Keywords

4.1 Introduction

Phytochemicals are defined as non-nutrient bioactive plant origin compounds naturally present in fruits, vegetables and whole grains (Liu 2004). Dietary intake of phytochemicals has been strongly linked to reduced risk of major chronic diseases such as cancer, diabetes and cardiovascular diseases (Liu 2003). Use of fruit and vegetables as health remedies traditionally is very common in a considerable part of world population (Salama and Marraiki 2010).

One group of sulfur forming in plants is known as organosulfur compounds. Organosulfur compounds, defined as organic molecules containing one or more carbon-sulfur bonds, are one of the groups present in foods as natural preservatives (Cremlyn 1996). These compounds are valued not only due to their rich and varied chemistry, but also for their many important biological properties (Polshettiwar and Kaushik 2004).

The families Alliaceae and Brassicaceae, two members of the plant kingdom, contain sulfur containing materials which possess biological properties (Stoewsand 1995). Today, it is well established that these vegetables are natural sources of a group of phytochemicals known as organosulfur compounds. Allium and Brassica vegetables especially garlic and onion have been known to be used as health remedies in ancient civilizations. Therapeutic application of these vegetables for the prevention of various diseases such as cancer and cardiovascular diseases has been well-studied (Vazquez-Prieto and Miatello 2010).

Organosulfur compounds in Allium and Brassica plants are called thiosulfinates and glucosinolates, respectively. These phytochemicals are characterized by the properties that give the plants their specific flavors and odors. Thiosulfinates and glucosinolates are also converted to various new sulfur containing materials which exhibit a variety of bioactive properties such as antimicrobial, anticarcinogenic, antitumor and pesticidal etc. via a number of biosynthetic reactions. This chapter outlines the antimicrobial properties of the organosulfur compounds present in Allium and Brassica plants and their derivatives on diverse microorganisms including bacteria, fungi and viruses.

4.2 Thiosulfinates

The major sources of organosulfur compounds are Allium vegetables. The Allium genus includes approximately 600 species, the most widely consumed of which are onions (Allium cepa), garlic (Allium sativum), leeks (Allium porrum), chives (Allium schoenoprasum), and shallots (Allium ascalonicum) (Bianchini and Vainio 2001; Benkeblia and Lanzotti 2007). Among the Allium species, onion and garlic are the oldest and most commonly cultivated plants by different cultures (Lanzotti 2006).

The earliest records of onion and garlic date back to the sixteenth century BC when several medicinal formulas based on garlic and onions as a curative agent for a variety of diseases such as heart problems, headache and tumors were mentioned in the Codex Ebers, the Egyptian medical papyrus (Block 1985). The Sumerians, Indians, Romans, Ancient Egyptians and Greeks are also all known to have consumed garlic and onions for thousands of years (Ahmad 1996). In China, tea made from garlic and onion has been used as a remedy for fever, headache, cholera and dysentery; garlic was utilized as an antiseptic in the treatment of gangrene in the World Wars (Corzo-Martinez et al. 2007). Garlic and onion are still used in folk medicine in all over the world. At present, these vegetables are among the most cultivated plants in the world with production amounts of 9 and 55 million tons per year, respectively (Peter 2000; FAO 2004).

4.2.1 Occurrence and Chemistry of Thiosulfinates

Not only do people consider Allium vegetables important because of their characteristic flavor that contributes to the savoriness of various dishes in world cuisine, they are also aware of the nutritional potential of these vegetables since they are rich in nutrients such as high levels of phosphor, potassium, zinc, vitamins A, C and D, folic acid, calcium and selenium etc. is also considered by people (Peter 2000; Rahman 2003). They are also rich sources of phenolic compounds (Vinson et al. 1998). Table 4.1 shows the approximate composition of fresh garlic and onion bulbs.

Table 4.1 Approximate composition of garlic and onion (Lawson 1996; Rahman 2003; Dini et al. 2008)
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As in most fresh foods, water is the main constituent of Allium vegetables (Table 4.1). Although garlic and onion contain similar compounds to a certain extent, the amounts of major components in onion are rather low compared to those of garlic. Fresh garlic contains a moisture level of 62–68% (Table 4.1), while it is even higher (88.6–92.8% of fresh weight) in onion than that in garlic (Peter 2000). When considering the biological importance of these vegetables, sulfur containing compounds attract the attention. As seen Table 4.1, the proportion of total sulfur compounds in garlic is 1.1–3.5%, meanwhile its amount in onion is only one third of that found in garlic (Benkeblia 2004).

Thiosulfinates are the most studied compounds among the active constituents of Allium vegetables. Their main structures were first reported by Wertheim and later by Semmler in garlic and onion oils in the nineteenth century (Benkeblia and Lanzotti 2007). Block (1992) identified eight different thiosulfinates from nine Allium vegetables including garlic and onion by HPLC analysis. Garlic was found to be the richest source of thiosulfinates with amounts ranging from 15 μmol/g (garlic grown at low temperature, 21°C) to 53 μmol/g (elephant garlic). The thiosulfinate contents of the onion species were all <0.35 μmol/g.

