727

Journal of Food Protection, Vol. 63, No. 6, 2000, Pages 727±734 Copyright ᮊ, International Association for Food Protection

Antibacterial Mechanism of Allyl Isothiocyanate²

CHIA-MIN LIN,1³ JAMES F. PRESTON III,2 AND CHENG-I WEI3*

1Food Science and Human Nutrition Department, P. O. Box 110370, University of Florida, Gainesville, Florida 32611-0370; 2Microbiology and Cell Science Department, P. O. Box 110700, University of Florida, Gainesville, Florida 32611-0700; and 3Nutrition and Food Science Department, 328 Spidle Hall, Auburn University, Auburn, Alabama 36849-5605, USA

MS 99-293: Received 4 October 1999/Accepted 28 January 2000

ABSTRACT Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/6/727/1673840/0362-028x-63_6_727.pdf by guest on 25 September 2021

Allyl isothiocyanate (AITC), a natural compound in plants belonging to the family Cruciferae, has been shown to have strong antimicrobial activity in liquid media as well as in its vapor form. To understand its antimicrobial mechanism, AITC was tested for bactericidal activities to Salmonella Montevideo, Escherichia coli O157:H7, and Listeria monocytogenes Scott A at different stages of growth and was compared with streptomycin, penicillin G, and polymyxin B, each of known antibac- terial mechanisms. Bactericidal activities were determined by measuring bacterial viability and leakage of metabolites. To determine its effects on membrane permeability, ␤-galactosidase activity was examined after exposure of E. coli K-12 strain 3.300 to the three antibiotics and to AITC. The two gram-negative bacteria, Salmonella Montevideo and E. coli O157:H7, were more sensitive to AITC and to polymyxin B than the gram-positive L. monocytogenes. AITC and polymyxin B were effective bactericidal agents to bacteria at all growth stages, whereas penicillin G and streptomycin did not exhibit bactericidal ␤ activity to stationary cells. High A260 and A280 values of cellular ®ltrate and -galactosidase activity were obtained after treatments of AITC and polymyxin B. These data indicated that AITC was most similar to polymyxin B with respect to its antibacterial effect on cell membranes and on leakage of cellular metabolites. Gaseous AITC caused metabolite leakages, measurable increases in ␤-galactosidase activity, and reduction of viable bacteria. The effectiveness of AITC in inhibiting bacteria at all growth stages and its strong activity in vapor phase support its application in food preservation.

Allyl isothiocyanate (AITC), a natural compound pre- tory concentration of 150 ␮g/ml on oxygen intake, which sent in all plants belonging to the family Cruciferae, has was about 100 and 50,000 times greater than the 50% in- been demonstrated to have a strong antimicrobial activity hibitory concentrations of KCN and antimycin A, respec- in both liquid and vapor forms (5, 8, 13, 15, 19, 22, 23). tively. These authors also determined the MIC of AITC on Interest in using AITC as an antibacterial agent in various the growth of tested yeasts. The MIC of AITC was about food systems has been increasing because of its natural or- 100 times less than the effective dose that inhibited oxygen igin and its generally recognized as safe status. intake. These data suggested that AITC was not a strong The antimicrobial mechanism(s) of AITC is not well respiratory inhibitor, and the inhibition of oxygen intake understood. Two studies demonstrated that AITC could al- was not the major contributing cause to the antimicrobial ter protein structures at a concentration that inhibited mi- function of AITC. crobial growth (9, 10). Kawakishi and Kaneko (9) showed Previous studies have shown that gram-negative bac- that oxidized glutathione was cleaved by AITC at the di- teria were more sensitive than gram-positive bacteria to in- sul®de bond. Proteins, such as insulin, bovine serum al- hibition by AITC (8, 21). Earlier data obtained in our lab- bumin, ovalbumin, and lysozyme, were shown to be at- oratory also showed that Listeria monocytogenes, a gram- tacked by AITC at the disul®de bonds, and thiourealike positive bacterium, was more resistant to AITC than Esch- derivatives were formed at lysine and arginine residues in erichia coli O157:H7 and Salmonella Montevideo, which these proteins (10). are gram-negative bacteria (15). The effects of AITC on Isothiocyanates were reported to affect the metabolic these two groups of bacteria, especially at different growth functions of microorganisms (11). The inhibition of oxygen stages, have not been reported. The purpose of this study uptake in three yeasts by AITC, methyl isothiocyanate, phe- was to determine the physiological responses of these bac- nyl isothiocyanate, and ␤-phenylethyl isothiocyanate was teria to AITC in order to gain insight into the molecular analyzed quantitatively. Among them, AITC was the most targets that could account for the antibacterial activity. This effective inhibitor. Compared with known respiratory in- was achieved by comparing the responses of these two hibitors, KCN and antimycin A, AITC had a 50% inhibi- groups of bacteria to AITC and three different antibiotics, polymyxin B, streptomycin sulfate, and penicillin G, which * Author for correspondence. Tel: (334) 844-4261; Fax: (334) 844-3268; are known to exert their antibacterial activities through spe- E-mail: [email protected]. ci®c effects on cell membranes, ribosomes, and cell wall ² Florida Agricultural Experiment Station Journal Series R-07177. ³ Present address: Center for Food Safety and Quality Enhancement, De- structure, respectively. In addition, these same agents were partment of Food Science and Technology, University of Georgia, Grif- compared to determine their effects on bacterial cell mem- ®n, GA 30223-1797, USA. brane permeability. 728 LIN ET AL. J. Food Prot., Vol. 63, No. 6

