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Antibacterial Mechanism of Allyl Isothiocyanate² 727 Journal of Food Protection, Vol. 63, No. 6, 2000, Pages 727±734 Copyright Q, 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, b-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 b 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 b-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 mg/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 b-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 mg/ml and to the L. monocytogenes suspension at 2,500 mg/ 8 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 mg/ml, respectively, and at 250, 500, or 500 mg/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 8 540 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 5 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 3 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 mg/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 378C with shaking at mined. Then, 10-ml aliquots were dispensed into each of six ster- m 200 rpm.
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