Intact garlic bulbs possess certain sulfur containing compounds known as S-alk(en)yl-L-cysteine sulphoxides (ACSOs). Alliin (S-allylcysteine sulfoxide), the major ACSO of these vegetables is a colorless, odorless water soluble amino acid so long as the bulb is intact or undamaged (Corzo-Martinez et al. 2007). These are present in all Allium spp. constituting 1–5% of dry weight of the plant. However, when the cells are damaged, the enzyme alliinase (or alliin lyase) releases and converts alliin into certain volatile and reactive components called thiosulfinates (Block 1985). Allicin (allyl 2-propene thiosulfinate) is the most abundant thiosulfinate produced (70% of overall garlic thiosulfinates), whereas allyl methyl thiosulfinate (AMTS) is the second (18%) and additional thiosulfinates are also formed in lower concentrations (Mazza 2002). Since the thiosulfinates as well as allicin are very unstable, they readily undergo various transformations to form more stable compounds, to alkyl sulfides such as di- and trisulfides, allyl sulfides, vinyl dithiins, ajoenes and mercaptocysteines (Shi et al. 2005). These compounds can be yielded in a variety of reactions such as dehydration, rearrangement, condensation, Diels-Alder reaction, hydrolysis and pyrolysis depending on conditions and still exhibit biological activity (Lanzotti 2006). Figure 4.1 illustrates chemical structures of alliin and sulfur containing compounds forming from alliin in Allium vegetables. Onions also produce these sulfides but only at the level of 4 mg/100 g of fresh weight which means barely any amount compared to garlic (Shi et al. 2005).

Fig. 4.1
figure 1_4

Chemical structures of alliin and its main hydrolysis products

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4.2.2 Antimicrobial Properties of Thiosulfinates

Even though it is common knowledge that Allium vegetables contain a variety of functional components, the beneficial aspects of the Allium species have been mainly attributed to specific organosulfur compounds. In addition to the traditional use of Allium vegetables for thousands of years, scientific studies carried out on these vegetables, their extracts or specific organosulfur compounds have also proved their diverse pharmacological and biological benefits such as antimicrobial, antioxidative, antitumor and antiasthmatic (Lanzotti 2006), while effects of various Allium products such as garlic oil and methanol extract to inhibit rumen fermentation and methane production were also discussed (Patra and Saxena 2010). In general, cooked or waited vegetables are recommended for human consumption due to their prolonged protective effect resulting from the increased level of bioactive compounds in the vegetal tissues (Goncagul and Ayaz 2010).

Allium vegetables have been shown to have antibacterial, antifungal, antiviral and antiprotozoal activities. This activity has been attributed to thiosulfinates and to the other sulfur containing compounds present in these vegetables. It has been found that the main antimicrobial agents are breakdown products of allicin such as diallyl disulfide (DADS), diallyl trisulfide (DATS), diallyl sulfide (DAS) and ajoene, antimicrobial strength of which is higher than that of allicin (Corzo-Martinez et al. 2007). It is obvious that the enzyme alliinase plays a critical role in antimicrobial action since thiosulfinates are generated following the activation of this enzyme after the vegetable tissues are injured. Inactivation of alliinase as a result of prolonged heating causes the failure of antimicrobial activity (Lawson 1996). In general, each sulfur compound has individually been found to exhibit weaker antimicrobial activity as compared to the crude extracts of Allium spp. (Lawson 1998). It is also known that some proteins, saponins and phenolic compounds can contribute to antimicrobial activity (Griffiths et al. 2002).

As in other natural antimicrobial compounds of plant origin (spice essential oils, phenolics and other plant extracts etc.), these compounds may generally show their antimicrobial activity by altering the permeability of microbial cell walls and the replacing of intracellular and extracellular materials with each other. Moreover, the main mechanism involved in the antimicrobial effect is assumed to be the inhibition of thiol-containing enzymes in microorganisms by the rapid reaction of thiosulfinates with thiol groups (Ankri and Mirelman 1999).

4.2.2.1 Antibacterial Activity

The antibacterial activities of the Allium species have been well documented. Historically, Louis Pasteur first noted the antibacterial activity of garlic (Block et al. 1993). By the 1900s, substances showing antibacterial action were beginning to be identified. Allicin was the first organosulfur compound to be isolated and defined as an antibacterial agent in garlic (Cavallito and Bailey 1944). The antibacterial activity of allicin is remarkably more bacteriostatic than bactericidal. Allicin in concentrations of 1:85.000 in broth is bactericidal against a wide variety of Gram-negative and Gram-positive organisms (Cavallito and Bailey 1944). Feldberg et al. (1988) suggested that the antibacterial action of allicin caused a quick and complete inhibition of RNA biosynthesis and additionally a partial inhibition of DNA and protein synthesis. In addition to allicin, other thiosulfinates have also been found to exhibit antibacterial activity. A great number of in vitro and in situ studies have been made in order to reveal the antibacterial properties of individual organosulfur compounds or various extracts of Allium vegetables.

