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Antimicrobial effect of spices and herbs on Vibrio parahaemolyticus
International Journal of Food Microbiology 111 (2006) 6 – 11 www.elsevier.com/locate/ijfoodmicro
Antimicrobial effect of spices and herbs on Vibrio parahaemolyticus Yutaka Yano ⁎, Masataka Satomi, Hiroshi Oikawa Seafood Safety Section, National Research Institute of Fisheries Science, 2-12-4 Fukuura, Yokohama 2368648, Japan Received 2 May 2005; received in revised form 1 March 2006; accepted 25 April 2006
Abstract The antimicrobial effects of spices and herbs from 18 plant species were examined on a foodborne pathogen, Vibrio parahaemolyticus, with the use of combinations of temperatures and nutrient levels. Basil, clove, garlic, horseradish, marjoram, oregano, rosemary, and thyme exhibited antibacterial activities at incubation of 30 °C, while with the exception of horseradish, the same spices and additional 7 species exhibited the activities at 5 °C. The lowest MIC (minimum inhibitory concentration) was 0.125% observed in clove and marjoram at 30 °C in a nutrient rich medium. Lowering of incubation temperature produced little effect on the MICs except for turmeric. The decreasing of the MIC in turmeric appeared to be basically attributed to the sensitivity of the bacterium to coldness. In nutrient poor medium, the lowest was 0.001 and 0.00025% in marjoram at 30 °C and at 5 °C, respectively. The sensitivity to several spices and herbs was similar among different clinical serotypes including the emerging strain O3:K6. These results suggest that the spices and herbs can be practical for protecting seafood from the risk of contamination by V. parahaemolyticus and used in hurdle technology with low temperature. © 2006 Elsevier B.V. All rights reserved. Keywords: Vibrio parahaemolyticus; Spices; Herbs; Antibacterial activity
1. Introduction Vibrio parahaemolyticus is a bacterium which causes mild gastroenteritis in humans on consumption of infected seafood (Oliver and Kaper, 1997). The bacterium generally inhabits coastal environments in tropical and temperate zones, and contaminates fishery products caught in these areas, especially during warm periods. In Japan, a variety of seafood has been traditionally consumed, and raw or lightly cooked seafood is favored. This eating habit seems to provide an explanation for many cases of foodborne disease by V. parahaemolyticus in the country. The foodborne diseases by the bacterium have recently also occurred in other regions of the world with the emerging of new pandemic clone O3:K6 and often has occurred on eating seafood (Matsumoto et al., 2000; Wong et al., 2000; Depaola et al., 2000). Some efforts are needed to enhance the safety of seafood. Antimicrobial activities of spices and herbs and essential oils have been well known for long time. Many studies reported the activities of spices and herbs or essential oils to foodborne ⁎ Corresponding author. Tel.: +81 45 788 7669; fax: +81 45 788 5001. E-mail address: [email protected] (Y. Yano). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.04.031
pathogenic bacteria (Deans and Ritchie, 1987; Shelef et al., 1980; Hirasa and Takemasa, 1998). A Japanese spice, wasabi (Wasabi japonica), is traditionally used on eating raw fish such as sushi in Japan. The spice has been known to have antimicrobial effect against several bacteria including V. parahaemolyticus and is believed to contribute to the safety of eating raw seafood (Hasegawa et al., 1999). However, a few studies have investigated the effects of other spices and herbs against marine pathogenic bacteria such as V. parahaemolyticus (Beuchat, 1976; Koga et al., 1999). The Japanese style of cuisine of eating raw and lightly cooked seafood is increasingly popular in Europe and the United States, and in Asian countries, and seems to be also adopted in the local cuisines with globalization of food. Many kinds of spices and herbs have been used for taste and preservation of various food and cuisine in the world and could be introduced to raw and lightly cooked seafood. For example, in Japan, raw fish slices with raw vegetables are covered with dressing (seasoning) containing spices and herbs like carpaccio in Italian cuisines and such dressings are going to be commercial. Thus, in this study, we screened the potential of whole spices and herbs to inhibit V. parahaemolyticus, investigated the minimum inhibitory concentrations
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(MICs) of these spices and herbs under different nutrient and thermal conditions, and compared the activities among different serotypes of the bacterium. 2. Materials and methods 2.1. Spices and herbs Dry spices and herbs for food were purchased from groceries and processing companies: anise (Pimpinella anisum), basil (Ocimum basilicum), carom (Trachyspermum ammi), clove (Syzygium aromaticum), coriander (Coriandrum sativum), cumin (Cuminum cyminum), garlic (Allium sativum), ginger (Zingiber officinale), horseradish (Armoracia rusticana), Japanese pepper (Zanthoxylum piperitum), marjoram (Origanum marjorana), oregano (Origanum vulgare), peppermint (Mentha piperita), rosemary (Rosmarinus officinalis), sage (Salvia officinalis), spearmint (Mentha spicata), thyme (Thymus vulgaris), and turmeric (Curcuma longa). Several spices and herbs were obtained from different processing companies. Each spice and herb was finely ground in a mortar and sterile distilled water was added to make a concentration of 5% (weight of dry matter/volume of water). The suspensions were kept for 24 h at room temperature for hydration and extraction, and then stored until experiments at − 20 °C. Prior to the tests, viable bacterial counts in the suspensions were examined with Marine agar (MA; Difco, USA) and thiosulfate–citrate–bile–sucrose agar (TCBS; Nissui Pharmacy, Japan) plates at 37 °C incubation. 2.2. Bacterial strains V. parahaemolyticus clinical strain VPY01 (serotype, O3:K6) which was positive in thermo stable direct hemolysin (TDH) production was used in all experiments. Clinical strains VPY12 (O3:K6), VPY21 (O4:K8), and VPY22 (O5:K68) which were positive in TDH production, and environmental strains A6 and B1 which were negative in PCR for tdh and trh genes were also used in this study. For the experiments, a loopful of the working stocks was transferred to 3 ml of heart infusion broth (HI broth; Difco) with NaCl added at a final concentration of 3% (Na–HI broth) and incubated at 37 °C for 18 h. The overnight cultures were used for the experiments. Escherichia coli non-pathogenic strain JCM109 was used in this study. E. coli was cultured using the same protocol for V. parahaemolyticus except for using HI broth only as the culture medium. 2.3. Screening of spices and herbs The antibacterial activity of spices and herbs was screened in nutrient rich medium (Na–HI broth). A 100 μl aliquot of 5% suspension of each spice or herb in Na–HI broth was put in each well of a 96 well micro-titer plate and kept for 30 min at 5 or 30 °C. The overnight culture of V. parahaemolyticus strain VPY01 was diluted with fresh Na–HI broth to a level of 104 CFU/ml. Immediately, a 100 μl aliquot of the bacterial dilution was inoculated to each well of the plates, and then