TABLE 1. Antibacterial mechanisms of polymyxin B, penicillin G, and streptomycin (13, 14) Antibiotic Attacking sites Mechanism Affected growth stages

Polymyxin B Cell membrane Creates pores on cell membrane and induc- All growth stages es leakage of cellular substances Penicillin G Cell wall Interferes with cell wall synthesis and trig- Exponential gers cell lysis Streptomycin Ribosome Causes misreading of mRNA sequences Exponential and produces malfunctional polypeptides

MATERIALS AND METHODS used to determine the bactericidal activities of AITC with strep- tomycin sulfate, polymyxin B, and penicillin G, purchased from Determination of bacterial growth. Antibiotic-sensitive

Sigma Chemical Co. (St. Louis, Mo.), for comparison of the sim- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/6/727/1673840/0362-028x-63_6_727.pdf by guest on 25 September 2021 strains of L. monocytogenes Scott A, Salmonella Montevideo ilarity of antibacterial mechanisms. The antibacterial mechanisms G4639, and E. coli O157:H7 ATCC 43895 were maintained on of these three antibiotics are listed in Table 1. AITC was added tryptic soy agar (TSA) plates. Overnight cultures of these three to E. coli O157:H7 and Salmonella Montevideo suspensions at species served as inocula for tryptic soy broth (TSB), which was 1,000 ␮g/ml and to the L. monocytogenes suspension at 2,500 ␮g/ Њ incubated at 37 C with shaking at 200 rpm. Bacterial growth was ml. Polymyxin B, streptomycin sulfate, or penicillin G in sterile monitored by measuring the optical density at 540 nm (OD540)in distilled water was added to L. monocytogenes, E. coli O157:H7, a DU-640 spectrophotometer (Beckman Instruments Inc., Fuller- and Salmonella Montevideo at the exponential growth stage at 10, ton, Calif.) every hour for the ®rst 12 h and then every4hfor 200, or 200 ␮g/ml, respectively, and at 250, 500, or 500 ␮g/ml later times. Numerical equations of the correlation of OD540 val- to cultures at the stationary growth phase. The test concentrations ues and bacterial populations, which were obtained by plating the of AITC and the three antibiotics were chosen based on the results serially diluted bacterial suspensions in Butter®eld's phosphate of our preliminary studies and published reports (13, 15). Viable buffer (BPB, pH 7.2 (7)) on TSA plates and then by incubating counts and OD values were determined at 30-min intervals for Њ 540 at 37 C for 48 h, were developed for each bacterial species and E. coli O157:H7 and Salmonella Montevideo at the exponential were used to prepare bacterial suspensions of desired concentra- growth stage and at 1-h intervals for these two cultures at the tions. stationary stage and for L. monocytogenes at both growth stages. Bactericidal activity of AITC at different growth stages. The experiments were repeated three times. The antibacterial activity of AITC (density ϭ 1.013 g/ml; Aldrich Leakage of cellular metabolites. Bacterial cultures (10 ml) Chemical Co, Inc., Milwaukee, Wis.) was tested with the three at the exponential growth stage were transferred into sterile cen- species at lag, early and late exponential, and stationary growth trifuge tubes and were centrifuged at 4,800 ϫ g for 15 min. After phases. A bacterial suspension in TSB at each growth stage was the supernatant was discarded, the pellet was resuspended in 10 obtained based on results from a preliminary study. In each ex- ml of BPB or Davis minimum broth (MB, pH 7.0; Difco Labo- periment, an overnight culture served as the inoculum. When ratories, Detroit, Mich. (1)) containing 1 g/liter glucose. The sus- OD540 readings indicated that they had reached the desired growth stage, bacterial suspensions were dispensed into eight sterile pension was centrifuged and resuspended twice in MB or BPB. ¯asks. AITC was added into each of two ¯asks at 500, 1,000, or Bacterial suspensions of the same species in all centrifuge tubes 2,500 ␮g/ml, respectively. Two ¯asks containing no AITC served were pooled, and the OD540 values and viable counts were deter- as controls. The ¯asks were incubated at 37ЊC with shaking at mined. Then, 10-ml aliquots were dispensed into each of six ster- ␮ 200 rpm. The experiment was repeated once. ile ¯asks (50 ml). AITC was added at 500, 1,000, or 2,500 g/ Each bacterial suspension was checked for viable counts and ml, whereas streptomycin and polymyxin B were added at 400 ␮ OD readings before and every hour after the addition of AITC. and 20 g/ml, respectively. Flasks containing only bacterial cul- 540 Њ Results from preliminary studies indicated that bacteria in the ear- tures served as controls. After the ¯asks were incubated at 37 C ly exponential growth phase were more sensitive to AITC and with shaking at 200 rpm for 1 h, 1-ml sample suspensions were that E. coli O157:H7 and Salmonella Montevideo were more sen- removed for determination of OD540 values and viable counts. The ␮ sitive than L. monocytogenes. Therefore, sampling intervals of 30 remaining suspensions were ®ltered through 0.45- m ®lters min were used for E. coli O157:H7 and Salmonella Montevideo (Nalge Co., Rochester, N.Y.), and the ®ltrates were used for de- at the early exponential growth stage, and 1-h intervals were used termination of A260 and A280 values. To eliminate the effect of test for L. monocytogenes. During the test, 1 ml of the bacterial sus- chemicals on A260 and A280 values, ¯asks containing BPB or MB pension was removed, placed in a 1.5-ml microcentrifuge tube, and AITC, polymyxin B, or streptomycin at test concentrations and centrifuged at 16,000 ϫ g for 3 min in a microcentrifuge served as additional controls. The extracellular solutions of these (model 5415; Eppendorf, Westbury, N.Y.). After the supernatant controls were collected similarly to the test groups. Differences in was discarded, the pellet was resuspended in 1 ml of BPB and A260 and A280 values between controls and test groups were used then was centrifuged to remove possible carryover of AITC. to estimate the release of metabolites. The experiments were re- Washed cells were resuspended in 1 ml of BPB. Viable counts peated once. were determined by plating each bacterial diluent in BPB on trip- ␤ licate TSA plates. Bacterial growth was also determined by mea- Assay of -galactosidase activity. E. coli K-12 strain 3.300, ␤ suring the OD values of bacterial suspensions in BPB. which contains a constitutively expressed -galactosidase gene, 540 was grown in TSB and was treated with AITC and the three an- Comparison of the bactericidal activity of AITC with tibiotics, as previously described. Since ␤-galactosidase exists in streptomycin sulfate, polymyxin B, and penicillin G. Bacterial the cytoplasm, the increase in enzyme activity of the culture me- cultures at both exponential and stationary growth stages were dium is a measure of the extent to which cells are rendered per- J. Food Prot., Vol. 63, No. 6 BACTERICIDAL ACTIVITY OF ALLYL ISOTHIOCYANATE 729 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/6/727/1673840/0362-028x-63_6_727.pdf by guest on 25 September 2021