It has been proven that the Allium species can inhibit both Gram (+) and Gram (−) bacteria as well as toxin production. Table 4.2 summarizes the in vitro studies made to identify antibacterial activities of Allium vegetables or their components. Garlic, the most studied vegetable, has been reported to be effective against strains of Pseudomonas, Proteus, Escherichia coli, Staphylococcus aureus, Klebsiella, Salmonella, Micrococcus, Bacillus subtilis, Mycobacterium and Clostridium. It may also inhibit beneficial intestinal microflora, but the potentially harmful species of the Enterobacteriaceae family are more sensitive to garlic compounds especially allicin (Corzo-Martinez et al. 2007). The sensitivity of Listeria monocytogenes, a psychrotrophic pathogen which can be found in foods, was measured against various extracts of Allium vinelae (wild garlic) by the disc diffusion method and methanol extract was found to be more active than ethanol and n-hexane extracts (Sagun et al. 2006). The efficiency of garlic shoot juice on L. monocytogenes was also shown (Kim et al. 2007). General lysis of the cytoplasm, lysis of the cell wall and cellular swelling were observed in bacterial cells by scanning electron microscopy in the same study. The inhibitory effect of garlic against S. aureus, Salmonella Typhi, E. coli and L. monocytogenes was measured using the turbidity method in another study. 80% inhibition level for E. coli, S. Typhi, S. aureus and L. monocytogenes were provided in the presence of garlic with levels of 3.95%, 7.0%, 5.0% and 8.8% in in vitro conditions, respectively (Kumar and Berwal 1998). Allicin and other Allium products were also considered as potential control agents for oral pathogens, causes of the dental diseases, such as Streptococcus mutans, S. sobrainus, Actinomyces oris, Act. viscosus and Porphyromonas gingivalis (Bakri and Douglas 2005; Bachrach et al. 2011).

Table 4.2 In vitro antibacterial activity of Allium vegetables or their components
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As can be seen in Table 4.2, the Bacillus species are the most sensitive bacteria against Allium compounds or extracts while ajoene has the highest antibacterial effect. The sensitivity of E. coli to allicin is higher compared to other sulfur containing compounds. It can also be seen that P. aeruginosa is the most resistant species against all organosulfur compounds in general. However, the different minimum inhibitory concentration (MIC) levels reported by researchers may arise from extraction method, final thiosulfinate concentration in the extracts and the method applied for the assessment of the inhibitory effect (Benkeblia and Lanzotti 2007).

Studies on the antibacterial activity of thiosulfinates have mainly focused on garlic and onion. These studies showed that garlic was more effective in bacterial inhibition than onion since the sulfur compound content of garlic is four times higher compared to the level found in onion. Furthermore, both onion and garlic are the most commonly used food ingredients for flavor enhancement purposes. Meat and dairy products are especially favorable for flavoring by these materials (Sagun et al. 2006; Salem et al. 2010). In addition to food flavoring, a number of biological effects, such as the antibacterial activity of Allium species in food systems, have been proven.

The addition of garlic essential oil to minced beef contributed to its storage stability by decreasing native bacterial counts (APC, Enterobacteriaceae, coliform bacteria and S. aureus) (Salem et al. 2010). However, all garlic oil supplements (0.5%, 1.0% and 1.5%) demonstrated fair enhancements of sensory attributes in storage periods of 3 days, while the samples treated with 0.5% garlic oil demonstrated the lowest sensory enhancement after 6 days. Wong and Kitts (2002) showed that the pre-seasoning of steak with garlic caused a reduction in the psychrotroph population in both non-irradiated and irradiated steaks after refrigerated storage for 2 weeks.

Yin and Cheng (2003) revealed the antibacterial properties of four thiosulfinates (diallyl sulfide, DADS, s-ethyl cysteine and n-acetyl cysteine) in minced beef. APC was significantly reduced and the growth of five inoculated pathogenic bacteria, S. Typhimurium, E. coli O157:H7, L. monocytogenes, S. aureus and Campylobacter jejuni was inhibited by the presence of DAS and DADS in ground beef. Park et al. (2008) reported an antimicrobial effect which inhibited the growth of APC and Enterobacteriaceae in pork belly and loin cuts by the injection of solutions containing 5% of garlic or onion powder into the cuts and the inhibition was superior compared to sodium ascorbate injection. Aydin et al. (2007) investigated the antimicrobial effect of chopped garlic in ground beef and raw meat balls (cig kofte, a traditional Turkish raw meat product). The addition of garlic at 5% or 10% to the raw meatball mix decreased the microbial count, in terms of APC and yeast and mold counts. However, it was not as effective in ground beef as much as it was in raw meat ball mix.

Various garlic products, garlic oil, garlic powder and fresh garlic were investigated for their antibacterial efficiency in chicken sausage. The addition of fresh garlic (30 g/kg) or garlic powder (9 g/kg) significantly reduced the APC and, subsequently, the shelf-life of the product was extended to 21 days. However, the addition of garlic oil resulted in no additional reduction in APC when compared with control samples (Sallam et al. 2004).

