Bactericidal Activity of Isothiocyanate Against Pathogens on Fresh Produce²

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Journal of Food Protection, Vol. 63, No. 1, 2000, Pages 25±30 Copyright ᮊ, International Association for Food Protection

Bactericidal Activity of Isothiocyanate against Pathogens on Fresh Produce²

CHIA-MIN LIN, JEONGMOK KIM, WEN-XIAN DU, AND CHENG-I WEI*

Food Science and Human Nutrition Department, P.O. Box 110370, University of Florida, Gainesville, Florida 32611-0370, USA

MS 99-87: Received 9 April 1999/Accepted 17 July 1999

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/1/25/1669963/0362-028x-63_1_25.pdf by guest on 28 September 2021 The bactericidal activity of allyl and methyl isothiocyanate (AITC and MITC) was tested with a rifampicin-resistant strain of Salmonella Montevideo and streptomycin-resistant strains of Escherichia coli O157:H7 and Listeria monocytogenes Scott A. Iceberg lettuce inoculated with high (107 to 108 CFU/g) and low (103 to 104 CFU/g) concentrations of bacterial pathogens was treated with AITC and MITC in sealed containers at 4ЊC for 4 days. AITC showed stronger bactericidal activity than MITC against E. coli O157:H7 and Salmonella Montevideo, whereas MITC showed stronger activity against L. monocytogenes than E. coli O157:H7 and Salmonella Montevideo. Up to 8-log reduction occurred with E. coli O157:H7 and Salmonella Montevideo on lettuce following treatment with vapor generated from 400 ␮l of AITC for 2 and 4 days, respectively. AITC was used to treat tomatoes inoculated with Salmonella Montevideo on stem scars and skin and apples inoculated with E. coli O157:H7 on stem scars. The bactericidal effect of AITC varied with bacteria species and exposure time. Salmonella Montevideo inoculated on tomato skin was more sensitive to AITC than that on stem scars. Treatment with vapor generated from 500 ␮l of AITC caused an 8-log reduction in bacteria on tomato skin but only a 5-log reduction on tomato stem scars. The bactericidal activity of AITC was weaker for E. coli O157:H7 on apple stem scars; only a 3-log reduction in bacteria occurred when 600 ␮l of AITC was used.

Antimicrobial agents and food preservatives are widely with two outbreaks of listeriosis (12, 19). Outbreaks of sal- used to inhibit foodborne bacteria. Isothiocyanates are a monellosis associated with fresh produce also have been group of compounds responsible for the pungent ¯avor of reported. Salmonella Miami and Salmonella Bareilly were all plants belonging to the family Cruciferae, such as cab- associated with outbreaks in precut watermelon (10). Sal- bage and horseradish. They are the key compounds respon- monella Montevideo and Salmonella Javiana were believed sible for the antimicrobial activity of Japanese green mus- to have caused the outbreaks associated with tomatoes in tard (wasabi) (6). Puri®ed allyl isothiocyanate (AITC) and 1990 and 1993, respectively (11). The outbreaks of Esch- methyl isothiocyanate (MITC) were shown to have strong erichia coli O157:H7 in the Western United States (4) and antimicrobial activity on culture media (7, 15, 21). Al- Connecticut (5) due to consumption of contaminated apple though the low water solubility of isothiocyanates has re- cider attracted the most attention related to the safety of stricted their application in food systems, people continue fresh produce. Lettuce contaminated with E. coli O157:H7 to apply the highly volatile and potent bactericidal isothio- was believed to be the source for an outbreak in Maine and cyanate vapors, such as AITC, for microbial controls in Montana in 1995 (24). various food products (7, 13, 18). Washing with chlorinated water is the usual procedure Fresh-cut, packaged, and ready-to-eat vegetables are used by the produce industry to remove bacterial contam- highly popular in today's markets. However, two foodborne inants from vegetables. However, studies have shown that illness surveys conducted by the Centers for Disease Con- washing with chlorinated water may not completely remove trol and Prevention during 1973 to 1987 and 1988 to 1991 or kill bacterial pathogens on vegetables (3). The stem scars revealed that fruits and vegetables account for 5 and 6.3%, and natural cracks on tomatoes were reported to provide respectively, of the total foodborne illness outbreaks (1, 2). protection for Salmonella Montevideo against the bacteri- Various bacterial pathogens were reported to survive and cidal effect of chlorinated water (26). L. monocytogenes grow on vegetables and fruits, and these have been asso- was shown to survive on lettuce leaves or tomato slices ciated with fresh produce±related outbreaks (17). Listeria that had been treated with 200 ppm of chlorinated water monocytogenes has been isolated from lettuce heads pur- (17). chased from retail outlets (23) and in prepackaged salads Isothiocyanates can be used as a processing aid to help (22, 25). In fact, vegetable products have been associated control potential pathogen contamination of the prepackaged vegetable salads applying their potent bactericidal * Author for correspondence. Present address: Nutrition and Food Science properties. Therefore, the objective of this study was to in- Department, 328 Spidle Hall, Auburn University, Auburn University, Alabama 36849-5605, USA. Tel: 352-392-1991 Ext. 251; Fax: 352-392- vestigate the bactericidal activity of AITC and MITC 9467. against three foodborne pathogensÐSalmonella Montevi- ² Florida Agricultural Experiment Station Journal Series no. R-06500. deo, L. monocytogenes, and E. coli O157:H7Ðadded to 26 LIN ET AL. J. Food Prot., Vol. 63, No. 1

