vanillin; Sigma-Aldrich, St. Louis, MO, USA) in 98% ethanol. In case of the individual treatments, 9.9 ml of soy sauce was used, and an aliquot (0.1 ml) of each EO stock solution was added to 9.8 ml of 0.85% saline to yield a final concentration of 1 mM. For combined treatment, an aliquot (0.1 ml) of each EO stock solution was added to 9.8 ml of soy sauce. All samples were prepared in 22 °C incubator or in a 4 °C refrigerator prior to the addition of 0.1 ml of bacterial cell

The pH of soy sauce and of soy sauce combined with EOs was measured at room temperature using a pH/Ion meter (S220 SevenCompact pH/Ion, Mettler-Toledo, Greifensee, Switzerland) calibrated with buffers at pH 4.0 and 7.0. 2.9. Sensory evaluation The odor of soy sauce samples was evaluated by a panel of 30 untrained volunteers (13 males and 17 females) ranging in age from 19 to 30. Testing was based on the nine point hedonic test. Samples (10 ml) of soy sauce with or without EOs were placed in small paper cups. The panel members were then asked to sniff the samples and

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score their preferences based on odor (a score of 1 was given to the most unpleasant, and a score of 9 to the most pleasant). 2.10. Statistical analysis Bacterial counts were converted to log CFU per ml. The counts, pH, and sensory evaluation scores were then subjected to analysis of variance (ANOVA). The significance of the differences was determined using Tukey's multiple comparison test. All analyses were performed using SAS version 9.4 (SAS Institute, Cary, NC, USA). A P-value of b0.05 was considered significant.

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7.0–7.6 log CFU/ml). Eugenol (1 mM) also increased the antibacterial activity of soy sauce at 22 °C, although less so than carvacrol or thymol; soy sauce containing 1 mM eugenol caused a ~ 1.0-, 4.4-, and 3.2-log CFU/ml reduction in the E. coli O157:H7, S. Typhimurium, and L. monocytogenes populations, respectively, at 22 °C. Eugenol did not cause a significant reduction in the E. coli O157:H7 or L. monocytogenes populations at 4 °C (P N 0.05), and only reduced the population of S. Typhimurium by about 0.5 log CFU/ml (P b 0.05). Transcinnamaldehyde, β-resorcylic acid, or vanillin (each at 1 mM) did not improve the antibacterial activity of soy sauce (P N 0.05).

3. Results

3.2. Antimicrobial efficacy of soy sauce containing carvacrol or thymol

3.1. Antimicrobial efficacy of soy sauce containing essential oils

The addition of carvacrol or thymol to soy sauce resulted in similar improvements in antibacterial activity (P N 0.05). Increasing concentrations of either carvacrol or thymol led to increasing reductions in the bacterial populations (P b 0.05). Soy sauce containing 0.25 mM carvacrol or thymol caused a slight reduction in the bacterial populations (to 5.5–6.3 log CFU/ml) after 10 min at 22 °C (Table 1). Soy sauce containing 0.5 mM carvacrol or thymol reduced the populations to b 2.0 log CFU/ml after 5 min, and to below the level of detection at 10 min at 22 °C (initial population of all three genera, 7.1–7.3 log CFU/ml; Table 1). Soy sauce containing either

Fig. 1 shows the bactericidal effects of each individual EO alone and when added to soy sauce. The EOs alone (1 mM) showed no significant antimicrobial effect against any of the test bacteria at either 22 °C or 4 °C (P N 0.05). Soy sauce alone had low bactericidal effects (P N 0.05); however, there was a marked increase in antimicrobial activity upon addition of 1 mM carvacrol or thymol. These preparations reduced inoculated E. coli O157:H7, S. Typhimurium, or L. monocytogenes to non-detectable levels at both 22 °C and 4 °C (initial population,

Fig. 1. Reduction of E. coli O157:H7, S. Typhimurium, and L. monocytogenes populations (log CFU/ml) after exposure to soy sauce alone, soy sauce containing ethanol (1%), essential oils alone, or soy sauce containing essential oils (1 mM) for 10 min at (a) 22 °C or (b) 4 °C. Data bars represent the mean ± standard error (n = 6). Data denoted by different superscripts (a–c, A–C, x–y) are significantly different (P b 0.05); *ND, not detected.