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incubated at 5 or 30 °C for 24 h. For a non-treated control, the bacterial dilution was inoculated to the same amount of Na–HI broth. In addition, fresh Na–HI broth was added to an equal volume of the 5% suspensions and incubated to check the background bacterial counts in the spices and herbs. After incubation, a 100 μl aliquot of the mixed solutions and of the 10-fold dilutions was spread onto TCBS agar plate and incubated at 37 °C for 18 h. The antibacterial activities of the spices and herbs against E. coli were also examined for comparison using HI broth, and the survival after the treatment was examined on DHL plates. Viable counts in non-treated control of V. parahaemolyticus were reduced by 1-log with the incubation at 5 °C for 24 h in this study. Viable counts in the control of E. coli were constant or increased only 0.1-log with the same incubation. Thus, the antibacterial activities to these bacteria at 5 °C were difficult to be evaluated. In this study, the absence of culturable bacteria after a 24 h incubation was defined as positive for the antibacterial activity. 2.4. Determination of MICs Each well of a sterile 96 well micro-titer plate was filled with a 100 μl aliquot of Na–HI broth. The first column of wells in each micro-titer plate received a 100 μl of the broth containing the spices or herbs to be tested. After mixing by pipetting, the mixtures (100 μl) were transferred to the next column of wells in a process of 1:1 serial dilution until column #12. Then, the plates were incubated at 5 or 30 °C for 30 min before bacterial inoculation. An inoculum was prepared by diluting the overnight culture of V. parahaemolyticus strain VPY01 to a level of 2 × 104 CFU/ml. Each well in the micro-titer plate was inoculated with a 100 μl of the inoculum and incubated at 5 or 30 °C for 24 h. Growth or survival of the bacterium after incubation was examined with following method because the bacterium does not grow at 5 °C. A 100 μl of the mixture from each well was spread onto TCBS and incubated at 37 °C for 24 h. The concentration in the lowest serial dilution of the spices and herbs at which growth did not occur on TCBS was recorded as the minimal inhibitory concentration (MIC). The MICs were also determined in sterile natural seawater (NSW) instead of Na–HI broth. 2.5. Changes of viable counts in the media added marjoram and turmeric The change of viable counts of V. parahaemolyticus strain VPY01 with the addition of marjoram was investigated. Overnight culture of the bacterium in Na–HI broth was diluted with Na–HI at a level of 105 CFU/ml. The bacterial solutions were kept at 5 or 30 °C for 30 min to adjust the temperatures, marjoram was added at concentrations of 0.063 to 1%, and incubated at 5 and 30 °C. The bacterial samples were collected at 3, 6, 9, and 24 h after the initiation of incubation, and diluted 10-fold in PBS. The dilutions were spread on TCBS and MA, incubated at 37 °C overnight, and then the colonies were counted. The survival curve was also examined in the medium with turmeric added using the same manner.
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2.6. Comparison of sensitivity among V. parahaemolyticus strains The sensitivity to several spices and herbs was compared among the clinical and environmental strains of V. parahaemolyticus. The MICs were determined at 30 °C incubation for clove, marjoram, oregano and rosemary, and at 5 °C incubation for turmeric in Na–HI broth using the same procedure as described above. The sensitivity to each spice and herb was defined as the reciprocal of the MIC. 3. Results 3.1. Screening of antibacterial activity of spices and herbs Twenty three spices and herbs of 18 species were examined for their antibacterial activities against V. parahaemolyticus at a final concentration of 2.5% (Table 1). In the incubation at 30 °C, basil, clove, garlic, horseradish, marjoram, oregano, rosemary, and thyme showed antibacterial activities. By decreasing the incubation temperature to 5 °C, carom, ginger, Japanese pepper, peppermint, sage, spearmint, and turmeric were additionally found to have antibacterial activities, but horseradish lost the activity. In these tests, the viable counts of the bacterium were
constant or reduced by 1-log after the incubation of low temperature (data not shown). Antibacterial activities against E. coli were detected for basil, clove, garlic, marjoram, oregano, and rosemary at 30 °C and for all of the same but basil and garlic at 5 °C. 3.2. Minimum inhibitory concentrations of spices and herbs MICs of basil, clove, garlic, horseradish, marjoram, oregano, rosemary, thyme, and turmeric were determined under several conditions (Table 2). In the nutrient rich medium (Na–HI broth), the lowest MIC at 30 °C was 0.125% in clove and marjoram, and at 5 °C was 0.063% in marjoram and turmeric. In other spices and herbs, the MICs ranged from 0.5% to >2.0% at 30 °C and 0.25% to > 2.0% at 5 °C. Reducing the incubation temperature produced little effect on the MICs of the spice and herbs except for turmeric, but the MIC of turmeric decreased from > 2% at 30 °C to 0.063% at 5 °C. In the nutrient poor medium (NSW), the MICs of spices and herbs were generally lower than those in the nutrient rich medium when compared between the same temperatures. Especially, the MICs of basil and marjoram were lowered to 0.016 and 0.001% in the poor medium at 30 °C incubation, respectively. At 5 °C incubation, the MIC of marjoram was much lowered to 0.00024% in the
Table 1 The results of screening for antibacterial activities of spices and herbs against Vibrio parahaemolyticus and Escherichia coli Spices and herbs
Scientific name
pHa
Growth or survival after 24 h incubation V. parahaemolyticus 30 °Cb
Anise seed Basil-I Basil-II Carom seed Clove Coriander seed Cumin seed-I Cumin seed-II Garlic Ginger Horseradish Japanese pepper Marjoram Oregano-I Oregano-II Peppermint Rosemary Sage Spearmint Thyme-I Thyme-II Turmeric-I Turmeric-II Control a
Pimpinella anisum Ocimum basilicum Ocimum basilicum Trachyspermum ammi Syzygium aromaticum Coriandrum sativum Cuminum cyminum Cuminum cyminum Allium sativum Zingiber officinale Armoracia rusticana Zanthoxylum piperitum Origanum marjorana Origanum vulgare Origanum vulgare Mentha piperita Rosmarinus officinalis Salvia officinalis Mentha spicata Thymus vulgaris Thymus vulgaris Curcuma longa Curcuma longa
5.7 5.9 6.0 5.7 3.6 4.7 5.8 5.7 5.8 5.7 4.3 4.8 5.9 5.6 5.4 6.2 5.8 6.0 5.7 5.8 5.8 6.1 5.8 7.2
c
+ − − + − + + + − + − + − − − + − + + − − + + +
E. coli 5 °C
30 °C
5 °C
± − − − − ± ± ± − − ± − − − − − − − − − − − − ±d
+ − − + − + + + − + + + − − − + − + + + + + + +
± ± ± ± − ± ± ± ± ± ± ± − − − ± − ± ± ± ± ± ± ±e
The pHs were determined in 10% solutions of the spices and herbs. Incubation temperature in the screening tests. c +; Growth, viable counts increased more than 106 CFU/ml after 24 h incubation. ±; Survival, viable counts were from 102 to 105 CFU/ml after 24 h incubation. −; Death, viable counts were reduced less than 101 CFU/ml after 24 h incubation. d Viable counts were reduced approximately by 1-log after 24 h incubation. e Viable counts were constant after 24 h incubation. b
Y. Yano et al. / International Journal of Food Microbiology 111 (2006) 6–11
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Table 2 Minimum inhibitory concentrations (%) of spices and herbs against Vibrio parahaemolyticus Spices and herbs Basil Clove Garlic Horseradish Marjoram Oregano Rosemary Thyme Turmeric
Na-HI brotha
NSWb
30 °Cc
5 °Cd
30 °C
5 °C
2 0.125 1 2 0.125 0.5 0.5 2 >2
1 0.25 1 >2 0.063 0.5 0.25 1 0.063
0.016 0.004 0.25 1 0.001 0.032 0.008 0.032 2
1 0.016 2 1 0.00025 0.25 0.063 0.5 0.004
a
Heart Infusion broth added NaCl. Sterile natural sea water. c Incubation temperature. d At 5 °C incubation, the viable counts in the broth with no spices and herbs (control) were constant or reduced by 1-log after 24 hr.