iceberg lettuce, which is the most common vegetable item population of native bacteria on lettuce following treatment with present in prepackaged salads. Tomatoes and apples also isothiocyanate. Both surface and pour plate methods were used, were used to represent fruit systems for testing the bacte- and plate count agar plates were incubated at room temperature ricidal ability of AITC against Salmonella Montevideo and for 3 days. E. coli O157:H7, respectively. Bactericidal activity of AITC against Salmonella Monte- MATERIALS AND METHODS video on the stem scars and skin of tomatoes. Ripe tomatoes were obtained from the Gulf Coast Research and Education Center Bacterial cultures and test chemical. A rifampicin-resistant at Bradenton, University of Florida. Before the experiment, to- strain of Salmonella Montevideo was prepared from Centers for matoes were washed under running tap water and wiped with pa- Disease Control and Prevention isolate G4639 obtained from a per towels. Twenty-®ve microliters of bacterial suspension at 9 to patient in Illinois in July 1993 (26). This culture was maintained 9.4 ϫ 106 CFU/ml in TSB was placed on the stem scars to yield on trypticase soy agar (TSA) (Difco Laboratories, Detroit, Mich.) a ®nal population of about 2.34 ϫ 105 CFU per stem scar. The that contained 0.1 g/liter of rifampicin (Sigma Chemical Co., St. bacterial suspension on the stem scars was dried at room temper-

