Original papers
Efficacy of Essential Oils on Inactivation of Escherichia coli O157:H7 in Vegetable Juice
2014 Volume 20 Issue 5 Pages 1043-1049
Details
2014 Volume 20 Issue 5 Pages 1043-1049
This study was conducted to investigate the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of selected essential oils (EOs) against Escherichia coli O157:H7 under neutral and acidic pH conditions, and to evaluate the efficacy of EOs on the inactivation of E. coli O157:H7 in vegetable juice samples stored at 5, 20 and 35°C or heated at 55°C. The growth and survival of E. coli O157:H7 were significantly affected by pH adjustment, additive concentration, storage temperature and time. A synergistic or hurdle effect was observed within EOs at pH 4.0 and pH 4.5, or with heat treatment. The calculated decimal reduction times (D-value, min) varied with the EOs concentration. Star anise oil (0.05%, vol/vol) provided the maximum reduction in D55 value, from 5.69 min to 0.38 min. The combination of mild heat treatment and EOs may be used to enhance the safety of juice products.
Escherichia coli O157:H7 has been identified as a common pathogenic bacterium in produce-associated outbreaks and is associated with a wide range of foods, such as fresh vegetables, alfalfa sprouts, radish sprouts, berries, and grapes (Abadiasa et al., 2012; Beuchat, 1996; Huang and Chen, 2011; Neill, 1989; Padhye and Doyle, 1992). Recently, there was a large outbreak of hemolytic uremic syndrome caused by Shiga toxin-producing E. coli O104:H4 linked to sprouts in Germany (Buchholz et al., 2011), and an outbreak of E. coli O157:H7 infection due to the consumption of lightly pickled Chinese cabbage in Japan (Ministry of Health, Labor and Welfare, Japan 2012). There have also been a number of foodborne disease outbreaks caused by E. coli O157:H7 linked to fruit and vegetable juices (Harris et al., 2003; Leizerson and Shimoni, 2005; McCarthy, 1996).
Numerous factors contribute to the high contamination risk of juice. For example, fruits and vegetables as raw materials are typically grown in open fields in close contact with soil, potentially contaminated with animal manure or feces and poor quality irrigation water (Steele and Odemuer, 2004). The transfer of pathogens may also occur directly from animals, birds, and insects, or through the handling of fruits and vegetables during harvest and postharvest (Doering et al., 2009). If the fruit and vegetable raw materials are contaminated with pathogens, the contaminants are released into the juice during pressing (Dock et al., 2000; Nguyen-the and Carlin, 1994; Zhao et al., 1993). Not only can E. coli O157:H7 cause disease with a low infection dose, this pathogen is acid-tolerant and can survive for extended periods in acidic foods such as apple juice (Splittstoesser et al., 1996) and at low temperatures (Zhao et al., 1993). As a result, it is extremely important to remove this pathogen from foods such as fruit and vegetable juices.
The U.S. Food and Drug Administration (FDA) has developed a hazard analysis and critical control point (HACCP) regulation to confirm juice safety as requiring > 5-log reduction of pathogenic bacteria in juice. Pasteurization and irradiation are the standard processes used in the juice industry. Although these methods are applicable to killing pathogenic bacteria present in juice, they are expensive and many believe that such treatment processes may deteriorate the nutritional and flavor qualities of juices (Knight and McKellar, 2007; Scaman et al., 2004).
In recent years, the growing demand for novel products that are safe, natural, and fresh has stimulated research into new processing methods to improve the microbiological quality of foods and beverages, with no marked changes in nutrient content and some sensory attributes (Yuste et al., 2002). Essential oils (EOs) of plants have been used in the flavor and fragrance industries; however, many EOs also exhibit antimicrobial activity (Knight and McKellar, 2007). Currently, plant EOs are being evaluated as alternative antibacterial treatments in fresh produce and their products. Many researchers have evaluated the antibacterial activities of EOs including lemon (Severino et al., 2014), cinnamon (Du et al., 2009; Knight and McKellar, 2007), clove (Du et al., 2009; Knight and McKellar, 2007), rosemary (Klancnik, 2009), oregano (Benavides et al., 2012; Gündüz et al., 2010), apricot (Friedman et al., 2004), Mentha piperita (Tyagi and Malik, 2011), persea (Joshi et al., 2010), Carum copticum (Hashemi et al., 2014) and Thymus vulgaris (Tsai et al., 2011), both in vitro and in food models such as fresh vegetables, fruit juices, etc. However, no studies have been conducted to evaluate the antibacterial effects of plant EOs (including star anise oil) in vegetable juice. The purpose of this study was to evaluate the antibacterial activities of cinnamon, clove, star anise oils and eugenol, a phenylpropene found in a number of EOs, against E. coli O157:H7 in broth at neutral and at acidic pHs, and in vegetable juice stored at various temperatures. We further investigated the influence of EOs and eugenol on the heat resistance (D-values) of E. coli O157:H7 in juice.