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H. Moon, M.S. Rhee / International Journal of Food Microbiology 217 (2016) 35–41

Table 1 Bacterial populations (log CFU/ml) after exposure to soy sauce containing carvacrol or thymol at 22 °C. Bacteria

Time (min)

Bacterial population (Log CFU/ml)a Controlb

E. coli O157:H7

S. Typhimurium

L. monocytogenes

0 1 5 10 0 1 5 10 0 1 5 10

c

Carvacrol (mM)

Thymol (mM)

0.25

0.5

1

0.25

0.5

1

6.9 ± 0.1a,b 6.3 ± 0.2b,c 5.5 ± 0.3c,d

5.0 ± 0.3d,e 1.0 ± 0.4g ND (− − +)

ND⁎,h(− + −) ND (− − −) ND (− − −)

6.7 ± 0.1a,b 5.4 ± 0.2d,e 4.6 ± 0.2e,f

3.5 ± 0.3f ND (+ − +) ND (− − −)

ND (− − −) ND (− − −) ND (− − −)

6.5 ± 0.1a,b 5.1 ± 0.2c 3.8 ± 0.3d,e

4.1 ± 0.2d 0.6 ± 0.3 ND (− − +)

NDf (− − −) ND (− − −) ND (− − −)

6.2 ± 0.1b 4.5 ± 0.2c,d 3.2 ± 0.3e

4.0 ± 0.2d,e 0.1 ± 0.1f ND (+ − +)

ND (+ − −) ND (− − −) ND (− − −)

7.1 ± 0.1a 6.8 ± 0.1a 6.2 ± 0.3a,b

4.5 ± 0.7c 2.0 ± 0.7d,e 0.7 ± 0.4e,f

NDf (− − −) ND (− − −) ND (− − −)

7.0 ± 0.1a 6.4 ± 0.2a,b 5.1 ± 0.6b,c

2.8 ± 0.5d ND (+ − +) ND (− − −)

ND (− − −) ND (− − −) ND (− − −)

a

7.2 ± 0.0 7.1 ± 0.1a 7.2 ± 0.1a 7.2 ± 0.1a 7.1 ± 0.0a 7.1 ± 0.1a 6.9 ± 0.1a,b 6.9 ± 0.0a,b 7.2 ± 0.2a 7.4 ± 0.2a 7.4 ± 0.2a 7.4 ± 0.2a

Data denoted by different superscript lower case letters are significantly different (P b 0.05). Results from enrichment test are expressed as + (growth on the agar) and − (no growth on the agar) (n = 3). a Bacterial populations are expressed as the mean ± standard error (n = 6). b Control, soy sauce containing 1% ethanol. c The population at 0 min represents the initial population. ⁎ ND, not detected.

EO at 1 mM completely eradicated all target bacteria within 5 min at 22 °C, and no growth was detected by enrichment (Table 1). The EOs had less effect on the antibacterial activity of soy sauce at 4 °C than at 22 °C. Soy sauce containing 0.25 mM carvacrol or thymol did not reduce the bacterial population at 4 °C (P N 0.05; Table 2); however, soy sauce containing 0.5 mM of either EO reduced the populations to b 4.4-log CFU/ml after 10 min. Soy sauce containing carvacrol or thymol (1 mM) had the greatest effect against L. monocytogenes, whereas it took 10 min to reduce the E. coli O157:H7 and S. Typhimurium populations to below detectable levels at 4 °C. L. monocytogenes were reduced to below detectable levels within 1 min (Table 2).

plating and enrichment method (data not shown). 2) Long time storage test. Soy sauce samples made 14 days ago induced the statistically same results as Tables 1 and 2 (data not shown).