Louis, Mo.) (TSA-RIF). Streptomycin-resistant strains of E. coli ature with circulating air in a laminar ¯ow hood. Three tomatoes Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/1/25/1669963/0362-028x-63_1_25.pdf by guest on 28 September 2021 O157:H7 and L. monocytogenes Scott A were isolated by streak- then were placed in the storage container with an open plastic bag ing the colonies of regular strains onto TSA plates containing 0.2 loaded with various amounts of AITC (100 to 500 ␮l). The con- g/liter of streptomycin (Sigma) (TSA-STR). The stability of these tainer was covered with a piece of Saran Wrap (Dow Brands), streptomycin-resistant strains was checked by streaking a single sealed tightly with a lid, and stored at 13ЊC for 2 days. Three colony of the resistant strains onto another set of TSA-STR plates containers were used for each test dose of AITC. The experiment Њ and incubating at 37 C for 24 h. This same procedure was re- was repeated once. peated twice. Streptomycin-resistant strains of E. coli O157:H7 Salmonella Montevideo suspension at 107 or 1010 CFU/ml and L. monocytogenes were maintained on TSA-STR plates. in TSB also was inoculated at 10-␮l aliquot onto the eight marked For testing, Salmonella Montevideo and E. coli O157:H7 circles (0.8 to 0.9 cm in diameter) on each tomato. After the bac- Њ were inoculated in trypticase soy broth (TSB) for 16 h at 37 C terial suspension was dried in a laminar ¯ow hood, three tomatoes and with shaking at 100 rpm, whereas L. monocytogenes was in- were packed in a food container and stored at 13ЊC as previously Њ oculated in TSB for 16 h at 30 C and with shaking at 100 rpm. described. Tomatoes inoculated with 105 CFU per circle of Sal- AITC (98% purity) and MITC (97% purity) were purchased from monella Montevideo were treated with AITC at 100, 150, 200, Aldrich Chemical Co. (Milwaukee, Wis.). 250, 300, or 400 ␮l loaded in a plastic bag. For tomatoes inocu- 8 Inoculation of bacteria on lettuce and assay for bacteri- lated with Salmonella Montevideo at 10 CFU per circle, AITC ␮ cidal activity of isothiocyanates. Iceberg lettuce was purchased at 150, 200, 250, 300, 400, 450, or 500 l was used. Duplicate from a local supermarket. After four outer layers of leaves were containers were used for each treatment dose of AITC. removed, the lettuce was cut into pieces using an alcohol-sterilized The bacterial numbers on the stem scars and tomato skin knife. Lettuce pieces were dipped in the bacterial suspension in were determined on days 0, 1, and 2 of AITC treatment. Stem TSB at 105 or 108 CFU/ml in a stainless steel pan for 30 s. After scars from three tomatoes were removed, pooled, weighed, and the inoculated lettuce pieces were placed on several layers of pa- homogenized with nine volumes (wt/vol) of BPB. For determi- per towels to remove excessive suspension, they were placed un- nation of bacterial numbers on tomato skin, 12 marked areas from der circulating air inside a laminar ¯ow hood for complete drying. two tomatoes were removed using a knife. They were pooled, The inoculated lettuce pieces (100 g) were placed in a 1- weighed, and homogenized with nine volumes of BPB (wt/vol). gallon freezer bag (Ziploc, Dow Brands, Indianapolis, Ind.) with The homogenate was diluted decimally, and diluent samples were a small bag (Glad-Lock Zipper, First Brands Co., Danbury, Conn.) surface plated onto four TSA-RIF plates. Pour plate method also loaded with various amounts (75 to 400 ␮l) of AITC or MITC. was used with some homogenates. The experiments were repeated The small bags were kept open for the entire treatment period by twice. stapling one end of the open side. For control groups, small empty bags were used. After the freezer bag was carefully sealed, it was Bactericidal activity of AITC against E. coli O157:H7 on placed in a 4-liter food storage container (Rubbermaid, Twinburg, the stem scars of apples. Fresh and unwaxed apples (Golden Ohio), which was then sealed tightly with a lid. Four containers Delicious) were obtained from North Carolina through the assis- stored at 4ЊC were used for each treatment dose. Bacterial num- tance of Wayne Mitchem, Department of Horticultural Sciences, bers were enumerated with the inoculated lettuce pieces and with North Carolina State University. Before the experiment, apples the treated samples on days 1, 2, and 4 of storage. On each test were washed under running tap water and wiped with paper tow- day, a 20-g portion of lettuce was picked randomly from the freez- els. Without removal of the pedicel, each apple stem scar was er bag and homogenized with nine volumes (wt/vol) of Butter- inoculated with 25 ␮l of bacterial suspension at 5 to 7 ϫ 106 ®eld's phosphate buffer (BPB, pH 7.2) in a sterile blender jar for CFU/ml in TSB. After the bacterial suspension was dried, three 30 s at low speed, followed by another 30 s at high speed. The apples were packed in a food container with a plastic bag loaded homogenate was diluted decimally, and samples were surface plat- with various amounts of AITC (150, 200, 250, 300, 350, 400, ed onto four TSA-RIF or TSA-STR plates, depending on the test 450, 500, 550, and 600 ␮l) as previously described. The container bacterial strain. Pour plate method also was done with sample was covered with a piece of Saran Wrap, sealed tightly with a lid, homogenates. Bacteria colonies were enumerated after incubation and stored at 4ЊC for 2 days. Triplicate containers were used for at 37ЊC for 48 h. The bactericidal assays were repeated twice for each AITC dose. The experiment was repeated twice. Bacterial all three species. numbers were determined on days 0, 1, and 2 of AITC treatment. The bactericidal effect of these isothiocyanates on the resi- Stem scars from three apples were removed, pooled, weighed, and dent bacteria of lettuce was also studied. Iceberg lettuce was pro- homogenized with nine volumes (wt/vol) of BPB for bacterial cessed as previously described except no arti®cial inoculation was enumeration using quadruplicate TSA-STR plates for each diluent performed. Standard plate count agar was used to estimate the sample. J. Food Prot., Vol. 63, No. 1 BACTERICIDAL ACTIVITY OF ISOTHIOCYANATE 27 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/1/25/1669963/0362-028x-63_1_25.pdf by guest on 28 September 2021