4.2.2.2 Antifungal Activity

The importance of fungi is highlighted by their harmful effects, such as food spoilage or toxin production as well as by their beneficial properties such as food fermentation. The antifungal activity of thiosulfinates has been extensively reported by a variety of in vitro and in vivo studies. Yamada and Azuma (1977) first reported the antifungal activity of allicin against Candida, Cryptococcus, Trichophyton, Epidermophyton and Microsporum. These researchers also stated that the MIC of allicin against various pathogenic fungi was affected considerably by differences in experimental conditions, e.g., incubation time, inoculum size, type of medium and medium pH. Moreover, alike in antibacterial activity, the breakdown products of allicin, including DATS, DADS, DAS and ajoene are the major active antifungal agents of onion and garlic extracts and have a greater antifungal effect than allicin (Tansey and Appleton 1975; Corzo-Martinez et al. 2007). Table 4.3 lists the results of a number of in vitro studies showing the antifungal properties of thiosulfinates in Allium vegetables.

Table 4.3 In vitro antifungal activity of thiosulfinates
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Several models for mode of antifungal action of thiosulfinates were suggested by various researchers. Yamada and Azuma (1977) reported that allicin in the concentration of 3.13 μg/mL inhibited swelling and germination of fungi spores in nutritious media. Barone and Tansey (1977) showed that garlic and allicin suppressed C. albicans cell metabolism by the inactivation of proteins, competitive inhibition of sulfhydryl compounds, or by the noncompetitive inhibition of enzyme function by oxidation. In another study, the deformation and distortion of yeast cells, cell collapse and cytoplasmic debris were observed in cells grown in the presence of garlic aqueous extract (Ghannoum 1988). Adetumbi et al. (1986) proposed that garlic extract inhibited the protein and nucleic acid synthesis of C. albicans in parallel with growth inhibition, but inhibited lipid synthesis completely.

The antifungal effects of Allium extracts have also been reported by many researchers. Yin and Tsao (1999) found that garlic bulb extract exhibited the best inhibitory effect among the extracts of seven different Allium vegetables on Aspergillus niger, A. flavus and A. fumigatus with the MIC values of 35, 75 and 104 μg/mL, respectively. Benkeblia (2004) showed the dose-dependent in vitro inhibition of five different essential oil extracts of three types of garlic and onion on A. niger, Penicillium cyclopium and Fusarium oxysporum. Lower concentrations of onion extracts (50 and 100 mL/L) did not cause a significant inhibitory effect compared to control. Shams-Ghahfarokhi et al. (2006) also investigated the antifungal activities of aqueous extracts of garlic and onion on Malassezia furfur (25 strains), C. albicans (18 strains), and other Candida spp. (12 strains) as well as 35 strains of various dermatophyte species. They showed that the extracts were able to inhibit the growth of all fungi tested in a dose-dependent manner. The activity of aqueous garlic extract against M. furfur, C. albicans, other Candida spp. and the dermatophytes was 64, 8, 4 and 32–128 fold that of the aqueous onion extract, respectively.

Heated garlic as well as fresh garlic and mustard is already used in some foods as preservatives. Kim and Kyung (2003) investigated the antifungal activity of heated garlic and alliin against different strains of C. albicans, C. utilis, Saccharo­myces cerevisiae, Pichia membranifaciens, Zygosaccharomyces bisporus and Z. rouxii. Alliin heated in distilled water showed an antifungal activity pattern similar to that of heated garlic, suggesting that the compound(s) thermally generated from alliin were the main antifungal compound(s) of heated garlic. The antifungal activity was increased as the time of heating increased up to 45 min at 121°C, and the activity did not change when garlic was further heated for up to 120 min.

4.2.2.3 Antiviral Activity

Relatively few studies have shown antiviral properties of thiosulfinates. Weber et al. (1992) tested the antiviral potentials of not only fresh garlic extract but also its sulfur components, allicin, allyl methyl thiosulfinate, methyl allyl thiosulfinate, ajoene, alliin, deoxyalliin, DADS, and DATS against selected viruses including, herpes simplex virus type 1, herpes simplex virus type 2, parainfluenza virus type 3, vaccinia virus, vesicular stomatitis virus, and human rhinovirus type 2. The order for virucidal activity was generally found as: ajoene≥allicin≥allyl methyl thiosulfinate≥methyl allyl thiosulfinate while no activity was observed for alliin, deoxyalliin, DADS or DATS. Ajoene, allyl alcohol and DADS were shown to be active against HIV (Human immunodeficiency virus) -infected cells (Tatarintsev et al. 1992; Shoji et al. 1993). Chen et al. (2011) studied the antiviral activity of the powders of five Allium plants (garlic, onion, leek, shallot and green onion) and allicin against two adenovirus isolates (ADV41 and ADV3). They found that shallots exhibited the highest level of antiviral activity for both ADV41 and ADV3, followed by garlic and onions. Shallots exhibited the highest level of antiviral activity against ADV3 and ADV41 infection from 0 to 2 h, during the early period of virus replication.