Microbial culture and inoculum preparation As the test inoculum, we used a mix of three rifampicin-resistant strains (CR-3, MN-28, MY-29) of enterohemorrhagic E. coli O157:H7 obtained from the National Food Research Institute, Tsukuba, Japan. The strains were transferred individually from nutrient agar slants (NA, Eiken Chemical Co., Ltd., Tokyo, Japan) into 10 mL of trypto soy broth (TSB, Eiken), and incubated at 37°C for 24 h. Each strain was subcultured again before being used as inoculum. The cells were harvested by centrifugation in a Kubota 6500 high-speed cooling centrifuge (Kubota Corp., Tokyo, Japan) at 4,293 × g for 10 min at 4°C. The pelleted cells were twice resuspended in 10 mL sterile phosphate-buffered saline (PBS, pH 7.2) and centrifuged as before. The cells were then suspended in 10 mL PBS. Equal volumes of each cell suspension were mixed and diluted to a final concentration of ∼8 log cfu/mL.
Plant essential oils (EOs) Pure cinnamon bark, clove bark and star anise EOs were obtained from Tree of Life Co., Ltd (Tokyo, Japan). Eugenol was obtained from Nacalai Tesque (Kyoto, Japan). All oils and eugenol were stored as per the manufacturers' instructions and diluted to 10% with 99.5% ethanol (Sigma Aldrich, Tokyo, Japan) before use in experiments.
Vegetable juice The sample vegetable juice used in this study was 100% mixed vegetable and fruit juice (Kagome Co., Ltd., Tokyo, Japan) purchased from a local grocery store. The juice contained 50% vegetable juice (including 21 kinds of vegetables such as carrot, spinach, cabbage, Chinese cabbage, kale, etc.) and 50% fruit juice (including 3 kinds of fruits: apple, orange and lemon). The pH of the mixed juice was 3.9 ± 0.1.
Determination of MIC and MBC The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of EOs and eugenol against E. coli O157:H7 were evaluated by the broth dilution method (Weerakkody et al., 2010) with some modifications. Two-fold serial dilutions of EOs and eugenol were prepared with neutral and acidic Mueller Hinton broth (MH, Eiken) ranging from 0.0031 to 0.2%. To prepare the acidic MH broth, we adjusted the pH of the MH broth to pH 4.5/4.0 using 35% (v/v) hydrochloric acid (HCl, Nacalai Tesque) before autoclaving. All tubes were inoculated with a 0.1 mL aliquots of the inoculum for a content of approximately 5.0 log cfu/mL. All the tubes were subsequently incubated at 35°C for 24 h. Comparing each tube to the control, the concentration of the lowest serial dilution of EOs and eugenol at which growth did not occur in broth was recorded as the MIC. A Mueller Hinton agar (Eiken) plate was divided into sections and inoculated with one loopful of all the tubes with no growth. After incubated at 35°C overnight, the lowest concentration among the negative tubes at which no growth was observed was recorded as the MBC.
Survival of E. coli O157:H7 in vegetable juice In order to evaluate the inactivation efficacy, EOs and eugenol were individually added to the vegetable juice at concentrations of 0.05% and 0.1%. Then, E. coli O157:H7 suspension was inoculated into the juice containing oils or component to obtain approximately 105 cfu/mL, respectively. A control of juice without oils or component was also inoculated. Inoculated samples were stored at 5°C and 20°C for 7 days, and at 35°C for 24 hours. Ten-fold serial dilutions were subsequently prepared with PBS. The number of surviving E. coli O157:H7 were enumerated on TSA.