FIGURE 1. The effect of dissolved AITC on the growth of E. coli FIGURE 2. The effect of dissolved AITC on the growth of Sal- O157:H7 at different growth stages. The sampling intervals were monella Montevideo at different growth stages. The sampling in- 30 min and 1 h, respectively, for cultures at early exponential and tervals were 30 min and 1 h, respectively, for cultures at early other phases. (A) lag, (B) early exponential, (C) late exponential, exponential and other phases. (A) lag, (B) early exponential, (C) and (D) stationary. late exponential, and (D) stationary. meable or lysed due to the effect of the test chemicals on the cell removed from the chambers and were placed in a laminar ¯ow membrane. Only bacteria at the exponential growth phase that hood without lids for 5 min. Bacteria were collected by gently were highly sensitive to test chemicals were used. ␤-Galactosidase rinsing the agar surfaces with BPB. The pooled suspensions from activity was assayed as described by Sambrook et al. (21). Un- two plates then were diluted with BPB to give an OD540 value of treated bacteria lysed by chloroform and 0.1% sodium dodecyl 0.7. After the bacterial suspension was dispensed into two sterile ␤ sulfate (SDS) were used as positive controls. The activity of ␤- tubes, viable bacterial counts, A260 and A280 values, and -galac- ␤ tosidase activity were determined, as previously described. The galactosidase was measured as A420 using o-nitrophenyl- -D-gal- actopyranoside as substrate. The incubation time was set at 15 experiments were repeated three times. min based on preliminary studies. The A550 and A600 values also Statistical analyses. Means and standard deviations were de- were obtained, but their readings were less than 0.01, con®rming termined using commercial spreadsheet software (Excel 97, Mi- that the A420 values were re¯ective of the formation of o-nitro- crosoft Corp., Seattle, Wash.). Microbiological counts were trans- phenyl. A group of ¯asks containing TSB and AITC, polymyxin formed into log data before statistical analysis. Signi®cant differ- B, penicillin G, or streptomycin at the same test concentrations ence (P Ͻ 0.05) was determined using the least signi®cant dif- served as additional controls. The experiments with AITC treat- ference test in the Statisca program (StatSoft Inc., Tulsa, Okla.). ment were repeated four times, whereas those with antibiotic treat- ments were repeated three times. RESULTS Exposure of E. coli K-12 to gaseous AITC. For each test, Bactericidal activity of AITC at different growth 100 ␮l of overnight culture was transferred into 20 ml of TSB stages. AITC showed dose- and time-related inhibitory/ and was incubated at 37ЊC for 5 h with shaking at 150 rpm. When bactericidal effects on E. coli O157:H7, Salmonella Mon- the OD540 reading reached 0.8, the bacterial culture was serially tevideo, and L. monocytogenes at four different growth diluted, and 0.1-ml aliquots of the diluents were spread on TSA stages (Figs. 1 through 3). E. coli O157:H7 was the most plates to determine bacterial counts and to provide plates that sensitive species and L. monocytogenes was the most resis- would contain approximately 105 CFU/plate for treatment with tant species to AITC. E. coli O157:H7 and Salmonella AITC vapor. After incubation at 37ЊC for 14 h, four open TSA glass plates were placed in a chromatography developing chamber Montevideo at the early and late exponential growth stages (Fisher Scienti®c Co., Pittsburgh, Pa.) along with four other open were more sensitive to AITC than at the lag and stationary glass dishes loaded with AITC. Four blank glass dishes were phases. placed in a control chamber. Glass petri dishes and chambers were AITC at 500 ␮g/ml did not dramatically affect the pop- used to avoid absorption of AITC. The chamber was sealed with ulations of the three bacterial species at any growth phase. vacuum grease, a glass lid, and two layers of Saran Wrap (Dow E. coli O157:H7 and Salmonella Montevideo at early and Brands, Indianapolis, Ind.). AITC loaded on the glass dishes evap- late exponential growth stages became undetectable follow- orated completely during treatment. The concentration of the ing treatment with AITC at 2,500 ␮g/ml for 30 min. How- AITC vapor (micrograms per milliliter of air) was calculated by ever, 1- and 2-h exposure times were needed for AITC to ϭ dividing the amount of AITC (microliters; density 1.013 g/ml) achieve the same inhibitory effect on E. coli O157:H7 and added to the glass dishes by the volume of the chamber (5.54 Salmonella Montevideo at lag phase, respectively. At 1,000 liters). The dose-related effect was studied by exposing the bac- ␮ terial cultures to AITC at 25, 50, and 100 ␮g/ml for 24 h at 20ЊC. g/ml, a 2-h exposure time was needed for AITC to reduce Furthermore, bacteria on TSA plates were exposed to AITC at E. coli O157:H7 at lag and exponential growth phases to 100 ␮g/ml for 4, 6, 8, 10, 12, and 24 h at 20ЊC to determine the nondetectable levels. It would take AITC at this level 1.5 time-related effect. Only one chamber was used for each treatment and 3 h to reduce Salmonella Montevideo at early expo- dose or time period. After exposure, the treated TSA plates were nential and other growth phases, respectively, to nonde- 730 LIN ET AL. J. Food Prot., Vol. 63, No. 6 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/6/727/1673840/0362-028x-63_6_727.pdf by guest on 25 September 2021