4.2.2.4 Antiparasitic Activity

The antiparasitic effects of freshly crushed garlic have been known since ancient times. Chinese people traditionally use the alcoholic extract of garlic cloves to cure intestinal diseases. Only a few reports are available regarding the antiparasitic activity of Allium vegetables or their sulfur compounds. It was reported that allicin (30 μg/mL) could efficiently inhibit the growth of some such as Giardia lamblia, Leishmania major, Leptomonas colosoma and Crithidia fasciculate (Ankri and Mirelman 1999). Ankri et al. (1997) also showed that allicin inhibited cysteine proteinases and the cytopathic effects of Entamoeba histolytica. Lun et al. (1994) tested the antiparasitic activity of DATS, known as dasuasu in China, against several important protozoal parasites in vitro. The IC50 (concentration to inhibit metabolism or growth of parasites by 50%) for Trypanosoma brucei brucei, T. b. rhodesiense, T. b. gambiense, T. evansi, T. congolense and T. equiperdum was found in the range of 0.8–5.5 μg/mL. IC50 values were 14 and 59 μg/mL for E. histolytica and G. lamblia, respectively.

4.3 Glucosinolates

Glucosinolates (GLSs) are a class of organic compounds which are formed from glucose and an amino acid and contain sulfur and nitrogen (Du and Halkier 1998). Cruciferous or Brassica vegetables are rich sources of GLSs and all the members of these plants contain these organosulfur compounds naturally (Song et al. 2005; Higdon et al. 2007). However, GLSs do not only exist in crucifers; at least 500 non-cruciferous species were reported to contain one or more forms of GLSs (Fahey et al. 2001). Similar to cruciferous vegetables, many plants belonging to other plant families such as Capparaceae also contain GLSs (Whitmore and Naidu 2000).

Brassicaceae is a large family including 338 genera and 3,709 species (Warwick et al. 2006). Most cultivated Brassica species include mustards, cabbage (derived from Brassica oleracea, Capitata, Pekinensis, Chinensis and Acephala groups), cauliflower (derived from B. oleracea var. botrytis), Brussels sprouts (B. oleracea var. gemmifera Zenker), broccoli (derived from B. oleracea), kohlrabi (B. oleracea L. var. gongylodes L.), kale (B. oleracea L. var. acephala group ornamental), horseradish (Armoracia rusticana G. M. Sch.), wasabi or Japanese horseradish (Wasabia japonica Matsum or Wasabia tenuis Matsum), and radishes (Raphanus sativus L. var. radicula Pers.) (Whitmore and Naidu 2000).

4.3.1 Occurrence and Chemistry of Glucosinolates

GLSs are amino acid derived secondary plant metabolites which contain a sulphate and a thioglucose (Halkier and Du 1997). These compounds are mainly found in the seeds, although they may also be present in other tissues of the plants. Various amino acids such as tyrosine, phenylalanine and tryptophan, are precursor components and are converted to GLSs by decarboxylation. Table 4.4 lists the GLSs, their degradation products and precursors.

Table 4.4 GLSs, their degradation products and precursors (Pengelly 2004)
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Cruciferous plants may contain total GLSs of about 1% of dry weight but the content is rather variable. For example, broccoli sprouts or seeds may possess 70–100 μmol/g total GLSs of fresh weight. More than 120 individual GLS compounds have been identified in different plant families so far (Fahey et al. 2001). More than 80% of all GLSs identified are found in the Brassicaceae family (Kjaer 1976; Whitmore and Naidu 2000) although Arabidopsis thaliana was found to contain 20 different GLSs (Brown et al. 2003).

GLSs are classified in aliphatic, aromatic and indolyl groups on the basis of whether they are derived from aliphatic amino acids (often methionine), phenylalanine or tyrosine, or tryptophan, respectively (Halkier and Du 1997). The structural variation of GLSs results from chain elongations of amino acids before the formation of the GLS core structure and secondary modifications of the GLSs side chain (e.g. thiol oxidation, desaturation, hydroxylation, and esterification) and/or glucose moiety (esterification) (Wittstock and Halkier 2002). Fahey et al. (2001) grouped GLSs under ten different chemical structures (sulfur-containing side-chains; aliphatic, straight-chain; aliphatic, branched-chain; olefins; aliphatic, straight and branched-chain alcohols; aliphatic, straight-chain ketones; aromatic; benzoates; indole; multiply glycosylated and others).

GLSs are chemically and thermally stable to a certain extent and therefore generally enzymatic hydrolysis occurs (Polat 2010). Myrosinase is a specific class of β-thioglucosidases located in idioblasts (myrosin cells) distributed in most tissues of GLS producing plants (Grubb and Abbel 2006). Reaction kinetics or the enzyme myrosinase may differ depending on the plant species and an individual plant may contain multiple forms of myrosinase (James and Rossiter 1991). By the enzyme myrosinase releases in consequence of tissue damage caused by bruising, wounding, mastication, harvest, shipping and/or handling, relatively nonreactive GLSs are hydrolyzed (Fahey et al. 2001) (Fig. 4.2). The hydrolysis products are thioglucose, sulphate and an unstable derivative which is converted to several degradation products, many of which have pronounced biological effects. These degradation products include isothiocyanates (ITCs), nitriles and thiocyanates. However, epithionitriles and oxazolidine-2-thiones are also produced depending on several factors such as pH and side-chain structures (Halkier and Du 1997) (Fig. 4.2). Stable ITCs are usually formed at pH 6–7, while nitriles are the main hyrolysis products under acidic conditions. Although the majority of hydrolysis products are relatively very stable, indole GLSs such as glucobrassicin yield unstable ITCs and further hydrolysis occurs to give 3-indolemethanol, 3-indoleacetonitrile and 3,3′-diindolylmethane and subsequently condenses into dimers, trimers or tetramers (Holst and Williamson 2004; Gilbert and Senyuva 2008).