▪
Fig. 2. Changes of viable counts of V. parahaemolyticus in Na–HI broth with turmeric added at concentrations of ○: 1%, ▵: 0.25%, □: 0.063%, and : 0% (control).
b
nutrient poor medium. When the MICs in the nutrient poor medium were compared between both temperatures, most of the spices and herbs exhibited higher MICs at 5 °C than at 30 °C. For example, the MIC of basil at 5 °C was 64 times higher than at 30 °C. On the other hand, the MIC of turmeric at 5 °C was one five-hundreds-twelfth of that at 30 °C. 3.3. Changes of viable counts in media with marjoram added The inhibitory effect of marjoram and turmeric against V. parahaemolyticus was confirmed in Na–HI broth (Fig. 1). In the Na–HI broth without marjoram (control), the bacterium which was inoculated at a level of 105 CFU/ml started to grow exponentially at 3 h and reached a level of 108 CFUs/ml at 9 h at 30 °C. In the broth with marjoram added and incubated at 30 °C, the bacterial level decreased below the detection limit within 10 min at concentrations of 1% and 0.25% and within 3 h at that of 0.125%. The addition at a concentration of 0.063% caused the delay of growth but did not stop the growth finally. At 5 °C incubation, the viable counts gradually decreased even in the control broth, and 1-log and 1.5-log decreases were observed at 9 and 24 h, respectively. With the addition of marjoram at a
Fig. 1. Changes of viable counts of V. parahaemolyticus in Na–HI broth with marjoram added at concentrations of ○: 1%, ▵: 0.25%, ▴: 0.125%, □: 0.063%, and : 0% (control).
▪
concentration of 1%, the bacterial level decreased to below detection limit within 10 min. Although the decrease of the population at 5 °C was slower than at 30 °C, the population was reduced to below the detection limit in cultures added with marjoram at all concentrations including 0.063% within 24 h. In the broth added turmeric (Fig. 2), the growth was delayed at concentrations of 1% and 0.25% when incubated at 30 °C but not stopped. On the other hand, the bacterial counts decreased just after addition at concentrations of 1 and 0.25%, and 3 h at a concentration of 0.063% when incubated at 5 °C. The bacterial counts were reduced less than detection limit at all concentrations for 24 h incubation. 3.4. Comparison of sensitivity among V. parahaemolyticus strains The sensitivity to spices and herbs was compared among clinical strains with different serotypes and environmental strains (Fig. 3). The sensitivity to each spice and herb was defined as the reciprocal of the MIC in the present study. It was generally similar among the strains, except for turmeric, and the difference was less than 2 times among the strains. In these tests, the inoculum sizes did not exceed 1-log among the strains used (data not shown). In addition, increasing by one order of magnitude in inoculum size did not affect the MICs or increased by only 2
Fig. 3. Variation in sensitivity of different strains of Vibrio parahaemolyticus to spices and herbs. The sensitivity is expressed as the reciprocal of the minimum inhibitory concentration (%). Strain VPY01 (O3:K6, tdh+), strain VPY12 (O3: K6, tdh+), strain VPY21 (O4:K8, tdh+), strain VPY22 (O5:K68, tdh+), strain A6 (environmental, tdh−), and strain B1 (environmental, tdh−).
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Table 3 Effect of inoculum size on minimum inhibitory concentrations (%) against Vibrio parahaemolyticus Inoculuma
1.0 × 105 1.0 × 104 1.0 × 103 a b c
Na-HI brothb
NSWc
Clove
Marjoram
Clove
Marjoram
0.125 0.061 0.061
0.061 0.061 0.031
0.008 0.008 0.004
0.0005 0.0005 0.00025
The bacterial number treated by the spices. Heart Infusion broth added NaCl. Sterile natural seawater.
times the MICs (Table 3). The sensitivity to turmeric was relatively variable in the strains and the largest difference was more than 10 times. 4. Discussion Basil, clove, garlic, horseradish, marjoram, oregano, rosemary, and thyme were found to exhibit inhibitory activities against V. parahaemolyticus in the incubation with nutrient rich medium and 30 °C which was good for the growth of the bacterium. Previous studies have reported the presence of antibacterial activities of oregano, rosemary, and thyme (Beuchat, 1976; Shelef et al., 1980). While antibacterial activity against V. parahaemolyticus has been unknown in basil, clove, and marjoram, these spices and herbs, and essential oils have been well known to have inhibitory effects against a variety of bacteria including Gram-negative bacteria (Deans and Ritchie, 1987; Suppakul et al., 2003). The results in the present study also indicate that these spices and herbs inhibited the growth of E. coli. The screening test was performed also at low temperature, and carom seed, ginger, Japanese pepper, sage, spearmint, and turmeric were additionally found to exhibit antibacterial activities against V. parahaemolyticus. This bacterium is well known to be sensitive to coldness (Oliver and Kaper, 1997) and, in fact, the viable counts were reduced by 1-log in the control broth with 5 °C incubation in the tests. Thus, these results don't indicate that these spices and herbs have antibacterial activities which are activated by low temperature. These spices and herbs seem to decrease the survival of the bacterium at low temperature. Although the following tests found that these spices and herbs except for turmeric had relatively high MICs at low temperature incubation, indicating weak antimicrobial activity, low temperature screening was effective for detecting the activity against the bacterium, compared with E. coli. The lowest MIC was 0.125% observed in clove and marjoram when tested in the nutrient rich medium and at 30 °C. These spices and herbs had 4 times stronger activities than oregano and rosemary which had the second lowest MICs in the tests. Beuchat (1976) reported that V. parahaemolyticus was highly sensitive to oregano at 35 °C in a nutrient rich medium. Shelef et al. (1980) reported that the growth of the bacterium was inhibited by rosemary at concentrations of 0.3 to 0.5% at 32 °C incubation. Essential oils of clove as well as those of oregano and rosemary are well known to have strong antimicrobial effects to a variety of bacteria, although marjoram relatively had weak activity against
Gram-negative bacteria (Smith-Palmer et al., 1998; Ueda et al., 1982). The oil of marjoram was also shown to be inhibitory to bacteria of the family Vibrio, while the activity was lower than oregano and clove (Deans and Ritchie, 1987). The present study suggests that marjoram itself has a strong activity against the bacterium at the same level as clove. Seafood, which is the main source in foodborne disease by V. parahaemolyticus, is generally stored in a refrigerator even for a short time, because seafood easily deteriorates in quality like its color and flavor. The major antibacterial components of spices and herbs have been known to be terpenes such as eugenol and carvacrol (Davidson, 1997). These are generally less dissolved at low temperature and the antibacterial activity of spices and herbs may be reduced when used in refrigerators. Previous studies have reported that temperature effects varied with the kind of spices and herbs, and bacteria tested (Ting and Deibel, 1991; Burt and Reinders, 2003). In the present study, decreasing the incubation temperature generally produced little effect to the MICs, or reduced MICs in the nutrient rich medium. The results seem to be basically attributed to the sensitivity of the bacterium to low temperature described above, and it is difficult to evaluate the true activity of the