FIGURE 2. The bactericidal activity of 400 ␮l of AITC and MITC to native bacteria on lettuce. The bars represent the standard deviation of the mean of eight replications.

The treatment with 400 ␮l of AITC and MITC of the lettuce inoculated with high levels (108 CFU/g) of the three pathogens also showed that E. coli O157:H7 was the most sensitive and L. monocytogenes was more resistant (data not shown). E. coli O157:H7 was not detected in treated lettuce after 2 days of exposure to AITC, whereas it took 4 days to achieve the same bactericidal effect against Sal- monella Montevideo. Although MITC at 400 ␮l was a more FIGURE 1. The bactericidal activity of 400 ␮l of AITC and MITC potent bactericidal agent than AITC against L. monocyto- to low levels (104 CFU/g) of Salmonella Montevideo, E. coli genes, it failed to inactivate 108 CFU/g of any of the three O157:H7, and L. monocytogenes inoculated onto lettuce. The species even after 4 days. bars represent the standard deviation of the mean of eight repli- Delaquis and Sholberg (7) showed that E. coli O157: cations. H7 recovered better than L. monocytogenes Scott A and Salmonella Typhimurium on TSA plates containing 5 g/ liter of yeast extract following treatment with AITC vapor. RESULTS AND DISCUSSION However, E. coli O157:H7 was the most sensitive bacteri- Bactericidal activity of AITC and MITC to L. mon- um to AITC vapor in this experiment with lettuce. There- ocytogenes, Salmonella Montevideo, and E. coli O157: fore, the differences in bacterial growth and survival in H7 on lettuce. The bactericidal activity of isothiocyanates TSA and lettuce contributed to their variation in sensitivity was affected by the concentration and exposure times. Va- to AITC vapor. Kyung and Fleming (15) also showed that pors of 400 ␮l of AITC and MITC showed potent bacte- AITC added to TSB had a higher minimum inhibitory con- ricidal effect against the three pathogens inoculated onto centration for L. monocytogenes than for Salmonella Ty- lettuce. Lettuce inoculated with low levels (104 CFU/g) of phimurium and E. coli. Salmonella Montevideo and E. coli O157:H7 showed no The bactericidal effect of these two isothiocyanates to detectable bacterial counts (Ͻ1 CFU/g based on pour plate the natural ¯ora on the lettuce also was examined. Initially, method) after exposure to 400 ␮l of AITC or MITC for 48 MITC had a greater bactericidal activity than AITC against h (Fig. 1). At this low inoculation level, E. coli O157:H7 the natural ¯ora (Fig. 2); however, these two compounds at was more sensitive to AITC than Salmonella Montevideo; 400 ␮l showed only a slight difference in their bactericidal no E. coli O157:H7 was detected on the treated lettuce after activity after 2 days. Extended exposure at 4 days did not 24 h, whereas some (Ͼ10 CFU/g based on pour plate meth- increase the bactericidal activity. The natural ¯ora on the od) Salmonella Montevideo remained. MITC was a more untreated lettuce continued to increase in numbers even at potent bactericidal agent than AITC against L. monocyto- 4ЊC. The rapid decrease in bacterial numbers in day 1 could genes. Lettuce inoculated with L. monocytogenes at 104 be due to the inactivation of the sensitive species of the CFU/g showed no bacterial counts after treatment with 400 natural ¯ora. Only the resistant species remained, which ␮l of MITC for 2 days. Exposure to 400 ␮l of AITC failed contributes to the leveled-off bactericidal effect as shown to achieve the same bactericidal effect. in Figure 2. 28 LIN ET AL. J. Food Prot., Vol. 63, No. 1 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/1/25/1669963/0362-028x-63_1_25.pdf by guest on 28 September 2021