Fig. 4.2
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Main hydrolysis mechanism of glucosinolates (Song et al. 2005)

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Sinigrin is one of the most common mustard oil GLSs found in many cruciferous species and a few other plant families. When plant tissues are damaged, this glucoside is hydrolyzed to release allyl thiocyanate (ATC), a volatile mustard oil (Erickson and Feeny 1974) (Fig. 4.3). Sinalbin is the predominant GLS of yellow mustard and yields mainly nonvolatile 4-hydroxybenzyl ITC responsible for the hot mouthfeel within the hydrolysis by myrosinase (Choubdar et al. 2010). Glucobrassicin and glucoiberin are found in all types of B. oleracea, although B. oleracea can contain a diversity of GLSs (Cartea and Velasco 2008) (Fig. 4.3).

Fig. 4.3
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The chemical structures of main glucosinolates and their hydrolysis products

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ITCs are the main hydrolysis products of GLSs. Figure 4.3 shows the chemical structures of main ITCs formed by the hydrolysis of GLSs. Cabbage species contain 4–146 ppm allyl isothiocyanate (AITC), 0–2.8 ppm and 1–6 ppm phenylethyl isothiocyanate (PEITC). The pulps and seeds of ripe papaya fruit contain 4 and 2,910 ppm benzyl isothiocyanate (BITC), respectively (Van Etten et al. 1976; Whitmore and Naidu 2000). Sulforaphane (SFN – generally known as a major ITC converted from glucoraphenine in radish) contents in Baemuchae and radish sprouts were found to be 172.6  ±  11.23 and 131.3  ±  4.08 l μg/g, respectively (Lim et al. 2009). BITC is produced by the hydrolysis of glucotropaeolin and the most abundant ITC found naturally in several plants such as Carica papaya L., Indian cress (Tropaeolum majus L.) and garden cress (Lepidium sativum L.) (Troncoso-Rojas et al. 2005) (Fig. 4.3).

Amounts of GLSs and ITCs in Brassica vegetables may be influenced from various storage, processing and cooking conditions. Storage at ambient temperature or in cold conditions does not cause significant loss of GLSs while finely shredded vegetables may be exposed to a remarkable decline in GLS levels. Cooking by steaming, microwaving and stir-fry does not result in significant loss of GLSs whereas boiling shows significant losses by leaching into cooking water (Song and Thornalley 2007).

4.3.2 Antimicrobial Properties of Glucosinolates and Isothiocyanates

The biological properties of cruciferous plants such as cabbage, broccoli, cauliflower, Brussels sprouts and kale, have mainly been associated with GLSs and their breakdown products such as ITCs, nitriles, thiocyanates and other indole compounds. First of all, GLSs are the defensive compounds of these plants against herbivores, insects and other harmful organisms (Fahey et al. 2001). Over the past 30 years, a number of secondary metabolites including GLSs, and specifically their breakdown products has been considered as important bioactive tools (i.e. anticarcinogenic, antioxidant) in the diet and therapy (Aires et al. 2009). Moreover, the pesticidal activity of crucifers primarily depends on the breakdown products, while intact GLSs have generally less biological activity (Vaughn 1999). Volatile ITCs are considered as the major inhibitor allelochemicals of Brassica plants. For example, methyl isothiocyanate (MITC) can inhibit the growth of various weeds such as pigweed, dandelion and crabgrass (Whitmore and Naidu 2000). AITC, ATC, allyl isocyanate (AIC) and allyl cyanide (AC) are all considered as potent fumigant materials against house fly (Tsao et al. 2002). On the other hand, in view of their antimicrobial activity, it is well recognized that GLSs and their breakdown products exhibit inhibitory effects against a wide spectrum of bacteria and fungi. Generally, GLSs are not themselves active; the hydrolysis products are the main antimicrobial agents. Moreover, chemical structure influences biological activities, particularly antimicrobial activity. For instance, aromatic ITCs are more effective than aliphatic ITCs (Aires et al. 2009).

4.3.2.1 Antibacterial Activity

Cruciferous compounds that possess inhibitory effects against spoilage and pathogenic bacteria as well as intestinal microbiota have been well studied in recent years. Table 4.5 summarizes the antibacterial activity of GLSs and their hydrolysis products, primarily ITCs. Previous studies showed that Gram-negative bacteria were more susceptible than Gram-positive against ITCs (Isshiki et al. 1992; Lin et al. 2000). Several of the antibacterial actions of ITCs have been reported. AITC alters protein structures and thereby inhibits microbial growth (Kawakishi and Kaneko 1987). Lin et al. (2000) observed the antibacterial mechanism of AITC against S. Montevideo, E. coli O157:H7 and L. monocytogenes Scott A. They showed that AITC was effective in both the exponential and stationary phases of the bacterial growth. AITC affected cell membranes causing leakage of cellular metabolites and increased β-galactosidase activity of bacteria. Delaquis and Mazza (1995) reported that AITC might damage the essential intracellular enzymes of microorganisms through oxidative cleavage of disulfide bonds.

