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

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 1mM 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 1mM (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 2mM was performed using the nine point hedonic test. Carvacrol or thymol (1mM) eliminated all the test bacteria (initial population, 7.0-7.5logCFU/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>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.

Moon H ，Rhee MS 《-》

Teriyaki sauce with carvacrol or thymol effectively controls Escherichia coli O157:H7, Listeria monocytogenes, Salmonella Typhimurium, and indigenous flora in marinated beef and marinade.

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 (3.1-3.5logCFU/g) were marinated in a teriyaki sauce with or without CV and TM (0.3 and 0.5%). After 1, 3, and 7days 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 7days (p<0.05). The pathogens relocated from the beef into the leftover marinade (3.0-3.4logCFU/mL) were also completely inactivated. The treatment inhibited growth of indigenous aerobic bacteria (p<0.05) and inactivated coliform bacteria. Physicochemical parameters were not significantly affected (p>0.05).

Moon H ，Kim NH ，Kim SH ，Kim Y ，Ryu JH ，Rhee MS ... -  《-》

Citrus fruit extracts with carvacrol and thymol eliminated 7-log acid-adapted Escherichia coli O157:H7, Salmonella typhimurium, and Listeria monocytogenes: A potential of effective natural antibacterial agents.

Despite the widespread belief that citrus fruit extracts (CFEs) are microbiologically safe due to their acidity, limited bactericidal effect results in low applicability as antibacterial agent and outbreaks occurred by acid-adapted pathogens. Here, we examined the antibacterial effects of CFEs [lime (Citrus medica), lemon (Citrus limon), calamansi (Citrus microcarpa)] combined with essential oil components (EOCs; carvacrol and thymol) against non-acid-adapted/acid-adapted Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes under 22 °C for 5 min. CFEs (<20%) alone or small amounts of EOCs (2.0 mM; 0.032%) alone could not inactivate the target bacteria effectively. However, combined treatments exhibited marked synergy: CFE + EOCs eliminated all the bacteria (>6.9 log CFU/ml). Among the CFEs tested, the highest synergism was shown by calamansi, an exotic citrus fruit previously unrecognized as an antibacterial agent. Although acid-adaptation improved bacterial survival, calamansi (<20%) + EOCs (<0.032%) completely inactivated even the most resistant pathogen (E. coli O157:H7). Validation test also showed that all tested commercial juice products also eliminated acid-adapted pathogens when used with EOCs. Physicochemical analysis of tested CFEs (pH measurement and HPLC analysis of components) revealed that low pH and flavanone (hesperidin) did not contribute to the synergistic bactericidal effects. Rather, the high citric acid content is likely to contribute to the strong synergistic effect with EOCs by damaging susceptible bacterial membranes. Sensory scores for CFEs were not altered by addition of EOCs at concentrations up to 1.5 mM. This study provides new insight into the utility of CFEs with EOCs to improve not only the microbiological safety of food products containing CFEs but also their applicability as natural antibacterial complex.

Chung D ，Cho TJ ，Rhee MS 《-》

Stress Resistance Development and Genome-Wide Transcriptional Response of Escherichia coli O157:H7 Adapted to Sublethal Thymol, Carvacrol, and trans-Cinnamaldehyde.

Thymol, carvacrol, and trans-cinnamaldehyde are essential oil (EO) compounds with broad-spectrum antimicrobial activities against foodborne pathogens, including Escherichia coli O157:H7. However, little is known regarding direct resistance and cross-resistance development in E. coli O157:H7 after adaptation to sublethal levels of these compounds, and information is scarce on microbial adaptive responses at a molecular level. The present study demonstrated that E. coli O157:H7 was able to grow in the presence of sublethal thymol (1/2T), carvacrol (1/2C), or trans-cinnamaldehyde (1/2TC), displaying an extended lag phase duration and a lower maximum growth rate. EO-adapted cells developed direct resistance against lethal EO treatments and cross-resistance against heat (58°C) and oxidative (50 mM H2O2) stresses. However, no induction of acid resistance (simulated gastric fluid, pH 1.5) was observed. RNA sequencing revealed a large number (310 to 338) of differentially expressed (adjusted P value [Padj ], <0.05; fold change, ≥5) genes in 1/2T and 1/2C cells, while 1/2TC cells only showed 27 genes with altered expression. In accordance with resistance phenotypes, the genes related to membrane, heat, and oxidative stress responses and genes related to iron uptake and metabolism were upregulated. Conversely, virulence genes associated with motility, biofilm formation, and efflux pumps were repressed. This study demonstrated the development of direct resistance and cross-resistance and characterized whole-genome transcriptional responses in E. coli O157:H7 adapted to sublethal thymol, carvacrol, or trans-cinnamaldehyde. The data suggested that caution should be exercised when using EO compounds as food antimicrobials, due to the potential stress resistance development in E. coli O157:H7.IMPORTANCE The present study was designed to understand transcriptomic changes and the potential development of direct and cross-resistance in essential oil (EO)-adapted Escherichia coli O157:H7. The results demonstrated altered growth behaviors of E. coli O157:H7 during adaptation in sublethal thymol, carvacrol, and trans-cinnamaldehyde. Generally, EO-adapted bacteria showed enhanced resistance against subsequent lethal EO, heat, and oxidative stresses, with no induction of acid resistance in simulated gastric fluid. A transcriptomic analysis revealed the upregulation of related stress resistance genes and a downregulation of various virulence genes in EO-adapted cells. This study provides new insights into microbial EO adaptation behaviors and highlights the risk of resistance development in adapted bacteria.

Yuan W ，Seng ZJ ，Kohli GS ，Yang L ，Yuk HG ... -  《-》

Use of enhanced nisin derivatives in combination with food-grade oils or citric acid to control Cronobacter sakazakii and Escherichia coli O157:H7.

Cronobacter sakazakii and Escherichia coli O157:H7 are well known food-borne pathogens that can cause severe disease. The identification of new alternatives to heating to control these pathogens in foods, while reducing the impact on organoleptic properties and nutritional value, is highly desirable. In this study, nisin and its bioengineered variants, nisin V and nisin S29A, are used alone, or in combination with plant essential oils (thymol, carvacrol and trans-cinnamaldehyde) or citric acid, with a view to controlling C. sakazakii and E. coli O157:H7 in laboratory-based assays and model food systems. The use of nisin variants (30 μM) with low concentrations of thymol (0.015%), carvacrol (0.03%) and trans-cinnamaldehyde (0.035%) resulted in extended lag phases of growth compared to those for corresponding nisin A-essential oil combinations. Furthermore, nisin variants (60 μM) used in combination with carvacrol (0.03%) significantly reduced viable counts of E. coli O157:H7 (3-log) and C. sakazakii (4-log) compared to nisin A-carvacrol treatment. Importantly, this increased effectiveness translated into food. More specifically, sub-inhibitory concentrations of nisin variants and carvacrol caused complete inactivation of E. coli O157:H7 in apple juice within 3 h at room temperature compared to that of the equivalent nisin A combination. Furthermore, combinations of commercial Nisaplin and the food additive citric acid reduced C. sakazakii numbers markedly in infant formula within the same 3 h period. These results highlight the potential benefits of combining nisin and variants thereof with carvacrol and/or citric acid for the inhibition of Gram negative food-borne pathogens.

Campion A ，Morrissey R ，Field D ，Cotter PD ，Hill C ，Ross RP ... -  《-》