FIGURE 3. The effect of dissolved AITC on the growth of L. monocytogenes at different growth stages. The sampling interval was 1 h. (A) lag, (B) early exponential, (C) late exponential, and (D) stationary. tectable levels. Exposure to AITC at 2,500 ␮g/ml did not completely inhibit the growth of L. monocytogenes at any growth phase. The treated bacteria, particularly those at the stationary FIGURE 5. The effect of streptomycin, penicillin G, polymyxin B, phase, did not dramatically decrease their OD540 values and AITC on the growth of Salmonella Montevideo at (A) expo- (data not shown). Since some of the bacterial suspensions nential and (B) stationary growth phases. Streptomycin, penicillin with high turbidity showed no presence of viable colonies G, polymyxin B, and AITC were tested at 200, 200, 10, and 1,000 on TSA plates, the nonviable bacteria did not undergo lysis. ␮g/ml, respectively, for the exponential-phase bacteria and at 500, 500, 250, and 1,000 ␮g/ml, respectively, for the stationary- phase bacteria.

Microscopic examination of these treated bacterial suspen- sions con®rmed the observations. Comparison of the bactericidal activity of AITC with streptomycin sulfate, polymyxin B, and penicillin G. Treatment of the bacterial cultures at the exponential growth phase with the three antibiotics and AITC caused decreases in OD540 readings. Polymyxin B, penicillin G, and AITC were more effective than streptomycin in reduc- ing the OD540 readings of the three bacterial species. How- ever, these test compounds failed to dramatically affect the OD540 readings of the cultures at the stationary phase (data not shown). The two gram-negative bacteria, E. coli O157:H7 and Salmonella Montevideo, at both exponential and stationary growth phases, were more susceptible to polymyxin B and AITC than the gram-positive bacterium, L. monocytogenes, at the same growth phases (Figs. 4 through 6). E. coli O157:H7 and Salmonella Montevideo at the exponential growth phase were completely killed following treatment for 2 h with 10 ␮g/ml polymyxin B or 1,000 ␮g/ml AITC. However, these two bacteria at the stationary phase were FIGURE 4. The effect of streptomycin, penicillin G, polymyxin B, less sensitive to polymyxin B and AITC; the increases of and AITC on the growth of E. coli O157:H7 at (A) exponential ␮ and (B) stationary growth phases. Streptomycin, penicillin G, polymyxin B concentration to 250 g/ml and the treatment polymyxin B, and AITC were tested at 200, 200, 10, and 1,000 time to 4 h failed to kill these two bacterial cultures. Ex- ␮g/ml, respectively, for the exponential-phase bacteria and at posure to AITC at 1,000 ␮g/ml for 4 h did not kill E. coli 500, 500, 250, and 1,000 ␮g/ml, respectively, for the stationary- O157:H7 or Salmonella Montevideo at the stationary phase bacteria. growth stage (Figs. 4 and 5). Streptomycin and penicillin J. Food Prot., Vol. 63, No. 6 BACTERICIDAL ACTIVITY OF ALLYL ISOTHIOCYANATE 731 A A A C A A A C B A 0.54 0.46 1.34 0.78 0.07 0.26 0.52 0.51 0.13 0.88 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Population 8.03 7.95 7.33 2.10 8.71 8.49 7.33 3.11 5.31 7.49 A A A A A A A A A B L. monocytogenes 0.08 0.02 0.07 0.05 0.01 0.12 0.08 0.09 0.12 0.16 260 A Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ in BPB and MB following treatments with Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/6/727/1673840/0362-028x-63_6_727.pdf by guest on 25 September 2021 0.32 0.26 0.22 0.33 0.25 0.26 0.19 0.25 0.26 0.41 A A B BC A A C D B C 4 for MB). 0.35 0.30 1.56 1.80 0.06 0.10 0.47 0.00 0.32 0.04 ϭ L. monocytogenes Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ n Population 8.88 8.40 3.77 1.27 8.80 8.02 2.71 0.00 5.51 1.74 Montevideo 2 for BPB,