FIGURE 3. Changes of the inactivation activity of AITC against the inoculated Salmonella Montevideo (A) or E. coli O157:H7 (B) on lettuce for 2 days. The bars represent the standard deviation of the mean of four replications.

Dose-related bactericidal activity of AITC against AITC did not affect the texture and produced no residual Salmonella Montevideo and E. coli O157:H7 inoculated pungent ¯avor of isothiocyanate following 1-h placement on lettuce. AITC showed a dose-related bactericidal activ- under ambient air. However, when higher doses of AITC ity against Salmonella Montevideo inoculated on lettuce at were used, the exposed lettuce had a strong pungent ¯avor both low (104 CFU/g) and high (108 CFU/g) levels (Fig. and gradually lost crispness. Lettuce exposed to AITC had 3). No Salmonella Montevideo was detected in lettuce that a stronger pungent ¯avor than that exposed to MITC, pos- had been inoculated at 104 CFU/g and then exposed to sibly due to the greater volatility of AITC than MITC. AITC at 250 to 400 ␮l for 48 h. Although 1-day exposure to 400 ␮l of AITC killed only 2.6-log units of Salmonella Bactericidal activity of AITC against Salmonella Montevideo inoculated on lettuce at 108 CFU/g, the 48-h Montevideo on the stem scars and skin of tomatoes and exposure caused more than 8-log reductions. Thus, for the E. coli O157:H7 on apple stem scars. Apple stem scars 48-h treatment, the minimum amount of AITC needed to provided a better protective environment for E. coli O157: cause 1-log (90%) reduction of Salmonella Montevideo on H7 than tomato stem scars for Salmonella Montevideo the lettuce at 108 CFU/g was about 200 ␮l. At the level of against the bactericidal activity of AITC. AITC at 500 ␮l 104 CFU/g, 150 ␮l of AITC was enough to reduce the caused a 5-log reduction of Salmonella Montevideo on to- bacteria by 2 logs (Ͼ99% reduction). mato stem scars. However, only a 3-log reduction of E. coli None of the inoculated E. coli O157:H7 at 7.4 ϫ 103 O157:H7 occurred in apple stem scars when 600 ␮lof CFU/g or 107 CFU/g was detected after the lettuce was AITC was used (Fig. 4). Differences in the physiological exposed for 2 days to AITC at 300 or 350 ␮l, respectively nature between apples and tomatoes and between bacterial (Fig. 3). Similarly, exposure for 2 days to AITC at 75 and species could explain why E. coli O157:H7 survived better 100 ␮l was adequate to reduce 90% of the inoculated bac- on apples than Salmonella Montevideo on tomatoes. Apple teria at 7.4 ϫ 103 CFU/g and 107 CFU/g, respectively. stem scars are much deeper than those of tomatoes. It took Treatment of lettuce with high doses of AITC caused a longer time for the inoculated bacterial suspension to dry changes in texture. Exposure of lettuce to 100 or 150 ␮lof on apple stem scars than on tomato stem scars. Multilayers J. Food Prot., Vol. 63, No. 1 BACTERICIDAL ACTIVITY OF ISOTHIOCYANATE 29 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/1/25/1669963/0362-028x-63_1_25.pdf by guest on 28 September 2021