Determinations of D-values EOs and eugenol were added at 0.05% and 0.01% as final concentrations in the vegetable juice and mixed well. The PBS bacterial suspension was added at a final concentration of 6.0 cfu/mL approximately. Then, 3 mL of the prepared juice was transferred to glass test tubes and heated to 55°C in a water bath (Yamato Thermo Mate; Yamato Scientific Co., Ltd., Tokyo, Japan). The juice temperature was monitored using a thermometer inserted in a control tube. Recording of time was initiated when the temperature had come within 2°C of the test temperature of 55°C. After heat treatment, tubes were immediately immersed in an ice bath and maintained there until enumeration of cell survival. Cell survival was enumerated by spread plating the serial dilutions onto TSA. Plates were incubated at 35°C for 24 – 48 h before colony enumeration. The decimal reduction times (D-value) of the test bacteria were then determined by plotting the log10 of the calculated colony-forming units versus heating time. The survivor curve (SC) was then determined by plotting the line of best fit on the survivor plots. The D-value was equivalent to the number of unit time of heating that resulted in 90% loss of viability of the test organism and graphically equivalent to the negative inverse of the slope of the SC (Gabriel and Nakano, 2009). If a survival curve was characterized with a lag time prior to log-linear inactivation, the time to 90% reduction was determined as the sum of the lag time and the D-value calculated from the succeeding loglinear inactivation curve.
Statistical analysis To test for differences (95% level of significance) between the measured values per treatments, all data were subjected to one-way ANOVA and Tukey's multiple range test (SPSS 17.0, IBM) at the 5% level of significance.
MICs and MBCs of EOs and eugenol against E. coli O157:H7 MICs and MBCs of cinnamon, clove, star anise oil and eugenol against E. coli O157:H7 under neutral and acidic conditions were determined (Table 1). All the EOs and eugenol, a major component of clove, used in this study showed high antibacterial activity against E. coli O157:H7 under both neutral and acidic conditions. The MICs were in the range of 0.025 to 0.05% at neutral pH, 0.0063 to 0.025% at pH 4.5 and less than 0.0031% at pH 4.0. The MBCs of E. coli O157:H7 ranged from 0.1 to 0.2% at neutral pH, 0.0125 to 0.1% at pH 4.5 and 0.0063 to 0.05% at pH 4.0. Under acidic conditions (pH 4.5 and pH 4.0), the EOs and eugenol showed a more effective result. Specifically, star anise oil showed the lowest MBC for E. coli O157:H7.
| pH | Test sample | MIC (%) | MBC (%) |
|---|---|---|---|
| 7.2 | Cinnamon oil | 0.025 | 0.1 |
| Clove oil | 0.05 | 0.1 | |
| Star anise oil | 0.05 | 0.2 | |
| Eugenol | 0.05 | 0.1 | |
| 4.5 | Cinnamon oil | 0.0063 | 0.05 |
| Clove oil | 0.025 | 0.1 | |
| Star anise oil | 0.0063 | 0.0125 | |
| Eugenol | 0.025 | 0.1 | |
| 4.0 | Cinnamon oil | <0.0031 | 0.0125 |
| Clove oil | <0.0031 | 0.05 | |
| Star anise oil | <0.0031 | 0.0063 | |
| Eugenol | <0.0031 | 0.05 |
MIC: Minimum inhibitory concentration; MBC: Minimum bactericidal concentration.
Inactivation of E. coli O157:H7 in vegetable juice The vegetable juice pH was 3.9 ± 0.1. Although the addition of EOs and eugenol did not change pH remarkably, it effectively reduced and inactivated E. coli O157:H7 in vegetable juice. At 5°C, E. coli O157:H7 counts in the control were remarkably unchanged, while in the juice with 0.05% EOs or eugenol, the counts decreased gradually, from 5.3 to 3.2 log cfu/mL over 7 days (Table 2). Increasing concentrations of oils or eugenol resulted in a concomitant increase in bacterial inactivation. Counts were not detected at day 1 in vegetable juice with 0.1% clove oil or eugenol, and at day 7 in juice with 0.1% cinnamon oil. The addition of 0.1% star anise oil also caused significant inactivation of E. coli O157:H7 compared with the control, a 3.7 log reduction at day 7.