FIGURE 6. The effect of streptomycin, penicillin G, polymyxin B, ϭ A A A A A A AB AB A B Montevideo, and and AITC on the growth of L. monocytogenes at (A) exponential n Salmonella

and (B) stationary growth phases. Streptomycin, penicillin G, 0.03 0.10 0.08 0.03 0.11 0.24 0.27 0.28 0.12 0.16 260 0.05; A

polymyxin B, and AITC were tested at 200, 200, 10, and 2,500 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ ␮g/ml, respectively, for the exponential-phase bacteria and at Ͻ P Salmonella 500, 500, 250, and 2,500 ␮g/ml, respectively, for the stationary- 0.39 0.32 0.31 0.39 0.26 0.30 0.51 0.61 0.30 0.80 phase bacteria. O157:H7,

G were only effective against the three bacterial species at A A B D A A BC D B C 0.01 0.03 0.00 0.00 0.09 0.17 1.71 0.00 0.11 0.11

the exponential growth phase. They did not interfere with E. coli

the growth of these species at the stationary phase, even at Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Population 500 ␮g/ml (Figs. 4 through 6). )of 260 8.42 8.33 6.45 0.00 8.82 8.28 3.67 0.00 5.21 1.86 Leakage of cellular metabolites. The effects of AITC A and two antibiotics on cellular leakage, in relation to chang- O157:H7

es in population of E. coli O157:H7, Salmonella Monte- a A A A A A A AB B A AB video, and L. monocytogenes in BPB and MB, are shown E. coli 0.05 0.01 0.06 0.03 0.01 0.31 0.29 0.18 0.09 0.26

in Table 2. The extent of cellular leakage of metabolites 260 A Ϯ Ϯ was monitored from the A260 and A280 readings of cell-free Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ C suspensions. Since the A260 readings of treated samples Њ 0.29 0.17 0.15 0.17 0.26 0.40 0.74 0.77 0.28 0.75 were proportional to the A280 readings, only the A260 values are shown in Table 2. E. coli O157:H7 and Salmonella Montevideo suspensions in MB were more sensitive to AITC than those in BPB. Increases in the AITC concentra- r1hat37 g/ml)

tion in MB suspensions caused increases in cellular leakage g/ml) ␮ and reductions in bacterial populations for E. coli O157:H7 ␮ g/ml) g/ml) g/ml) g/ml) ␮ ␮ ␮ ␮ and Salmonella Montevideo. Although dramatic reductions g/ml) g/ml) in the populations of E. coli O157:H7 and Salmonella Mon- ␮ ␮ tevideo in BPB occurred following treatment with 2,500 ␮ g/ml AITC, the increase in A260 and A280 values was only moderate. Signi®cant increases in A and A values only Streptomycin (400 Polymyxin B (20 AITC (2,500 AITC (2,500 AITC (1,000 AITC (1,000 260 280 AITC (500 AITC (500 occurred after E. coli O157:H7 and Salmonella Montevideo were exposed to 1,000 and 2,500 ␮g/ml AITC in MB. L. Bacterial population (log CFU/ml) and released metabolite ( monocytogenes was more resistant to AITC than the other two bacterial species. Only AITC at 2,500 ␮g/ml was ca- Numbers within each column of the same medium with different letters are signi®cantly different ( AITC, polymyxin B, and streptomycin fo pable of causing a reduction in the L. monocytogenes pop- TABLE 2. MediumBPB Test chemical Control MB Control a 732 LIN ET AL. J. Food Prot., Vol. 63, No. 6