Table 4.5 Antibacterial activity of GLSs and their derivatives
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SFN was found to be effective in the inhibition of Helicobacter pylori, the cause of human gastritis and stomach cancer (Fahey et al. 2002; Haristoy et al. 2003, 2005; Yanaka et al. 2009). SFN was also reported as both bacteriostatic and bactericidal against all strains of bacteria. Broccoli sprouts are relatively rich sources of ITCs with a content of 20–50 times more precursor GLSs than mature broccoli. Broccoli sprouts were suggested as a potential digestive eradicator of H. pylori from the human gastrointestinal system (Galan et al. 2004).

Sinigrin, a thioglucoside of cruciferous plants and its hydrolysis products AITC, AC, ATC and 1-cyano-2,3-epithiopropane (CETP) were tested for their MIC in broth against 9 species of bacteria including the strains of E. coli, Pseudomonas fluorescens, Aeromonas hydrophila, S. aureus, B. subtilis, Pediococcus pentosaceus, Leu. mesenteroides, Lb. brevis and Lb. plantarum (Shofran et al. 1998). Sinigrin, AC and CETP were found to be ineffective in the inhibition of any of the bacteria. ATC showed an inhibitory effect against only E. coli, Pseudomonas fluorescens and S. aureus. E. coli was the most sensitive pathogen to AITC with the MIC value of 100 ppm. In this study, the effect of pH on the antimicrobial activity of AITC was also tested and the MIC values of AITC varied from 60 to 140 ppm against E. coli and 120 to 220 ppm for S. aureus, respectively. Kyung and Fleming (1997) tested the susceptibility of 15 different bacterial strains to sinigrin and some other sulfur compounds. As same in the previous study, they found sinigrin to be inactive against the bacteria while the MIC values of AITC varied from 50 to 500 ppm.

The balance of intestinal microflora is of importance because it helps to maintain human health, contributes to pathogen resistance and interacts with the host immune system (Lahtinen et al. 2009). Several studies have been conducted on the effect of GLSs and derivatives on intestinal microflora. Kim and Lee (2009) reported that PEITC isolated from Sinapis alba L. (white mustard) strongly inhibited the growth of C. difficile and C. perfringens at 1 mg/disc and moderately inhibited the growth of E. coli at a dose of 2 mg/disc in vitro while it did not exhibit any inhibitory effect on the growth of beneficial microbiota including bifidobacteria and lactobacilli. Aires et al. (2009) showed in vitro antibacterial activity of GLS hydrolysis products and specifically SFN and BITC against Gram-positive and Gram-negative pathogenic bacteria isolated from the human intestinal tract. On the other hand, several bacterial strains associated with GLS hydrolysis and belonging to E. coli, Bifidobacterium longum, Enterobacter cloacae, Bacteroides thetaiotaomicron and Bacteroides vulgatus species have been identified in the intestinal flora. These strains degrade GLS during 24–48 h cultivation and cause a decline in medium pH from 7.1 to 5.2 (Traka and Mithen 2009).

Ogawa et al. (2000) investigated the combined effects of hydrostatic pressure, temperature and addition of AITC on the inactivation of different strains of E. coli in vitro. They found that the antibacterial effects of pressurization with the addition of AITC at 4°C and 40°C were greater than at 20°C and all bacteria tested were effectively killed at 200 or 250 MPa with 10–80 μg/mL of AITC.

The favorableness of GLSs and hydrolysis products for extending the shelf-life of foods, especially meat products, has been investigated. Muthukumarasamy et al. (2003) reported that AITC (about 1,300 ppm) completely eliminated E. coli O157:H7 in low inoculated ground beef and reduced the viability >4.5 log cfu/g at high inoculated beef sample by the end of the storage period for 25 days at 4°C. Nadarajah et al. (2005) also obtained similar results in ground beef patties. In another study, Chacon et al. (2006) used microencapsulated AITC to eliminate E. coli O157:H7 and total aerobic bacteria (TAB) in finely chopped beef samples. AITC levels lower than 1,000 ppm were ineffective in reducing E. coli O157:H7 numbers while both low and high levels of inoculated E. coli O157:H7 were completely eliminated by 4,980 ppm AITC addition after 15 and 18 days of storage, respectively. 4,980 ppm AITC kept APC levels <3 log cfu/g during 18 days of storage. Shin et al. (2010) investigated the efficiency of the AITC-MAP (modified atmosphere packaging) system to control the growth of L. monocytogenes and S. Typhimurium on raw chicken breast during refrigerated storage for up to 21 days. Both release rates of 0.6 and 1.2 μg/h plus MAP systems significantly reduced the population of S. Typhimurium whereas L. monocytogenes was weakly inhibited at the lower release rate of AITC.