spices and herbs at low temperature. It is, however, obvious that these spices and herbs decreased the survival of the bacterium at low temperature. Figs. 1 and 2 also indicated that the viable counts were much more reduced in the broth added spices and herbs than those in the control broth at 5 °C. In other words, the spices and herbs can enhance the destructive effects of low temperature to the bacterium. The present study suggests that some of these spices and herbs can be practical for protecting seafood from the risk of the bacterium and can be used in hurdle technology with low temperature. The MIC of turmeric was as low as that of marjoram at low temperature in the nutrient rich medium. Little has been examined on the antibacterial activity of turmeric, although the spice was reported to exhibit weak antimicrobial activity against several bacteria (Lutomski et al., 1974). In the present study also, the MIC of turmeric was more than 2% at 30 °C, and that of marjoram was 0.125% at high temperature. The results in Figs. 2 and 3 also indicated the difference of both spices in temperature effect of the antibacterial activity. These results suggest the presence of anti-Vibrio substances in turmeric which may be different from the ones in marjoram. The antibacterial activity of turmeric may be able to act synergistically with those of other spices and herbs. Further study is needed to understand the nature of the antibacterial activity of spices and herbs. Spices and herbs are generally applied to food which is a nutrient rich environment for the bacterium. The antibacterial activity, however, could be also used in nutrient poor environments, for example, cleaning of food processing devices and depuration of shellfish (Birkenhauer and Oliver, 2003). In this study, the MICs of spices and herbs were generally decreased in NSW than those in Na–HI broth. The growth of V. parahaemolyticus delays in NSW at 30 °C, compared with Na–HI broth. In addition, some substances like fatty acids in broth are known to protect the bacteria from attack of the antimicrobial activity of spices and herbs (Keweloh et al., 1991). Thus, the MICs were expected to be decreased in the nutrient poor medium, but the very
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low MIC of marjoram was interesting. The spice can decrease the bacterial survival even at a concentration of 2.5 ppm at low temperature. The MICs of other spices and herbs were increased with the decreasing of temperature except for marjoram and turmeric, and the increase may be due to the lower solubility of antibacterial substances such as terpenes at low temperature. Marjoram and turmeric may contain substances differing from those of others, or could contain any nutrients for the bacterium at high temperature. These spices and herbs could be used for the control of V. parahaemolyticus in nutrient poor environments. The presence of various serotypes is known in the bacterium V. parahaemolyticus. In Japan, serotype O4:K8 was the main causative agent of the infections in 1994, and since 1996 the emerging pathogenic strain O3:K6 has been responsible for many of the recent outbreaks (Matsumoto et al., 2000). The strain also caused outbreaks in Asia, India, and North America (Wong et al., 2000; Depaola et al., 2000). In the present study, the pathogenic serotype strains including O3:K6 and O4:K8 were sensitive to a similar extent in each spice or herb except for turmeric. Shelef et al. (1980) reported that the pathogenic strains O4:K8 and O4:K11 were more sensitive to rosemary than a nonpathogenic one. Hasegawa et al. (1999) showed that pathogenic strains including O4:K8 were of similar sensitivity, but did not examine the emerging strain O3:K6. The present study indicates that the strain O3:K6 is possibly as sensitive as the strains reported before. Some spices and herbs could contribute to reducing the risk of the emerging pathogenic strain O3:K6. Acknowledgements This study was carried out with financial support (MAFF) from the Ministry of Agriculture, Forestry, and Fisheries. We thank Mrs. Y. Shimono and N. Hatano for technical assistance. References Beuchat, L.R., 1976. Sensitivity of Vibrio parahaemolyticus to spices and organic acids. Journal of Food Science 41, 899–902. Birkenhauer, J.M., Oliver, J.D., 2003. Use of diacetyl to reduce the load of Vibrio vulnificus in the eastern oyster, Crassostrea virginica. Journal of Food Protection 66, 38–43. Burt, S.A., Reinders, R.D., 2003. Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7. Letters in Applied and Microbiology 36, 162–167.
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Davidson, P.M., 1997. Chemical preservatives and natural antimicrobial compounds. In: Doyle, M., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington D.C., pp. 520–556. Deans, S.G., Ritchie, G., 1987. Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5, 165–180. Depaola, A., Kaysner, C.A., Bowers, J., Cook, D.W., 2000. Environmental investigations of oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Applied and Environmental Microbiology 66, 4649–4654. Hasegawa, N., Matsumoto, Y., Hoshino, A., Iwashita, K., 1999. Comparison of effects of Wasabi japonica and allyl isothiocyanate on the growth of four strains of Vibrio parahaemolyticus in lean and fatty tuna meat suspensions. International Journal of Food Microbiology 49, 27–34. Hirasa, K., Takemasa, M., 1998. Antimicrobial and antioxidant properties of spices. In: Hirasa, K., Takemasa, M. (Eds.), Spice Science and Technology. Marcel Dekker Inc., New York, pp. 163–200. Keweloh, H., Diefenbach, R., Rehm, H.J., 1991. Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids. Archives of Microbiology 157, 49–53. Koga, T., Hirota, N., Takumi, K., 1999. Bactericidal activities of essential oils of basil and sage against a range of bacteria and the effect of these essential oils on Vibrio parahaemolyticus. Microbiological Research 154, 267–273. Lutomski, J., Kedzia, B., Debska, W., 1974. Effect of the alcohol extract and active ingredients from Curcuma longa on bacteria and fungi. Planta Medica 2, 9. Matsumoto, C., Okuda, J., Ishibashi, M., Iwanaga, M., Garg, P., Rammamurthy, T., Wong, H.C., Depaola, A., Kim, Y.B., Albert, M.J., Nishibuchi, M., 2000. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses. Journal Clinical Microbiology 38, 578–585. Oliver, J.D., Kaper, J.B., 1997. Vibrio species. In: Doyle, M., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington D.C., pp. 228–264. Shelef, L.A., Naglik, O.A., Bogen, D.W., 1980. Sensitivity of some common foodborne bacteria to the spices sage, rosemary, and allspice. Journal of Food Science 45, 1042–1044. Smith-Palmer, A., Stewart, J., Fyfe, L., 1998. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Letters in Applied Microbiology 26, 118–122. Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W., 2003. Antimicrobial properties of basil and its possible application in food packaging. Journal of Agricultural and Food Chemistry 51, 3197–3207. Ting, W.T.E., Deibel, K.E., 1991. Sensitivity of Listeria monocytogenes to spices at two temperatures. Journal of Food Safety 12, 129–137. Ueda, S., Yamashita, H., Nakajima, M., Kuwabara, Y., 1982. Inhibition of microorganisms by spice extracts and flavoring compounds. Nippon Shokuhin Kogyo Gakkaishi 29, 111–116. Wong, H., Liu, S., Ku, L., Lee, I., Wang, T., Lee, Y., Lee, C., Kuo, L., Shin, D.Y., 2000. Characterization of Vibrio parahaemolyticus isolates obtained from foodborne illness outbreaks during 1992 through 1995 in Taiwan. Journal of Food Protection 63, 900–906.