␤ TABLE 3. -Galactosidase activity (A420)ofE. coli K-12 strain related study. Signi®cant increases in A260, A280, and A420 3.300 following treatment with AITC, polymyxin B, streptomycin, values were shown after a 4-h exposure to AITC at 100 and penicillin G for1hat37ЊC ␮g/ml, but 8 h was needed to achieve a signi®cant reduc-

Test chemical A420 tion of the bacterial population (Table 5). The values of released metabolites and ␤-galactosidase activities were not Control 0.18 Ϯ 0.14 Aa signi®cantly different after6hofexposure. However, larger b Ϯ CHCl3 and SDS 1.51 0.21 D reductions of bacterial populations were achieved with lon- ␮ Ϯ AITC (500 g/ml) 0.28 0.14 A ger exposure times. AITC (1,000 ␮g/ml) 0.56 Ϯ 0.09 C AITC (2,500 ␮g/ml) 0.99 Ϯ 0.38 BC DISCUSSION Polymyxin B (20 ␮g/ml) 0.84 Ϯ 0.20 BC Streptomycin (400 ␮g/ml) 0.27 Ϯ 0.12 A AITC in both liquid and gaseous forms showed similar Penicillin G (400 ␮g/ml) 1.45 Ϯ 0.52 BD antibacterial activities against the three test species. E. coli

O157:H7 was the most susceptible to AITC and L. mono- Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/6/727/1673840/0362-028x-63_6_727.pdf by guest on 25 September 2021 a Numbers with different letters are signi®cantly different (P Ͻ cytogenes was the most resistant. Gaseous AITC had a 0.05; n ϭ 4 for AITC treatments, n ϭ 3 for antibiotic treat- ments). higher antibacterial potency than the liquid form (5, 8). The b 50% inhibitory concentration values of liquid soluble AITC Bacteria from the control group lysed with CHCl3 and SDS served as a positive control for the ␤-galactosidase assay. for E. coli O157:H7, Salmonella Montevideo, and L. mon- ocytogenes determined in our laboratory were 81, 121, and 142 ␮g/ml, respectively (data not shown). Similar results ulation. It did not cause increases in the cellular leakage of were found by Kyung and Fleming (13) with E. coli B34, metabolites. Although streptomycin at 400 ␮g/ml caused Salmonella Typhimurium, and L. monocytogenes. The signi®cant reductions of the populations of the three spe- MICs of AITC for these three species were 50, 100, and cies, it did not increase cellular leakage. 200 ␮g/ml, respectively. However, Shofran et al. (22) re- ported a higher MIC value of 200 ␮g/ml for E. coli B34. Assay of ␤-galactosidase activity. Signi®cant ␤-ga- The MICs of AITC vapor against E. coli, Salmonella Ty- lactosidase activities (A ) were shown following treat- 420 phimurium, and Salmonella Enteritidis were determined to ments of E. coli K-12 with AITC, polymyxin B, and pen- be 34, 54, and 110 ng/ml, respectively. AITC also had a icillin G. Activities were correlated with concentrations of MIC of 54 ng/ml against two other gram-negative bacteria, AITC. Those activities found following exposure to high Vibrio parahaemolyticus and Pseudomonas aeruginosa. concentrations of AITC were closer to those detected fol- Gram-positive bacteria were more resistant to AITC vapor lowing exposure to polymyxin B than following exposure than gram-negative bacteria; the MICs ranged from 90 to to streptomycin and penicillin G. The highest values were 110 ng/ml for Bacillus subtilis, Bacillus cereus, Staphylo- obtained from the treatments with penicillin G, and total coccus epidermidis, and Staphylococcus aureus (8). Dela- lysis of bacterial cells occurred (Table 3). AITC, polymyxin quis and Sholberg (5) observed the bactericidal activity of B, penicillin G, and streptomycin in TSB, without bacterial gaseous AITC against E. coli O157:H7, Salmonella Typhi- culture at the tested concentrations, did not show any in- murium, and L. monocytogenes at 1 ␮g/ml. Our results also crease in A (␤-galactosidase activity). 420 showed that vapor AITC could damage the cell membrane Exposure of E. coli K-12 to gaseous AITC. After a and could reduce by 1,000 fold viable counts at 25 ␮g/ml. 24-h exposure, signi®cant increases of released metabolites The greater antibacterial activity of AITC in gaseous versus ␤ (A260 and A280) and -galactosidase activity (A420) were liquid form may be due to several reasons: (i) low solubility determined at the lowest concentration of AITC used (25 in broth and high volatility of AITC, (ii) possible degra- ␮g/ml). At the same concentration, the viable count was dation of AITC in aqueous solution (20), or (iii) better con- reduced 1,000 fold (Table 4). Exposure to higher concen- tact and/or absorption of bacterial cells to gaseous than to trations of AITC did not result in signi®cantly higher A260, liquid AITC. A280, and A420 values, but larger reductions in viable counts Our observation with the three antibiotics indicated an were obtained. Similar results were obtained with the time- action consistent with their reported mechanisms (4, 12,