Fresh fruit and vegetables are at the risk of outbreaks of foodborne illnesses resulting from the contamination of pathogens during pre- and post-harvest stages. ITCs have been investigated as natural antimicrobial agents to sanitize fresh produce. Obadiat and Frank (2009a) compared the effectiveness of AITC, carvacrol and cinnamaldehyde in the vapor phase to eliminate strain cocktails of Salmonella and E. coli O157:H7 from sliced and whole tomatoes. AITC exhibited the highest antimicrobial activity and lowest level of AITC (8.3 μL/liter of air) inactivated Salmonella on sliced tomatoes by 1.0 and 3.5 log at 4°C and 10°C, respectively, in 10 days and by 2.8 log at 25°C in 10 h. AITC also reduced the E. coli O157:H7 level on sliced tomatoes by 3.0 log at 4°C and 10°C in 10 days, but there was no inactivation at 25°C in 10 h. The same researchers also tested those antimicrobial compounds in vapor phase on the intact and damaged portions of lettuce and spinach leaves against E. coli O157:H7 (Obadiat and Frank 2009b). On intact lettuce surface, 4 μL/liter of air AITC inactivated >4 log of E. coli O157:H7 at 0°C and 4°C in 4 days and at 10°C in 2 days. Pathogen inactivation on spinach surface was lower than on lettuce by 1 log. Higher concentrations of AITC were required when the surfaces were damaged by cutting. Lin et al. (2000) showed that lettuce inoculated with E. coli O157:H7 and S. Montevideo (each 104 cfu/g) showed no detectable bacterial counts after exposure to 400 μL of AITC and MITC for 24 and 48 h, respectively. L. monocytogenes was reduced to undetectable levels after the treatment with 400 μL of MITC for 2 days whereas 400 μL of MITC failed to achieve the same bactericidal effect.

4.3.2.2 Antifungal Activity

Although cruciferous plants produce inducible chemical defensive systems, they may be exposed to infection by a wide range of fungi. It was demonstrated that GLSs and their breakdown products, the major defensive compounds of crucifers, exhibit antimicrobial activity against fungal pathogens both in vitro and in planta (Sellam et al. 2007). The inhibitory effects of GLSs and their derivatives against various fungal species are shown in Table 4.6.

Table 4.6 Antifungal activity of GLSs and their derivatives
Full size table

Postharvest fungal growth becomes a threat for many crops during their storage under unfavorable conditions and causes considerable economic losses worldwide. The antifungal effects of vapor-phase ITCs have been demonstrated in many studies. Mari et al. (2002) reported that the best control of blue mold (P. expansum) on pears was obtained by exposing fruits in a 5 mg/L AITC-enriched atmosphere for 24 h. Wu et al. (2011) tested the potential use of AITC and EITC as fumigants in in vitro and in vivo trials to determine their effects on P. expansum Link and Botrytis cinerea Persl. infection in apples. A 3:1 ratio of AITC:EITC showed the best inhibitory activity on in vitro spore germination of P. expansum and B. cinerea. In in vivo trials on artificially infected apples, AITC, EITC and their combination reduced the incidence by more than 85% after 3–4 days of apple incubation at 20°C. Kurt et al. (2011) investigated in vitro and in vivo antifungal activity of synthetic pure ITCs against Sclerotinia sclerotiorum, a necrotrophic pathogen causing Sclerotinia stem and root rot of tomato and other economically important crops. MITC, AITC and BITC completely inhibited mycelial growth of the mold in the volatile phase. Butyl ITC (BUITC) and BITC reduced the apothecial production of S. sclerotiorum at their highest concentrations. In in vivo assay, AITC and PEITC reduced disease incidence by 76.7% and 70%, respectively.

Goncalves et al. (2009) investigated the antifungal efficiency of AITC sachets in cottage cheese preservation. The sachets produced by the incorporation of AITC were attached to the inner surface of the lids of cups containing cheese and the cups were stored for 35 days at 5  ±  2°C and 10  ±  2°C. After 7 days, the AITC sachets were found to be effective in reducing yeast and mold counts.

4.4 Conclusion

Organosulfur compounds in intact vegetables of the Allium and Brassica spp. and particularly their hydrolysis products are considered as key compounds of these vegetables. They give bioactive properties to these vegetables such as antimicrobial, anticarcinogenic, antitumor etc. while also giving them their pungent flavor and odor. Garlic is the most studied Allium vegetable as regards the antimicrobial activity since it is the richest source of antimicrobial organosulfur compounds among the Allium ssp. whereas all species of crucifers incorporate organosulfur materials. Allicin and other thiosulfinates such as DAS, DADS, DATS and ajoene present in Allium plants are the principal organosulfur compounds to exhibit antimicrobial activity. In the Brassica family, GLSs are not generally active themselves; their hydrolysis products are the main antimicrobial agents. Volatile ITCs like AITC, ATC, MITC and SFN have been shown to be the most effective antimicrobial compounds formed by the hydrolysis of GLSs in cruciferous plants.

Overall, it can be concluded that organosulfur compounds have the importance not only for the plant but also in the food industry in virtue of their health benefits. These compounds as well as various extracts of edible parts of organosulfur-source plants might be used in food product formulations for health and/or antimicrobial purposes. Although specific flavors/odors of these materials adversely affect organoleptic properties of most foods used, they might be considered as convenient ingredients of especially some meat and dairy products. Furthermore, several studies should be focused on preventing the come into forefront of the negative flavor/odors of them in foods when they are consumed.