Antimicrobial effect of spices and herbs on Vibrio parahaemolyticus Yutaka Yano ⁎, Masataka Satomi, Hiroshi Oikawa Seafood Safety Section, National Research Institute of Fisheries Science, 2-12-4 Fukuura, Yokohama 2368648, Japan Received 2 May 2005; received in revised form 1 March 2006; accepted 25 April 2006
Abstract The antimicrobial effects of spices and herbs from 18 plant species were examined on a foodborne pathogen, Vibrio parahaemolyticus, with the use of combinations of temperatures and nutrient levels. Basil, clove, garlic, horseradish, marjoram, oregano, rosemary, and thyme exhibited antibacterial activities at incubation of 30 °C, while with the exception of horseradish, the same spices and additional 7 species exhibited the activities at 5 °C. The lowest MIC (minimum inhibitory concentration) was 0.125% observed in clove and marjoram at 30 °C in a nutrient rich medium. Lowering of incubation temperature produced little effect on the MICs except for turmeric. The decreasing of the MIC in turmeric appeared to be basically attributed to the sensitivity of the bacterium to coldness. In nutrient poor medium, the lowest was 0.001 and 0.00025% in marjoram at 30 °C and at 5 °C, respectively. The sensitivity to several spices and herbs was similar among different clinical serotypes including the emerging strain O3:K6. These results suggest that the spices and herbs can be practical for protecting seafood from the risk of contamination by V. parahaemolyticus and used in hurdle technology with low temperature. © 2006 Elsevier B.V. All rights reserved. Keywords: Vibrio parahaemolyticus; Spices; Herbs; Antibacterial activity
1. Introduction Vibrio parahaemolyticus is a bacterium which causes mild gastroenteritis in humans on consumption of infected seafood (Oliver and Kaper, 1997). The bacterium generally inhabits coastal environments in tropical and temperate zones, and contaminates fishery products caught in these areas, especially during warm periods. In Japan, a variety of seafood has been traditionally consumed, and raw or lightly cooked seafood is favored. This eating habit seems to provide an explanation for many cases of foodborne disease by V. parahaemolyticus in the country. The foodborne diseases by the bacterium have recently also occurred in other regions of the world with the emerging of new pandemic clone O3:K6 and often has occurred on eating seafood (Matsumoto et al., 2000; Wong et al., 2000; Depaola et al., 2000). Some efforts are needed to enhance the safety of seafood. Antimicrobial activities of spices and herbs and essential oils have been well known for long time. Many studies reported the activities of spices and herbs or essential oils to foodborne ⁎ Corresponding author. Tel.: +81 45 788 7669; fax: +81 45 788 5001. E-mail address: [email protected] (Y. Yano). 0168-1605/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ijfoodmicro.2006.04.031
pathogenic bacteria (Deans and Ritchie, 1987; Shelef et al., 1980; Hirasa and Takemasa, 1998). A Japanese spice, wasabi (Wasabi japonica), is traditionally used on eating raw fish such as sushi in Japan. The spice has been known to have antimicrobial effect against several bacteria including V. parahaemolyticus and is believed to contribute to the safety of eating raw seafood (Hasegawa et al., 1999). However, a few studies have investigated the effects of other spices and herbs against marine pathogenic bacteria such as V. parahaemolyticus (Beuchat, 1976; Koga et al., 1999). The Japanese style of cuisine of eating raw and lightly cooked seafood is increasingly popular in Europe and the United States, and in Asian countries, and seems to be also adopted in the local cuisines with globalization of food. Many kinds of spices and herbs have been used for taste and preservation of various food and cuisine in the world and could be introduced to raw and lightly cooked seafood. For example, in Japan, raw fish slices with raw vegetables are covered with dressing (seasoning) containing spices and herbs like carpaccio in Italian cuisines and such dressings are going to be commercial. Thus, in this study, we screened the potential of whole spices and herbs to inhibit V. parahaemolyticus, investigated the minimum inhibitory concentrations
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(MICs) of these spices and herbs under different nutrient and thermal conditions, and compared the activities among different serotypes of the bacterium. 2. Materials and methods 2.1. Spices and herbs Dry spices and herbs for food were purchased from groceries and processing companies: anise (Pimpinella anisum), basil (Ocimum basilicum), carom (Trachyspermum ammi), clove (Syzygium aromaticum), coriander (Coriandrum sativum), cumin (Cuminum cyminum), garlic (Allium sativum), ginger (Zingiber officinale), horseradish (Armoracia rusticana), Japanese pepper (Zanthoxylum piperitum), marjoram (Origanum marjorana), oregano (Origanum vulgare), peppermint (Mentha piperita), rosemary (Rosmarinus officinalis), sage (Salvia officinalis), spearmint (Mentha spicata), thyme (Thymus vulgaris), and turmeric (Curcuma longa). Several spices and herbs were obtained from different processing companies. Each spice and herb was finely ground in a mortar and sterile distilled water was added to make a concentration of 5% (weight of dry matter/volume of water). The suspensions were kept for 24 h at room temperature for hydration and extraction, and then stored until experiments at − 20 °C. Prior to the tests, viable bacterial counts in the suspensions were examined with Marine agar (MA; Difco, USA) and thiosulfate–citrate–bile–sucrose agar (TCBS; Nissui Pharmacy, Japan) plates at 37 °C incubation. 2.2. Bacterial strains V. parahaemolyticus clinical strain VPY01 (serotype, O3:K6) which was positive in thermo stable direct hemolysin (TDH) production was used in all experiments. Clinical strains VPY12 (O3:K6), VPY21 (O4:K8), and VPY22 (O5:K68) which were positive in TDH production, and environmental strains A6 and B1 which were negative in PCR for tdh and trh genes were also used in this study. For the experiments, a loopful of the working stocks was transferred to 3 ml of heart infusion broth (HI broth; Difco) with NaCl added at a final concentration of 3% (Na–HI broth) and incubated at 37 °C for 18 h. The overnight cultures were used for the experiments. Escherichia coli non-pathogenic strain JCM109 was used in this study. E. coli was cultured using the same protocol for V. parahaemolyticus except for using HI broth only as the culture medium. 2.3. Screening of spices and herbs The antibacterial activity of spices and herbs was screened in nutrient rich medium (Na–HI broth). A 100 μl aliquot of 5% suspension of each spice or herb in Na–HI broth was put in each well of a 96 well micro-titer plate and kept for 30 min at 5 or 30 °C. The overnight culture of V. parahaemolyticus strain VPY01 was diluted with fresh Na–HI broth to a level of 104 CFU/ml. Immediately, a 100 μl aliquot of the bacterial dilution was inoculated to each well of the plates, and then
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incubated at 5 or 30 °C for 24 h. For a non-treated control, the bacterial dilution was inoculated to the same amount of Na–HI broth. In addition, fresh Na–HI broth was added to an equal volume of the 5% suspensions and incubated to check the background bacterial counts in the spices and herbs. After incubation, a 100 μl aliquot of the mixed solutions and of the 10-fold dilutions was spread onto TCBS agar plate and incubated at 37 °C for 18 h. The antibacterial activities of the spices and herbs against E. coli were also examined for comparison using HI broth, and the survival after the treatment was examined on DHL plates. Viable counts in non-treated control of V. parahaemolyticus were reduced by 1-log with the incubation at 5 °C for 24 h in this study. Viable counts in the control of E. coli were constant or increased only 0.1-log with the same incubation. Thus, the antibacterial activities to these bacteria at 5 °C were difficult to be evaluated. In this study, the absence of culturable bacteria after a 24 h incubation was defined as positive for the antibacterial activity. 2.4. Determination of MICs Each well of a sterile 96 well micro-titer plate was filled with a 100 μl aliquot of Na–HI broth. The first column of wells in each micro-titer plate received a 100 μl of the broth containing the spices or herbs to be tested. After mixing by pipetting, the mixtures (100 μl) were transferred to the next column of wells in a process of 1:1 serial dilution until column #12. Then, the plates were incubated at 5 or 30 °C for 30 min before bacterial inoculation. An inoculum was prepared by diluting the overnight culture of V. parahaemolyticus strain VPY01 to a level of 2 × 104 CFU/ml. Each well in the micro-titer plate was inoculated with a 100 μl of the inoculum and incubated at 5 or 30 °C for 24 h. Growth or survival of the bacterium after incubation was examined with following method because the bacterium does not grow at 5 °C. A 100 μl of the mixture from each well was spread onto TCBS and incubated at 37 °C for 24 h. The concentration in the lowest serial dilution of the spices and herbs at which growth did not occur on TCBS was recorded as the minimal inhibitory concentration (MIC). The MICs were also determined in sterile natural seawater (NSW) instead of Na–HI broth. 2.5. Changes of viable counts in the media added marjoram and turmeric The change of viable counts of V. parahaemolyticus strain VPY01 with the addition of marjoram was investigated. Overnight culture of the bacterium in Na–HI broth was diluted with Na–HI at a level of 105 CFU/ml. The bacterial solutions were kept at 5 or 30 °C for 30 min to adjust the temperatures, marjoram was added at concentrations of 0.063 to 1%, and incubated at 5 and 30 °C. The bacterial samples were collected at 3, 6, 9, and 24 h after the initiation of incubation, and diluted 10-fold in PBS. The dilutions were spread on TCBS and MA, incubated at 37 °C overnight, and then the colonies were counted. The survival curve was also examined in the medium with turmeric added using the same manner.
