M: Food Microbiology & Safety JFS M: Food Microbiology and Safety Effect of Free-SH Containing Compounds on Allyl Isothiocyanate Antimicrobial Activity against Escherichia coli O157:H7 F.B. LUCIANO, F.S. HOSSEINIAN, T. BETA, AND R.A. HOLLEY ABSTRACT: Escherichia coli O157:H7 contamination is a significant meat safety issue in many countries. Allyl isothiocyanate (AIT) is a natural compound found to limit the survival of E. coli O157:H7 and other pathogens in meat and meat products. In the present study, it was found that glutathione and cysteine naturally present in meat can interfere with AIT antimicrobial activity. Spectroscopy, HPLC, and LC-MS were used to confirm that glutathione was able to react with AIT and formed a conjugate with no or low bactericidal activity against the tested organisms. The same reaction also occurred at pH values of 4.9 and 5.8 at 25 and 4 ◦ C, respectively, which broadly represent storage conditions in raw beef (pH 5.8) and during fermented sausage (pH 4.9) manufacture. Reactions observed help to explain reduction in antimicrobial potency of AIT in food (meat) systems. Keywords: allyl isothiocyanate, conjugation, E. coli O157:H7, glutathione, meat safety Introduction G lucosinolates are a group substances found within cell com- partments of plants that belong to the family Cruciferae (for example, broccoli, mustard, horseradish, wasabi) (Nielsen and Rios 2000). When these intracellular compartments are disrupted af- ter damage to some extent, glucosinolates are hydrolyzed by the membrane-bound enzyme myrosinase, resulting on the forma- tion of 3 main groups of substances: nitriles, thiocyanates, and isothiocyanates (Delaquis and Mazza 1995). The last group con- tains diverse compounds with well-established antimicrobial ac- tivity, such as benzyl isothiocyanate, phenethyl isothiocyanate, and, more important for this study, allyl isothiocyanate (AIT) (Delaquis and Sholberg 1997; Hashem and Saleh 1999; Shin and others 2004). These substances have a characteristic strong flavor and high volatility (Kawakishi and Kaneko 1987). The interest in antimicrobials derived from natural sources has increased in the past few years due to the usually accepted safe status of these compounds (Lin and others 2000). Additionally, the utilization of AIT in food systems has been approved in Japan pro- vided it has a natural plant source (Sekiyama and others 1994). On the other hand, Canada and the United States have not yet permit- ted the use of isothiocyanates as food antimicrobials, but they can be found unintentionally in foods with added condiments such as mustard, wasabi, and horseradish (Delaquis and Sholberg 1997). It has been estimated that 76 million people fall ill from foodborne pathogens in the United States each year. Of these, 325000 are hos- pitalized and more than 5000 die. This is estimated to cost US$5 to 6 billion in medical expenses and lost productivity (Deisingh and Thompson 2004). E. coli O157:H7 causes about 20000 illnesses and 250 deaths yearly in the United States, and it is also an impor- tant foodborne pathogen in Canada and some European countries (Peacock and others 2001). To date, human infections attributed to MS 20070900 Submitted 12/4/2007, Accepted 3/11/2008. Authors are with Dept. of Food Science, Faculty of Agricultural and Food Sciences, Univ. of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada. Direct inquiries to au- thor Holley (E-mail: rick holley@umanitoba.ca). E. coli O157:H7 have been reported in over 30 countries, with most outbreaks associated with the consumption of undercooked meats, especially ground beef or hamburger patties (Nadarajah and others 2005). Allyl isothiocyanate has been demonstrated to have high bac- tericidal activity in vapor and liquid forms against various food pathogens of concern, including Listeria monocytogenes, Staphy- lococcus aureus, Salmonella Typhimurium, and E. coli O157:H7 (Isshiki and others 1992; Lin and others 1999; Rhee and others 2003). Lin and others (2000) tested liquid AIT against 3 differ- ent bacterial species and found that 500 μg/mL (approximately 0.5 μL/mL) was needed to achieve a 3 to 4 log reduction of E. coli O157:H7 (in vitro) at different stages of bacterial growth. This level had a similar effect when applied against Salmonella Montevideo; however, 2500 μg/mL of AIT were required for L. monocytogenes. Delaquis and Sholberg (1997) evaluated the activity of AIT in the vapor form and showed that 1000 μg/L of air were needed to yield a ≥ 5.65 log reduction of E. coli O157:H7, S. Typhimurium, and L. monocytogenes growth on tryptic soy agar disks at 35 ◦ C. Isothiocyanates are well known as powerfully reactive species that go through nucleophilic addition reactions (Verma 2003). The isothiocyanate group has a highly electrophilic central carbon atom (—–N== = C== = S) which can react readily and under mild conditions with hydroxyls, amines, and thiols, generating products such as car- bamates, thiourea, and thiocarbamates, respectively (Zhang and Talalay 1994). In this context, AIT reacted with glutathione, amino acids, proteins, water, alcohol, and sulfites (Kawakishi and Namiki 1969; Cejpek and others 2000) and was able to disintegrate the cys- tine disulfide bond through an oxidative process (Kawakishi and Namiki 1982). The antimicrobial effects of AIT have been largely reported in raw meat and meat products (Ward and others 1998; Muthuku- marasamy and others 2003; Nadarajah and others 2005; Chacon and others 2006a, 2006b). Compounds containing free thiol, amine, and hydroxyl groups are commonly present in meat (Faustman and Cassens 1991) and theoretically can react with AIT. Ward and others (1998) and Nadarajah and others (2005) showed that the percentage M214 JOURNAL OF FOOD SCIENCE—Vol. 73, Nr. 5, 2008 C  2008 Institute of Food Technologists doi: 10.1111/j.1750-3841.2008.00762.x Further reproduction without permission is prohibited