3.4. pH of the soy sauce solutions The pH of non-treated soy sauce (no EOs) was about 4.6, whereas that of soy sauce containing EOs ranged from 4.5 to 4.7. Thus, adding EOs did not lead to a significant change in the pH of soy sauce (P N 0.05; Table 3). 3.5. Sensory evaluation

3.3. Stability of effects of carvacrol and thymol in soy sauce 1) Long time exposure test. All three pathogens were not detected from soy sauce containing carvacrol and thymol after 14 days by both

The sensory score for soy sauce without EOs was 4.6 ± 0.2. There were no significant differences in the sensory scores after the addition of EO at 0.25, 0.5, and 1.0 mM (P N 0.05). The scores for soy sauce

Table 2 Bacterial populations (log CFU/ml) after exposure to soy sauce containing carvacrol or thymol at 4 °C. Bacteria

Time (min)

Bacterial population (Log CFU/ml)a Controlb

E. coli O157:H7

S. Typhimurium

L. monocytogenes

0c 1 5 10 0 1 5 10 0 1 5 10

7.2 ± 0.0a 7.2 ± 0.0a 7.2 ± 0.0a 7.1 ± 0.0a 7.3 ± 0.1a 7.3 ± 0.1a 7.3 ± 0.1a 7.2 ± 0.1a 7.3 ± 0.1a 7.3 ± 0.1a 7.4 ± 0.0a 7.3 ± 0.1a

Carvacrol (mM)

Thymol (mM)

0.25

0.5

1

0.25

0.5

1

7.0 ± 0.1a 7.0 ± 0.1a 6.6 ± 0.0a,b

6.9 ± 0.1a 5.6 ± 0.3c 4.1 ± 0.2d

6.4 ± 0.1a,b,c 0.7 ± 0.4e ND⁎,e (− − +)

7.1 ± 0.1a 6.7 ± 0.0a 6.5 ± 0.1a,b,c

6.9 ± 0.1a 5.7 ± 0.4b,c 4.3 ± 0.1d

6.4 ± 0.1a,b,c 0.7 ± 0.5e ND (− − −)

7.2 ± 0.1a 7.0 ± 0.1a 6.6 ± 0.1a,b

7.0 ± 0.1a 5.6 ± 0.3c 4.0 ± 0.3d

6.8 ± 0.1a,b 1.1 ± 0.3f ND g (− − −)

7.3 ± 0.1a 7.1 ± 0.1a 6.8 ± 0.1a,b

7.1 ± 0.1a 6.2 ± 0.1b,c 4.0 ± 0.3d

6.9 ± 0.1a,b 2.2 ± 0.3e ND (− − −)

7.1 ± 0.1a 7.3 ± 0.2a 7.2 ± 0.1a

6.7 ± 0.1a,b 5.8 ± 0.2b 3.3 ± 0.3c

NDe (− + −) ND (− − −) ND (− − −)

7.2 ± 0.2a 7.3 ± 0.2a 7.1 ± 0.2a

6.7 ± 0.1a,b 4.3 ± 0.5c 1.4 ± 0.5d

ND (− − −) ND (− − −) ND (− − −)

Data denoted by different superscript lower case letters are significantly different (P b 0.05). Results from enrichment test are expressed as + (growth on the agar) and − (no growth on the agar) (n = 3). a Bacterial populations are expressed as the mean ± standard error (n = 6). b Control, soy sauce containing 1% ethanol. c The population at 0 min represents the initial population. ⁎ ND, not detected.

H. Moon, M.S. Rhee / International Journal of Food Microbiology 217 (2016) 35–41 Table 3 pH values of soy sauce and soy sauce containing essential oils. Essential oil

Concentration (mM)

Concentration (%)

pHa

Controlb Carvacrol

– 0.25 0.5 1 0.25 0.5 1 1 1 1 1

– 0.0039 0.0078 0.0157 0.0039 0.0078 0.0157 0.0156 0.0127 0.0159 0.0146

4.62 ± 0.03 4.63 ± 0.02 4.64 ± 0.03 4.63 ± 0.02 4.63 ± 0.02 4.63 ± 0.03 4.63 ± 0.02 4.60 ± 0.02 4.61 ± 0.01 4.60 ± 0.03 4.62 ± 0.02

Thymol

Eugenol Trans-cinnamaldehyde β-resorcylic acid Vanillin

a Data are expressed as the mean ± standard error (n = 6). There was no statistically significant difference between any of the pH values (P N 0.05). b Control, soy sauce without EO.