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2.6. Comparison of sensitivity among V. parahaemolyticus strains The sensitivity to several spices and herbs was compared among the clinical and environmental strains of V. parahaemolyticus. The MICs were determined at 30 °C incubation for clove, marjoram, oregano and rosemary, and at 5 °C incubation for turmeric in Na–HI broth using the same procedure as described above. The sensitivity to each spice and herb was defined as the reciprocal of the MIC. 3. Results 3.1. Screening of antibacterial activity of spices and herbs Twenty three spices and herbs of 18 species were examined for their antibacterial activities against V. parahaemolyticus at a final concentration of 2.5% (Table 1). In the incubation at 30 °C, basil, clove, garlic, horseradish, marjoram, oregano, rosemary, and thyme showed antibacterial activities. By decreasing the incubation temperature to 5 °C, carom, ginger, Japanese pepper, peppermint, sage, spearmint, and turmeric were additionally found to have antibacterial activities, but horseradish lost the activity. In these tests, the viable counts of the bacterium were
constant or reduced by 1-log after the incubation of low temperature (data not shown). Antibacterial activities against E. coli were detected for basil, clove, garlic, marjoram, oregano, and rosemary at 30 °C and for all of the same but basil and garlic at 5 °C. 3.2. Minimum inhibitory concentrations of spices and herbs MICs of basil, clove, garlic, horseradish, marjoram, oregano, rosemary, thyme, and turmeric were determined under several conditions (Table 2). In the nutrient rich medium (Na–HI broth), the lowest MIC at 30 °C was 0.125% in clove and marjoram, and at 5 °C was 0.063% in marjoram and turmeric. In other spices and herbs, the MICs ranged from 0.5% to >2.0% at 30 °C and 0.25% to > 2.0% at 5 °C. Reducing the incubation temperature produced little effect on the MICs of the spice and herbs except for turmeric, but the MIC of turmeric decreased from > 2% at 30 °C to 0.063% at 5 °C. In the nutrient poor medium (NSW), the MICs of spices and herbs were generally lower than those in the nutrient rich medium when compared between the same temperatures. Especially, the MICs of basil and marjoram were lowered to 0.016 and 0.001% in the poor medium at 30 °C incubation, respectively. At 5 °C incubation, the MIC of marjoram was much lowered to 0.00024% in the
Table 1 The results of screening for antibacterial activities of spices and herbs against Vibrio parahaemolyticus and Escherichia coli Spices and herbs
Scientific name
pHa
Growth or survival after 24 h incubation V. parahaemolyticus 30 °Cb
Anise seed Basil-I Basil-II Carom seed Clove Coriander seed Cumin seed-I Cumin seed-II Garlic Ginger Horseradish Japanese pepper Marjoram Oregano-I Oregano-II Peppermint Rosemary Sage Spearmint Thyme-I Thyme-II Turmeric-I Turmeric-II Control a
Pimpinella anisum Ocimum basilicum Ocimum basilicum Trachyspermum ammi Syzygium aromaticum Coriandrum sativum Cuminum cyminum Cuminum cyminum Allium sativum Zingiber officinale Armoracia rusticana Zanthoxylum piperitum Origanum marjorana Origanum vulgare Origanum vulgare Mentha piperita Rosmarinus officinalis Salvia officinalis Mentha spicata Thymus vulgaris Thymus vulgaris Curcuma longa Curcuma longa
5.7 5.9 6.0 5.7 3.6 4.7 5.8 5.7 5.8 5.7 4.3 4.8 5.9 5.6 5.4 6.2 5.8 6.0 5.7 5.8 5.8 6.1 5.8 7.2
c
+ − − + − + + + − + − + − − − + − + + − − + + +
E. coli 5 °C
30 °C
5 °C
± − − − − ± ± ± − − ± − − − − − − − − − − − − ±d
+ − − + − + + + − + + + − − − + − + + + + + + +
± ± ± ± − ± ± ± ± ± ± ± − − − ± − ± ± ± ± ± ± ±e
The pHs were determined in 10% solutions of the spices and herbs. Incubation temperature in the screening tests. c +; Growth, viable counts increased more than 106 CFU/ml after 24 h incubation. ±; Survival, viable counts were from 102 to 105 CFU/ml after 24 h incubation. −; Death, viable counts were reduced less than 101 CFU/ml after 24 h incubation. d Viable counts were reduced approximately by 1-log after 24 h incubation. e Viable counts were constant after 24 h incubation. b
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Table 2 Minimum inhibitory concentrations (%) of spices and herbs against Vibrio parahaemolyticus Spices and herbs Basil Clove Garlic Horseradish Marjoram Oregano Rosemary Thyme Turmeric
Na-HI brotha
NSWb
30 °Cc
5 °Cd
30 °C
5 °C
2 0.125 1 2 0.125 0.5 0.5 2 >2
1 0.25 1 >2 0.063 0.5 0.25 1 0.063
0.016 0.004 0.25 1 0.001 0.032 0.008 0.032 2
1 0.016 2 1 0.00025 0.25 0.063 0.5 0.004
a
Heart Infusion broth added NaCl. Sterile natural sea water. c Incubation temperature. d At 5 °C incubation, the viable counts in the broth with no spices and herbs (control) were constant or reduced by 1-log after 24 hr.