␤ TABLE 4. Viable count (log CFU/ml), released metabolites (A260, A280), and -galactosidase activity (A420)ofE. coli K-12 strain 3.300 following treatment with gaseous AITC at 25, 50, and 100 ␮g/ml for 24 h at 20ЊC

a Treatment A260 A280 A420 Viable count

Control 0.22 Ϯ 0.12 Ab 0.18 Ϯ 0.10 A 0.25 Ϯ 0.19 A 8.82 Ϯ 0.04 A AITC (25 ␮g/ml) 0.79 Ϯ 0.14 B 0.46 Ϯ 0.08 B 1.12 Ϯ 0.30 B 5.64 Ϯ 0.43 B AITC (50 ␮g/ml) 0.83 Ϯ 0.18 B 0.48 Ϯ 0.04 B 1.79 Ϯ 0.38 BC 3.54 Ϯ 0.30 C AITC (100 ␮g/ml) 0.92 Ϯ 0.10 B 0.52 Ϯ 0.01 B 2.13 Ϯ 0.04 C 1.98 Ϯ 0.02 D a ␤ ␤ Bacteria from the control group lysed with CHCl3 and SDS served as a positive control for the -galactosidase assay. -Galactosidase Ϯ activity (A420) of the positive control was 1.49 0.42. b Numbers within each column with different letters are signi®cantly different (P Ͻ 0.05, n ϭ 3). J. Food Prot., Vol. 63, No. 6 BACTERICIDAL ACTIVITY OF ALLYL ISOTHIOCYANATE 733

␤ TABLE 5. Viable count (log CFU/ml), released metabolites (A260, A280), and -galactosidase activity (A420)ofE. coli K-12 strain 3.300 following treatment with gaseous AITC at 100 ␮g/ml for 4, 6, 8, 10, 12, and 24 h at 20ЊC

a Time Treatment A260 A280 A420 Viable count

4h Control 0.25 Ϯ 0.06 Ab 0.16 Ϯ 0.02 A 0.12 Ϯ 0.01 A 8.86 Ϯ 0.04 A AITC 0.46 Ϯ 0.13 B 0.32 Ϯ 0.07 B 0.46 Ϯ 0.15 B 8.44 Ϯ 0.59 A 6h Control 0.25 Ϯ 0.20 A 0.20 Ϯ 0.06 A 0.29 Ϯ 0.14 A 8.98 Ϯ 0.05 A AITC 1.67 Ϯ 0.20 C 1.22 Ϯ 0.28 C 0.84 Ϯ 0.51 C 7.80 Ϯ 0.98 A 8h Control 0.27 Ϯ 0.03 A 0.23 Ϯ 0.02 A 0.12 Ϯ 0.06 A 8.62 Ϯ 0.55 A AITC 1.51 Ϯ 0.43 C 1.10 Ϯ 0.39 C 0.71 Ϯ 0.28 C 3.91 Ϯ 1.93 B 10 h Control 0.16 Ϯ 0.02 A 0.13 Ϯ 0.02 A 0.16 Ϯ 0.03 A 8.74 Ϯ 0.17 A AITC 2.00 Ϯ 0.06 C 1.37 Ϯ 0.01 C 1.19 Ϯ 0.18 C 2.90 Ϯ 0.80 BC 12 h Control 0.27 Ϯ 0.12 A 0.23 Ϯ 0.10 A 0.12 Ϯ 0.08 A 8.87 Ϯ 0.12 A Ϯ C Ϯ C Ϯ D Ϯ BC

AITC 1.61 0.42 1.31 0.32 1.54 0.29 2.80 1.52 Downloaded from http://meridian.allenpress.com/jfp/article-pdf/63/6/727/1673840/0362-028x-63_6_727.pdf by guest on 25 September 2021 24 h Control 0.36 Ϯ 0.08 A 0.36 Ϯ 0.11 A 0.17 Ϯ 0.07 A 8.77 Ϯ 0.23 A AITC 1.17 Ϯ 0.21 C 1.67 Ϯ 0.13 C 1.40 Ϯ 0.57 CD 1.14 Ϯ 0.16 C a ␤ ␤ Bacteria from the control group lysed with CHCl3 and SDS served as positive controls for the -galactosidase assay. -Galactosidase Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ activities (A420) of the positive controls were 1.07 0.04, 1.17 0.38, 1.04 0.17, 1.26 0.47, 1.34 0.39, and 1.05 0.17 at 4, 6, 8, 10, 12, and 24 h, respectively. b Numbers within each column with different letters are signi®cantly different (P Ͻ 0.05, n ϭ 6).