containing 2 mM carvacrol and thymol were 3.1 ± 0.2 and 2.9 ± 0.2, respectively. See Fig. 2. 4. Discussion Previous studies show that the combination of food preservation methods and EOs results in increased bactericidal activity (Burt, 2004; Marino et al., 2001). Here, we showed that the antibacterial activity of soy sauce was markedly increased by the addition of a small amount of carvacrol or thymol (0.25, 0.5, or 1 mM; final concentration b 0.0157%) through synergism. These concentrations were less than the minimal inhibitory concentration (MIC) (Burt, 2004; Kim et al., 1995) for each EO; neither of these EOs showed bactericidal effects when used alone (i.e., in the absence of soy sauce). Besides, through the long time exposure and storage tests, it was concluded that the effects of carvacrol and thymol were stable for long time. In some points (i.e., E. coli O157:H7 with 1 mM carvacrol and S. Typhimurium with 1 mM thymol after 1 min at 22 °C), the recovery of the bacteria was observed despite the negative results from direct plating. It is thought that injured cells were recovered during enrichment; however, several conditions eradicated completely all the three bacteria from only 1 to 10 min. Thus, we suggest that the optimal conditions to completely inactivate all the three pathogens are carvacrol and thymol in N0.5 mM at 22 °C and thymol in 1 mM at 4 °C. The use of EOs as food preservatives is limited because, when used at effective doses, they impart unpleasant smells/flavors to food (Angienda and Hill, 2011; Lambert et al., 2001). However, we found

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that the addition of small amounts of EO to soy sauce led to a marked increase in bactericidal activity, but had no effect on sensory scores. We believe that it is because EOs acted synergistically with factors present in soy sauce, meaning that only small doses were required to control foodborne pathogens. Furthermore, the flavor of carvacrol, thymol, or any other EOs would be masked by the natural flavor of the soy sauce. We assumed that major antibacterial factors present in soy sauce (i.e., high NaCl levels, low water activity, and low pH) were responsible for these synergistic bactericidal effects. Soy sauce usually contains 15–17% of NaCl, which develops low water activity and places bacterial cells under hypertonic stress (Kim and Lee, 2008; Lioe et al., 2007; Luh, 1995). The high salt concentration withdraws water from the microorganisms, causing the cells to shrink and wilt (Marquis, 1968); the cells then become more susceptible to membrane attack (Angienda and Hill, 2011). Several studies show that carvacrol and thymol exert antimicrobial effects by disintegrating the outer membrane and disrupting the cytoplasmic membrane of Gram negative bacteria (Helander et al., 1998; Lambert et al., 2001; Ultee et al., 2002). Also, carvacrol causes sub-lethal injury to cells by altering the fatty acid composition of the membrane (Luz et al., 2014). Therefore, we believe that the combination of salt-induced hypertonic stress and membrane attack by carvacrol or thymol acts synergistically to improve the antibacterial activity of soy sauce. We also assumed that carvacrol and thymol act similarly once added to soy sauce because a previous study shows that both EOs are isomeric and have a similar mechanism of action (Marino et al., 2001). Although eugenol, trans-cinnamaldehyde, and vanillin are known as membrane-active compounds besides carvacrol and thymol (Fitzgerald et al., 2004; Gill and Holley, 2004), this low concentration (1 mM) of the present study was not enough to show bactericidal synergism with soy sauce. Carvacrol and thymol are generally the most bactericidal EO compounds (Oussalah et al., 2007). Otherwise, EOs such as eugenol, transcinnamaldehyde, β-resorcylic acid, and vanillin have much higher MIC and minimal bactericidal concentration (MBC) (Di Pasqua et al., 2006; Fitzgerald et al., 2004; Kim et al., 1995; Pei et al., 2009), so it was assumed that these EOs show weak synergism with soy sauce compared to carvacrol and thymol. In other words, the trends in combined bactericidal effects coincide with the individual effects of each EO. The pH of soy sauce ranges from 4.5 to 5.0; this low pH is attributed to the diverse array of organic acids, largely lactic acid, present in the product (Kim and Lee, 2008; Lioe et al., 2007; Luh, 1995). Zhou et al. (2007) reported that these organic acids act synergistically with carvacrol or thymol, and showed that the increased hydrogen ion (H+) concentration prevents carvacrol and thymol from dissociating into ions in

Fig. 2. Sensory scores from nine point hedonic test. Data bars represent the mean ± standard error (n = 30). Data denoted by different superscripts (a–c) are significantly different (P b 0.05). A score of 1 indicates the least preferred, and a score of 9 represents the most preferred.