▪
Fig. 2. Changes of viable counts of V. parahaemolyticus in Na–HI broth with turmeric added at concentrations of ○: 1%, ▵: 0.25%, □: 0.063%, and : 0% (control).
b
nutrient poor medium. When the MICs in the nutrient poor medium were compared between both temperatures, most of the spices and herbs exhibited higher MICs at 5 °C than at 30 °C. For example, the MIC of basil at 5 °C was 64 times higher than at 30 °C. On the other hand, the MIC of turmeric at 5 °C was one five-hundreds-twelfth of that at 30 °C. 3.3. Changes of viable counts in media with marjoram added The inhibitory effect of marjoram and turmeric against V. parahaemolyticus was confirmed in Na–HI broth (Fig. 1). In the Na–HI broth without marjoram (control), the bacterium which was inoculated at a level of 105 CFU/ml started to grow exponentially at 3 h and reached a level of 108 CFUs/ml at 9 h at 30 °C. In the broth with marjoram added and incubated at 30 °C, the bacterial level decreased below the detection limit within 10 min at concentrations of 1% and 0.25% and within 3 h at that of 0.125%. The addition at a concentration of 0.063% caused the delay of growth but did not stop the growth finally. At 5 °C incubation, the viable counts gradually decreased even in the control broth, and 1-log and 1.5-log decreases were observed at 9 and 24 h, respectively. With the addition of marjoram at a
Fig. 1. Changes of viable counts of V. parahaemolyticus in Na–HI broth with marjoram added at concentrations of ○: 1%, ▵: 0.25%, ▴: 0.125%, □: 0.063%, and : 0% (control).
▪
concentration of 1%, the bacterial level decreased to below detection limit within 10 min. Although the decrease of the population at 5 °C was slower than at 30 °C, the population was reduced to below the detection limit in cultures added with marjoram at all concentrations including 0.063% within 24 h. In the broth added turmeric (Fig. 2), the growth was delayed at concentrations of 1% and 0.25% when incubated at 30 °C but not stopped. On the other hand, the bacterial counts decreased just after addition at concentrations of 1 and 0.25%, and 3 h at a concentration of 0.063% when incubated at 5 °C. The bacterial counts were reduced less than detection limit at all concentrations for 24 h incubation. 3.4. Comparison of sensitivity among V. parahaemolyticus strains The sensitivity to spices and herbs was compared among clinical strains with different serotypes and environmental strains (Fig. 3). The sensitivity to each spice and herb was defined as the reciprocal of the MIC in the present study. It was generally similar among the strains, except for turmeric, and the difference was less than 2 times among the strains. In these tests, the inoculum sizes did not exceed 1-log among the strains used (data not shown). In addition, increasing by one order of magnitude in inoculum size did not affect the MICs or increased by only 2
Fig. 3. Variation in sensitivity of different strains of Vibrio parahaemolyticus to spices and herbs. The sensitivity is expressed as the reciprocal of the minimum inhibitory concentration (%). Strain VPY01 (O3:K6, tdh+), strain VPY12 (O3: K6, tdh+), strain VPY21 (O4:K8, tdh+), strain VPY22 (O5:K68, tdh+), strain A6 (environmental, tdh−), and strain B1 (environmental, tdh−).
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Table 3 Effect of inoculum size on minimum inhibitory concentrations (%) against Vibrio parahaemolyticus Inoculuma
1.0 × 105 1.0 × 104 1.0 × 103 a b c
Na-HI brothb
NSWc
Clove
Marjoram
Clove
Marjoram
0.125 0.061 0.061
0.061 0.061 0.031
0.008 0.008 0.004
0.0005 0.0005 0.00025
The bacterial number treated by the spices. Heart Infusion broth added NaCl. Sterile natural seawater.
times the MICs (Table 3). The sensitivity to turmeric was relatively variable in the strains and the largest difference was more than 10 times. 4. Discussion Basil, clove, garlic, horseradish, marjoram, oregano, rosemary, and thyme were found to exhibit inhibitory activities against V. parahaemolyticus in the incubation with nutrient rich medium and 30 °C which was good for the growth of the bacterium. Previous studies have reported the presence of antibacterial activities of oregano, rosemary, and thyme (Beuchat, 1976; Shelef et al., 1980). While antibacterial activity against V. parahaemolyticus has been unknown in basil, clove, and marjoram, these spices and herbs, and essential oils have been well known to have inhibitory effects against a variety of bacteria including Gram-negative bacteria (Deans and Ritchie, 1987; Suppakul et al., 2003). The results in the present study also indicate that these spices and herbs inhibited the growth of E. coli. The screening test was performed also at low temperature, and carom seed, ginger, Japanese pepper, sage, spearmint, and turmeric were additionally found to exhibit antibacterial activities against V. parahaemolyticus. This bacterium is well known to be sensitive to coldness (Oliver and Kaper, 1997) and, in fact, the viable counts were reduced by 1-log in the control broth with 5 °C incubation in the tests. Thus, these results don't indicate that these spices and herbs have antibacterial activities which are activated by low temperature. These spices and herbs seem to decrease the survival of the bacterium at low temperature. Although the following tests found that these spices and herbs except for turmeric had relatively high MICs at low temperature incubation, indicating weak antimicrobial activity, low temperature screening was effective for detecting the activity against the bacterium, compared with E. coli. The lowest MIC was 0.125% observed in clove and marjoram when tested in the nutrient rich medium and at 30 °C. These spices and herbs had 4 times stronger activities than oregano and rosemary which had the second lowest MICs in the tests. Beuchat (1976) reported that V. parahaemolyticus was highly sensitive to oregano at 35 °C in a nutrient rich medium. Shelef et al. (1980) reported that the growth of the bacterium was inhibited by rosemary at concentrations of 0.3 to 0.5% at 32 °C incubation. Essential oils of clove as well as those of oregano and rosemary are well known to have strong antimicrobial effects to a variety of bacteria, although marjoram relatively had weak activity against
Gram-negative bacteria (Smith-Palmer et al., 1998; Ueda et al., 1982). The oil of marjoram was also shown to be inhibitory to bacteria of the family Vibrio, while the activity was lower than oregano and clove (Deans and Ritchie, 1987). The present study suggests that marjoram itself has a strong activity against the bacterium at the same level as clove. Seafood, which is the main source in foodborne disease by V. parahaemolyticus, is generally stored in a refrigerator even for a short time, because seafood easily deteriorates in quality like its color and flavor. The major antibacterial components of spices and herbs have been known to be terpenes such as eugenol and carvacrol (Davidson, 1997). These are generally less dissolved at low temperature and the antibacterial activity of spices and herbs may be reduced when used in refrigerators. Previous studies have reported that temperature effects varied with the kind of spices and herbs, and bacteria tested (Ting and Deibel, 1991; Burt and Reinders, 2003). In the present study, decreasing the incubation temperature generally produced little effect to the MICs, or reduced MICs in the nutrient rich medium. The results seem to be basically attributed to the sensitivity of the bacterium to low temperature described above, and it is difficult to evaluate the true activity of the spices and herbs at low temperature. It is, however, obvious that these spices and herbs decreased the survival of the bacterium at low temperature. Figs. 1 and 2 also indicated that