| Test sample | Conc (%) | Storage time (days) | ||||
|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 7 | ||
| Control | 5.3 ± 0.03Aa | 5.2 ± 0.02Aa | 5.3 ± 0.11Aa | 5.3 ± 0.04Aa | 5.3 ± 0.16Aa | |
| Cinnamon | 0.05 | 5.3 ± 0.07Aa | 5.3 ± 0.05Aa | 5.2 ± 0.03Aa | 5.2 ± 0.10Aa | 4.1 ± 0.01Bb |
| 0.1 | 5.3 ± 0.02Aa | 5.3 ± 0.04Aa | 4.7 ± 0.11Bbe | 2.7 ± 0.07Cb | NDDc | |
| Clove | 0.05 | 5.3 ± 0.03Aa | 5.3 ± 0.04Aa | 5.2 ± 0.01Aa | 5.2 ± 0.02Aa | 5.0 ± 0.11Aa |
| 0.1 | 5.0 ± 0.21Aa | NDBb | NDBc | NDBc | NDBc | |
| Star anise | 0.05 | 5.3 ± 0.06Aa | 5.0 ± 0.17Aa | 4.5 ± 0.06Bb | 4.3 ± 0.21Bd | 3.2 ± 0.07Cd |
| 0.1 | 5.3 ± 0.13Aa | 4.0 ± 0.16Bc | 3.0 ± 0.05Cd | 2.5 ± 0.14Db | 1.6 ± 0.05Ee | |
| Eugenol | 0.05 | 5.3 ± 0.10Aa | 5.1 ± 0.12Aa | 4.9 ± 0.09Be | 4.9 ± 0.17Bd | 3.7 ± 0.10Cf |
| 0.1 | 4.8 ± 0.25Aa | NDBb | NDBc | NDBc | NDBc | |
ND = not detected. Within a row, means not followed by the same letters (A through E) are significantly different (p < 0.05). Within a column, means not followed by the same letters (a through f) are significantly different (p < 0.05).
At 20°C, E. coli O157:H7 counts in the control did not change significantly at day 3 and declined by 1.5 log at day 7 (Table 3). No growth was obtained in the vegetable juice with the addition of oils or eugenol during storage. Counts were not detected from day 1 in the juice with additions of 0.1% clove oil or eugenol, day 2 with the addition of 0.1% cinnamon oil, day 3 with the addition of 0.1% star anise oil and day 7 with the addition of 0.05% EOs or eugenol.
| Test sample | Conc (%) | Storage time (days) | ||||
|---|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 7 | ||
| Control | 5.3 ± 0.02Aa | 5.0 ± 0.21Aa | 5.3 ± 0.10Aa | 5.3 ± 0.02Aa | 3.8 ± 0.05Ba | |
| Cinnamon | 0.05 | 5.3 ± 0.04Aa | 5.3 ± 0.03Aa | 4.8 ± 0.05Bb | 3.5 ± 0.02Cb | NDDb |
| 0.1 | 5.3 ± 0.12Aa | 4.1 ± 0.04Bb | NDCc | NDCc | NDCb | |
| Clove | 0.05 | 5.3 ± 0.05Aa | 5.1 ± 0.08Aa | 4.9 ± 0.12Ab | 4.5 ± 0.15Bd | NDCb |
| 0.1 | 4.9 ± 0.21Aa | NDBc | NDBc | NDBc | NDBb | |
| Star anise | 0.05 | 5.2 ± 0.06Aa | 4.2 ± 0.12Bb | 3.0 ± 0.03Cd | 2.5 ± 0.07De | NDEb |
| 0.1 | 5.3 ± 0.09Aa | 2.4 ± 0.13Bd | 1.0 ± 0.12Ce | NDDc | NDDb | |
| Eugenol | 0.05 | 5.3 ± 0.08Aa | 4.3 ± 0.06Bb | 3.9 ± 0.08Cf | 3.4 ± 0.09Db | NDEb |
| 0.1 | 4.9 ± 0.20Aa | NDBc | NDBc | NDBc | NDBb | |
ND = not detected. Within a row, means not followed by the same letters (A through E) are significantly different (p < 0.05). Within a column, means not followed by the same letters (a through f) are significantly different (p < 0.05).