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solution; thus the intact EOs diffuse more readily across the bacterial cell membrane. Previous studies show that carvacrol or thymol do not act synergistically with lactic acid as they do with soy sauce (de Oliveira et al., 2010; Zhou et al., 2007). Hence, as Masuda et al. (1998) demonstrated, the ability of soy sauce to retard food deterioration is based on the combination of high NaCl, low pH, ethanol, and preservatives. We also believe that it is the combination of a wide variety of bacterial stressors within soy sauce that promote its antimicrobial activity. Further studies should attempt to identify these synergistic factors in more detail. Soy sauce containing carvacrol or thymol was less effective against E. coli O157:H7 and S. Typhimurium at 4 °C than at 22 °C; however, it was more effective at controlling L. monocytogenes at 4 °C. There are two possible explanations for this: first, it is well-known that Gram positive bacteria are more susceptible to carvacrol and thymol than Gram negative bacteria (Burt, 2004); second, when bacteria grow at low temperatures, the membrane loses its flexibility and becomes rigid — this rigidity is thought to make E. coli O157:H7 and S. Typhimurium less susceptible to the membrane-based attack of carvacrol and thymol in soy sauce (Murata and Los, 1997). L. monocytogenes, meanwhile, is demonstrated to grow refrigeration temperatures, and it has the ability to maintain sufficiently fluid membrane other than E. coli O157:H7 and S. Typhimurium (Annous et al., 1997; Bayles et al., 1996). At low temperatures, the proportion of unsaturated fatty acids in the cell wall of L. monocytogenes increases significantly, allowing the membrane to maintain flexibility (Marr and Ingraham, 1962; Murata and Los, 1997). As Annous et al. (1997) reported, anteiso-C15:0 fatty acid plays a key role in the growth of L. monocytogenes at low temperatures. Besides, L. monocytogenes employs various methods to survive at lower temperatures such as formation of cold-shock protein and activation of stress factors (Bayles et al., 1996; Farber and Peterkin, 1991; Najjar et al., 2007; Walker et al., 1990). This, however, makes the bacteria more susceptible to membrane attack by carvacrol and thymol in soy sauce. Since soy sauce contains components with potential antimicrobial activity, many researchers have attempted to examine its antibacterial effects (Kataoka, 2005; Kim and Lee, 2008; Lioe et al., 2007; Luh, 1995; Masuda et al., 1998). Masuda et al. (1998) examined the effects of soy sauce against E. coli O157:H7; it took 9 days of exposure at 30 °C before the tested E. coli O157:H7 population fell below the detection limit (20 CFU/ml). No reduction in the bacterial population was observed at 4 °C. Salmonella spp. and Shigella spp. also required more than 24 h exposure to soy sauce at room temperature (Kataoka, 2005). In addition, these reported pathogen-controlling effects were mainly attributed to the preservatives added to the soy sauce (Kataoka, 2005; Masuda et al., 1998). Thus, it was unclear whether soy sauce could prolong the shelf-life of foods. The present study, however, examined the bactericidal activity of soy sauce containing small amounts of carvacrol or thymol; the results showed that these combinations eliminated more than 7-log CFU/ml E. coli O157:H7, S. Typhimurium, and L. monocytogenes within 10 min at both room and refrigeration temperatures. Hence, this new method may facilitate the production of safe marinades and guarantee the microbiological safety of marinated foods stored at refrigeration temperatures (as recommended by the USDA and USFDA (USDA, 2013; USFDA, 2014)). To the best of our knowledge, this is the first study to report that EOs markedly increase the antibacterial activity of soy sauce. The addition of carvacrol or thymol at non-lethal concentrations (b0.0157%) led to a marked increase in the antibacterial activity of soy sauce, most likely via synergism between antibacterial factors present in soy sauce and the EO. Moreover, the EO had no sensory impact on the sauce. All three major foodborne pathogens tested herein (more than 7 log CFU/ml) were successfully controlled within 10 min at both 4 °C and 22 °C. In conclusion, the combination of high NaCl levels, low pH, and other (unknown) ingredients in soy sauce acts synergistically with carvacrol or thymol to increase the antibacterial effects of the product. Therefore, we suggest that preservatives such as sodium benzoate can be replaced

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