14). As expected, penicillin G and streptomycin had mini- terial populations (3, 16, 17). The treatment of E. coli mal bactericidal activity for the test bacteria at the station- O157:H7 and Salmonella Montevideo with AITC or poly- ary phase (Figs. 4 through 6). However, they showed mod- myxin B resulted in a reduction in the bacterial population erate bactericidal activity to the bacteria at the exponential that was inversely related to the increased A260 and A280 growth phase. Polymyxin B was highly effective against E. values of the extracellular solutions. The same pattern of coli O157:H7 and Salmonella Montevideo at the exponen- results was obtained with ␤-galactosidase studies. In the tial growth phase. It also showed potent bactericidal activity study of exposing gaseous AITC, a dramatic reduction of against bacteria at the stationary phase. Polymyxin B was the bacterial population was achieved, although the turbid- more toxic to gram-negative bacteria (E. coli O157:H7 and ity of the bacterial suspension did not change. Similar re- Salmonella Montevideo) than to gram-positive bacteria (L. sults were obtained when stationary-phase bacteria were monocytogenes) because of differences in cellular mem- treated with liquid AITC. These results indicated that the brane structures and cell wall composition between the two loss of bacterial viability was not due to cell lysis, and groups (18). Based on our results, AITC showed effective leakage of cellular metabolites may account for the bacte- bactericidal activity against the test bacteria at all growth ricidal activities. Short exposure times and low concentra- ␤ stages. This indicated that its bactericidal mechanism was tions resulted in high A260 and A280 values and -galacto- not limited to its inhibitory effect on the biosynthesis of sidase activity but insigni®cant population reductions. Dur- macromolecules, which occurs actively during the expo- ing these studies, many small colonies appeared on the TSA nential growth phase. In addition, AITC was more effective plates for the treated groups. These indicated that slightly on gram-negative than on gram-positive bacteria. The order injured bacteria were recovered under the experimental of sensitivity to AITC and polymyxin B was the same conditions. among the three test bacteria. Bacterial OD540 values de- Bacteria in BPB were more resistant to AITC and an- creased with the treatments of AITC and polymyxin B at tibiotics than bacteria in MB, perhaps due to the availability the exponential growth phase. Therefore, the antibacterial of more nutrients in MB than in BPB to support active mechanism(s) of AITC more closely resembled that of bacterial growth. These results also supported the obser- polymyxin B than that of penicillin G or streptomycin. vation that bacteria at the exponential growth phase were Polymyxin B has a hydrophilic heptapeptide ring and more sensitive to AITC than at the stationary phase. a hydrophobic chain that give it an amphiphilic property, Our study showed that AITC possessed potent killing like a nonionic detergent. This detergent property may al- activity against bacteria at both exponential and stationary low it to disaggregate the membrane structure (14). AITC growth phases. Bacteria at the stationary phase were not also has a polar isothiocyanate end and a nonpolar allyl affected by penicillin G or streptomycin. In addition, AITC side chain (2, 6). Some aromatic alcohols, such as phene- was also inhibitory to fungi and yeast (13, 19, 22). Collec- thyl alcohol, shown to be more effective on gram-negative tively, these studies indicate that inhibition of biosynthesis than on gram-positive bacteria, were found to have similar of bacterial cells is not the major mechanism of the bac- amphiphilic structures and to exert their antibacterial activ- tericidal activity of AITC. The inhibition of stationary cells ities by acting on cell membranes (3, 16). High A260 and by AITC is a promising result for its application to food A280 values of the extracellular solution of bacteria treated systems. In fact, AITC has been reported to be an effective with polymyxins or phenethyl alcohol were reported, and bacteriostatic and bactericidal agent for lettuce and pre- these values were proportional to the reduction of the bac- cooked roast beef (15, 23). Stationary-phase cells display 734 LIN ET AL. J. Food Prot., Vol. 63, No. 6 less metabolic activity. Bacteria in food systems are, in gen- 10. Kawakishi, S., and T. Kaneko. 1987. Interaction of proteins with allyl eral, low in metabolic activity due to processing or low- isothiocyanate. J. Agric. Food Chem. 35:85±88. 11. Kojima, M., and K. Ogawa. 1971. Studies on the effect of isothio- temperature storage. Thus, the bactericidal or bacteriostatic cyanates and their analogues on microorganisms: (I) effects of iso- activities of AITC to all growth stages provide a strong case thiocyanates on the oxygen uptake of yeasts. J. Ferment. Technol. for its application as a food preservative. However, some 49:740±746. agricultural commodities, such as alfalfa sprouts, are sen- 12. Kucers, A., S. W. Crowe, M. L. Grayson, and J. F. Hoy. 1997. Pen- icillin G, p. 3±70; polymyxin, p. 667±675; streptomycin, p. 429± sitive to the toxic effect of AITC when this compound is 438. In The use of antibiotics: a clinical review of antibacterial, used as an antibacterial agent against E. coli O157:H7. antifungal and antiviral drugs, 5th ed. Butterworth-Heinemann, Ox- ford, UK. 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