At 35°C, no growth was observed in the control stored for 1 day (Table 4). E. coli O157:H7 counts were not detected in the vegetable juice with 0.05% cinnamon, clove, star anise oils and eugenol after 1 day of storage.
| Test sample | Conc (%) | Storage time (days) | |
|---|---|---|---|
| 0 | 1 | ||
| Control | 5.3 ± 0.03A | 5.2 ± 0.22A | |
| Cinnamon | 0.05 | 5.3 ± 0.11 | ND |
| 0.1 | 5.3 ± 0.07 | ND | |
| Clove | 0.05 | 5.3 ± 0.05 | ND |
| 0.1 | 5.0 ± 0.18 | ND | |
| Star anise | 0.05 | 5.2 ± 0.03 | ND |
| 0.1 | 5.3 ± 0.05 | ND | |
| Eugenol | 0.05 | 5.3 ± 0.04 | ND |
| 0.1 | 5.0 ± 0.16 | ND | |
ND = not detected. Within a row, means not followed by the same letters are significantly different (p < 0.05).
Heat inactivation The D-values of E. coli O157:H7 heated at 55°C in vegetable juice with or without EOs are presented in Table 5. EOs or eugenol significantly decreased (p < 0.05) the heat resistance of E. coli O157:H7 compared to the non-addition control juice with a D55 value of 5.69 min. When the concentrations of added oils or eugenol in the vegetable juice were increased from 0.01 to 0.05%, the D55 value of E. coli O157:H7 showed an approximately 5-fold decrease. The E. coli O157:H7 in the vegetable juice with star anise oil had the lowest heat resistance. Moreover, the D55 values of E. coli O157:H7 exposed to 0.01% or 0.05% star anise oil were 2.95 and 0.38 min, respectively.
| Test samples | D55-values (min) in different concentrations: | |
|---|---|---|
| 0.01% | 0.05% | |
| Control | 5.69 ± 0.12a | 5.69 ± 0.12a |
| Cinnamon | 3.41 ± 0.09Ab | 0.79 ± 0.03Bb |
| Clove | 4.09 ± 0.05Ac | 0.67 ± 0.02Bbc |
| Star anise | 2.95 ± 0.15Ad | 0.38 ± 0.10Bd |
| Eugenol | 4.05 ± 0.14Ac | 0.60 ± 0.05Bc |
Min = minutes; D-values in the same row not followed by the same letters (A through B) are significantly different (p < 0.05).
D-values in the same column not followed by the same letters (a through d) are significantly different (p < 0.05).
In this study, the MIC and MBC values, as well as the inactivation efficacy, indicated that the EOs and eugenol have significant bactericidal activity against E. coli O157:H7. Since the acidic pH of the vegetable juice could contribute to the activities of the test sample, we also evaluated the MICs and MBCs under acidic conditions (pH 4.0 and pH 4.5). The MIC and MBC values were significantly influenced by acidic pH. On the other hand, the effect of ethanol, in which the EOs were dissolved, was suggested to be negligible in this study, since at an EO concentration of up to 0.1%, the ethanol concentration in the system was only 1%; the growth and survival of E. coli O157 was not affected (data not shown). Cinnamon oil and star anise oil showed higher levels of bactericidal activity under acidic conditions. The major components of cinnamon, clove and star anise oils are cinnamaldehyde, eugenol and anethole, respectively. The antibacterial action of these components remains to be clarified. More than one mechanism has been proposed for the bactericidal activity of these EOs or the component (Burt, 2004; Moreira et al., 2005), such as inhibition of enzyme production, cell or bacterial cytoplasmic membrane damage, interference with energy metabolism, disruption of electron transport and nutrient uptake, and nucleic acid synthesis effects (Burt, 2004; Cutter, 2000). Thus, combinations of EOs and acidic pH or temperature could have a synergistic effect against pathogenic organisms (Yossa et al., 2012). Enhanced antibacterial activity of the oils obtained under acidic conditions may be attributed to the observation that phenolic constituents of plant EOs become more hydrophobic at an acidic pH and thus dissolve more readily in the lipid phase of bacterial membranes (Knight and McKellar, 2007; Tassou et al., 1995). Knight and McKellar (2007) evaluated the antibacterial activity of nine EOs and components against E. coli O157:H7 in neutral and acidic culture broth. Cinnamon and clove oils displayed the lowest MICs of 0.025% and 0.075%, respectively. Smith-Palmer et al. (1998) reported the MICs for cinnamon and clove oils against E. coli O157:H7 were 0.05% and 0.04%, respectively, and the MBCs were both up to 0.1%. In general, MBC was higher than MIC (Table 1). Although there are few reports dealing with star anise oil or its major component, anethole, it exhibits the same or even higher antibacterial activity as cinnamon and clove oils in this study.