FIGURE 4. Time- and dose-related bactericidal activity of AITC against Salmonella Montevideo inoculated onto tomato stem scars FIGURE 5. The dose-related bactericidal activity of AITC for 2 at 2.34 ϫ 105 CFU per scar and E. coli O157:H7 inoculated onto days against Salmonella Montevideo inoculated onto tomato apple stem scars at 1.25 to 1.75 ϫ 105 CFU per scar. The bars skins. The bars represent the standard deviation of the mean of represent the standard deviation of the mean of six replications. six replications. of bacterial bio®lm could have formed on apple stem scars h for treated fruits when less than 300 ␮l of AITC was after the bacterial suspension dried. The negative numbers used. When 500 ␮l of AITC was applied, the pungent odor for the control tomatoes resulted because of increases in remained for about 18 to 24 h. Salmonella Montevideo population. Based on the results of this study, AITC vapor at 76.0 Salmonella Montevideo inoculated onto tomato skin to 101.3 mg/liter (300 to 400 ␮l ϫ 1.013 g/ml [density of was more sensitive to the bactericidal activity of AITC than AITC]/4 liters [volume of the food container]) could elim- on the stem scars. Whether the tomato skins were inocu- inate Salmonella Montevideo or E. coli O157:H7 inoculat- lated with a high (4.1 ϫ 108 CFU per circle) or low (5.9 ed at 104 to 105 CFU/g on lettuce. Therefore, AITC vapor ϫ 105 CFU per circle) concentration of Salmonella Mon- at about 100 ppm could kill about 5 logs of a bacterial tevideo, a 5-log inactivation of the bacteria occurred when population in 24 h. Since the population of pathogenic bac- 400 ␮l of AITC was used (Fig. 5). None of the inoculated teria on contaminated produce is much less than 105 CFU/ Salmonella Montevideo at 4.1 ϫ 108 CFU/g was detected g in most cases and bacterial resistance to AITC vapor was after 48 h of treatment with 500 ␮l of AITC. Salmonella lower when the population was small, AITC at a lower Montevideo did not survive as well on tomato skin as on application rate can be used to kill those pathogens (Sal- stem scars. A nearly 1-log reduction in bacterial population monella Montevideo and E. coli O157:H7) that could be occurred with the untreated tomatoes inoculated either at present on ready-to-eat produce such as lettuce and toma- high or low concentrations of Salmonella Montevideo. To- toes or on apples to be used for cider production. mato stem scars are the main access for air exchange; they AITC is a ``generally recognized as safe'' substance do not have the cuticle layer of natural wax that covers the and is exempted from the requirement for a residue toler- tomato skin. This structural difference thus affected bacte- ance in or on all raw agricultural commodities by the En- rial attachment to these two areas and their survival (26). vironmental Protection Agency (8). The strong odor of The texture of tomatoes and apples did not change fol- AITC could disappear if a permeable packaging material is lowing exposure to low levels of AITC. However, they be- used. However, this must be evaluated to better control gas came softer following 2 days of treatment with high levels exchanges. Furthermore, the acceptability of using AITC of AITC (Ͼ300 ␮l). The residual pungent ¯avor lasted 12 vapor for commercial control of potential bacterial patho- 30 LIN ET AL. J. Food Prot., Vol. 63, No. 1 gens in ready-to-eat vegetable salads remains to be deter- 12. Ho, J. L., K. N. Shands, G. Friedland, P. Eckind, and D. W. Fraser. mined. In Japan, puri®ed AITC is permitted for use as a 1986. An outbreak of type 4b Listeria monocytogenes infection in- volving patient from eight Boston hospitals. Arch. Intern. Med. 146: food preservative (20). Capsulelike devices to allow slow 520±524. release of AITC vapor have been developed for industrial 13. Isshiki, K., K. Tokuoka, R. Mori, and S. Chiba. 1992. Preliminary use as microbicides, deodorizers, preservatives, and insect examination of allyl isothiocyanate vapor for food preservation. repellents (9, 14, 16, 27). Ogawa and Isshiki (18) showed Biosci. Biotech. Biochem. 9:1476±1477. that citrus and vanilla ¯avor could mask the odor of AITC 14. Kataoka, H., and M. Murata. 14 January 1997. Manufacture of sus- tained-release microcapsules containing volatile substances and dis- without reducing its bactericidal activity. persion of the microcapsules. Jpn. Kokai Tokkyo Koho, patent 97 12447 (Dai Ichi Kogyo Seiyaku Co. Ltd.), 10 p. ACKNOWLEDGMENTS 15. Kyung, K. H., and H. P. Fleming. 1997. 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