the viable counts were much more reduced in the broth added spices and herbs than those in the control broth at 5 °C. In other words, the spices and herbs can enhance the destructive effects of low temperature to the bacterium. The present study suggests that some of these spices and herbs can be practical for protecting seafood from the risk of the bacterium and can be used in hurdle technology with low temperature. The MIC of turmeric was as low as that of marjoram at low temperature in the nutrient rich medium. Little has been examined on the antibacterial activity of turmeric, although the spice was reported to exhibit weak antimicrobial activity against several bacteria (Lutomski et al., 1974). In the present study also, the MIC of turmeric was more than 2% at 30 °C, and that of marjoram was 0.125% at high temperature. The results in Figs. 2 and 3 also indicated the difference of both spices in temperature effect of the antibacterial activity. These results suggest the presence of anti-Vibrio substances in turmeric which may be different from the ones in marjoram. The antibacterial activity of turmeric may be able to act synergistically with those of other spices and herbs. Further study is needed to understand the nature of the antibacterial activity of spices and herbs. Spices and herbs are generally applied to food which is a nutrient rich environment for the bacterium. The antibacterial activity, however, could be also used in nutrient poor environments, for example, cleaning of food processing devices and depuration of shellfish (Birkenhauer and Oliver, 2003). In this study, the MICs of spices and herbs were generally decreased in NSW than those in Na–HI broth. The growth of V. parahaemolyticus delays in NSW at 30 °C, compared with Na–HI broth. In addition, some substances like fatty acids in broth are known to protect the bacteria from attack of the antimicrobial activity of spices and herbs (Keweloh et al., 1991). Thus, the MICs were expected to be decreased in the nutrient poor medium, but the very
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low MIC of marjoram was interesting. The spice can decrease the bacterial survival even at a concentration of 2.5 ppm at low temperature. The MICs of other spices and herbs were increased with the decreasing of temperature except for marjoram and turmeric, and the increase may be due to the lower solubility of antibacterial substances such as terpenes at low temperature. Marjoram and turmeric may contain substances differing from those of others, or could contain any nutrients for the bacterium at high temperature. These spices and herbs could be used for the control of V. parahaemolyticus in nutrient poor environments. The presence of various serotypes is known in the bacterium V. parahaemolyticus. In Japan, serotype O4:K8 was the main causative agent of the infections in 1994, and since 1996 the emerging pathogenic strain O3:K6 has been responsible for many of the recent outbreaks (Matsumoto et al., 2000). The strain also caused outbreaks in Asia, India, and North America (Wong et al., 2000; Depaola et al., 2000). In the present study, the pathogenic serotype strains including O3:K6 and O4:K8 were sensitive to a similar extent in each spice or herb except for turmeric. Shelef et al. (1980) reported that the pathogenic strains O4:K8 and O4:K11 were more sensitive to rosemary than a nonpathogenic one. Hasegawa et al. (1999) showed that pathogenic strains including O4:K8 were of similar sensitivity, but did not examine the emerging strain O3:K6. The present study indicates that the strain O3:K6 is possibly as sensitive as the strains reported before. Some spices and herbs could contribute to reducing the risk of the emerging pathogenic strain O3:K6. Acknowledgements This study was carried out with financial support (MAFF) from the Ministry of Agriculture, Forestry, and Fisheries. We thank Mrs. Y. Shimono and N. Hatano for technical assistance. References Beuchat, L.R., 1976. Sensitivity of Vibrio parahaemolyticus to spices and organic acids. Journal of Food Science 41, 899–902. Birkenhauer, J.M., Oliver, J.D., 2003. Use of diacetyl to reduce the load of Vibrio vulnificus in the eastern oyster, Crassostrea virginica. Journal of Food Protection 66, 38–43. Burt, S.A., Reinders, R.D., 2003. Antibacterial activity of selected plant essential oils against Escherichia coli O157:H7. Letters in Applied and Microbiology 36, 162–167.
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Davidson, P.M., 1997. Chemical preservatives and natural antimicrobial compounds. In: Doyle, M., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington D.C., pp. 520–556. Deans, S.G., Ritchie, G., 1987. Antibacterial properties of plant essential oils. International Journal of Food Microbiology 5, 165–180. Depaola, A., Kaysner, C.A., Bowers, J., Cook, D.W., 2000. Environmental investigations of oysters after outbreaks in Washington, Texas, and New York (1997 and 1998). Applied and Environmental Microbiology 66, 4649–4654. Hasegawa, N., Matsumoto, Y., Hoshino, A., Iwashita, K., 1999. Comparison of effects of Wasabi japonica and allyl isothiocyanate on the growth of four strains of Vibrio parahaemolyticus in lean and fatty tuna meat suspensions. International Journal of Food Microbiology 49, 27–34. Hirasa, K., Takemasa, M., 1998. Antimicrobial and antioxidant properties of spices. In: Hirasa, K., Takemasa, M. (Eds.), Spice Science and Technology. Marcel Dekker Inc., New York, pp. 163–200. Keweloh, H., Diefenbach, R., Rehm, H.J., 1991. Increase of phenol tolerance of Escherichia coli by alterations of the fatty acid composition of the membrane lipids. Archives of Microbiology 157, 49–53. Koga, T., Hirota, N., Takumi, K., 1999. Bactericidal activities of essential oils of basil and sage against a range of bacteria and the effect of these essential oils on Vibrio parahaemolyticus. Microbiological Research 154, 267–273. Lutomski, J., Kedzia, B., Debska, W., 1974. Effect of the alcohol extract and active ingredients from Curcuma longa on bacteria and fungi. Planta Medica 2, 9. Matsumoto, C., Okuda, J., Ishibashi, M., Iwanaga, M., Garg, P., Rammamurthy, T., Wong, H.C., Depaola, A., Kim, Y.B., Albert, M.J., Nishibuchi, M., 2000. Pandemic spread of an O3:K6 clone of Vibrio parahaemolyticus and emergence of related strains evidenced by arbitrarily primed PCR and toxRS sequence analyses. Journal Clinical Microbiology 38, 578–585. Oliver, J.D., Kaper, J.B., 1997. Vibrio species. In: Doyle, M., Beuchat, L.R., Montville, T.J. (Eds.), Food Microbiology: Fundamentals and Frontiers. ASM Press, Washington D.C., pp. 228–264. Shelef, L.A., Naglik, O.A., Bogen, D.W., 1980. Sensitivity of some common foodborne bacteria to the spices sage, rosemary, and allspice. Journal of Food Science 45, 1042–1044. Smith-Palmer, A., Stewart, J., Fyfe, L., 1998. Antimicrobial properties of plant essential oils and essences against five important food-borne pathogens. Letters in Applied Microbiology 26, 118–122. Suppakul, P., Miltz, J., Sonneveld, K., Bigger, S.W., 2003. Antimicrobial properties of basil and its possible application in food packaging. Journal of Agricultural and Food Chemistry 51, 3197–3207. Ting, W.T.E., Deibel, K.E., 1991. Sensitivity of Listeria monocytogenes to spices at two temperatures. Journal of Food Safety 12, 129–137. Ueda, S., Yamashita, H., Nakajima, M., Kuwabara, Y., 1982. Inhibition of microorganisms by spice extracts and flavoring compounds. Nippon Shokuhin Kogyo Gakkaishi 29, 111–116. Wong, H., Liu, S., Ku, L., Lee, I., Wang, T., Lee, Y., Lee, C., Kuo, L., Shin, D.Y., 2000. Characterization of Vibrio parahaemolyticus isolates obtained from foodborne illness outbreaks during 1992 through 1995 in Taiwan. Journal of Food Protection 63, 900–906.