The growth and survival of E. coli O157:H7 in vegetable juice with or without cinnamon, clove, star anise oils and eugenol were also evaluated in this study. Despite being acid-tolerant, no growth was observed for E. coli O157:H7 at all storage points. Bacterial counts were stable at 5°C for 7 days and 20°C for 3 days. Oils typically promoted the progress of inactivation. Higher concentrations were needed to obtain similar effects in food systems as obtained in experiments in vitro (Karatzas et al., 2001). This may be related to the physicochemical properties of the suspending media (culture broth or juice). In addition, the bactericidal effects depended upon the type and concentration of EOs or component, as well as storage temperature and time. The results of this study supported this observation. With increasing storage temperature or time, the necessary concentration of added oils or eugenol for the same level of pathogen inactivation was decreased (Tables 2–4). In general, higher concentrations of EOs and eugenol were required to reduce or inactivate E. coli O157:H7 in the vegetable juice stored at 5°C than at 20 or 35°C. Ceylan et al. (2004) found that E. coli O157:H7 counts showed negligible changes in apple juice (pH 3.75) stored at 8°C for 14 days. Better inactivation results were obtained at room temperature (20°C) or higher (35°C). In this study, counts greater than 3 log cfu/mL were detected at day 7 in the vegetable juice with 0.05% EOs stored at 5°C. However, E.coli O157:H7 was not detected at day 7 in the vegetable juice with 0.05% EOs stored at 20°C and at day 1 at 35°C. Temperature has a significant influence on membrane fluidity properties. At low temperatures, phospholipids are closely packed into a rigid gel structure, while at high temperatures they are less ordered and the membrane has a liquid-crystalline structure (Raybaudi-Massilia, 2009).
The D-value of E. coli O157:H7 in vegetable juice was significantly reduced by adding 0.01% or 0.05% cinnamon, clove, star anise oils and eugenol. Similar results were reported by Knight and McKellar (2007), who concluded that D-values for apple cider alone were higher than those for cider plus either 0.01% cinnamon or clove oil. The combination of EOs or component and mild heat indicated a potential synergistic or hurdle effect. Not only does this effectively ensure the microbiological safety of vegetable or fruit juices, it can also prevent nutrient loss due to the low temperature treatment. Specifically, star anise oil was more effective in reducing the heat resistance of E. coli O157:H7 than the other samples. Star anise, which imparts a sweet smelling flavor similar to black licorice, is a common spice used in both curries and Chinese stews, as are cinnamon and clove. Moreover, there have been no reports of its toxicity. While star anise is commonly associated with black licorice, it is widely used in foods, medicines, toothpastes and other flavored products. It is often used as a breath freshener and a remedy for a nervous stomach, is a popular addition to blends for aches and pains, and has an affinity with the female cycle. It is widely known that clove and cinnamon oils are often added to spiced apple drinks and other juice products. Hence, it is possible that star anise oil can be used as substitute flavor or antibacterial material in the juice industry.
In conclusion, the selected EOs and eugenol exhibited bactericidal activity at low concentrations against E. coli O157:H7 in vegetable juice, and markedly decreased the heat resistance of E. coli O157:H7 in combination with mild heat treatment. Thus, the addition of cinnamon or clove oil, and especially star anise oil could enhance the safety of juice products. Further studies on the effect of the oils on the sensory qualities of food products are needed. According to the properties and current usage of star anise oil, it is not thought to affect food quality, especially of spiced drinks.
Acknowledgments We gratefully thank the National Food Research Institute, Tsukuba, Japan, for providing the E. coli O157:H7 strain.
