alliin

ANTIMICROBIAL ACTIVITIES OF SULFUR COMPOUNDS DERIVED FROM S-ALK(EN)YL-L-CYSTEINE

SULFOXIDES IN ALLIUM AND BRASSICA

K. H. Kyung1 and Y. C. Lee2

1Dept. of Food Sci., Sejong Univ., Seoul 143-747, Korea

2Dept. of Food Sci. & Technol., Chung-Ang Univ., Ansung 456-756, Korea

ABSTRACT

Allium and Brassica vegetables have long been known for their antimicrobial activity against various

microorganisms, including Gram-positive and Gram-negative bacteria and fungi. Most of microorganisms

tested were sensitive to extracts of the Allium and Brassica vegetables and the degree of sensitivity

varied depending on the strain under study and test conditions. Among the vegetables, garlic showed

the most potent activity, followed by onion. Brassica including cabbage showed the least potent activity.

The principal antimicrobial compounds of Allium and Brassica have been elucidated as allicin (S-allyl-L-

propenethiosulfinate) and methyl methanethiosulfinate, respectively. Both compounds belong to the

same chemical group, thiosulfinate, generated from S-allyl and S-methyl derivatives of L-cysteine

sulfoxide, respectively, existing in Allium and Brassica as major non-protein sulfur containing amino

acids. There have been only few applications of garlic as a natural food preservative, in spite of

numerous studies on antimcirobial activity of the vegetables. Relative instability of the antimicrobial

compounds and strong odor of their mother plants seem to limit the use of them as a practical food

preservative.

KEY WORDS: Antimicrobial activity, S-alk(en)yl-L-cysteine sulfoxide, garlic, cabbage, Allium and Brassica

INTRODUCTION

The antimicrobial activities of plant extracts, especially from Allium and Brassica have been recognized

for many years, since Walton et al. (1) and Sherman and Hodge (2) scientifically demonstrated the

presence of antimicrobial activity of garlic (Allium sativum) and cabbage (Brassica oleracae),

respectively. The major antimicrobial activity of Allium (3,4) and Brassica (5) vegetables is proven to be

due to volatile sulfur compounds derived from S-alk(en)yl-L-cysteine sulfoxide, as well as glucosinolates

in case of Brassica (6-8). The presence of S-alkenyl-L-cysteine sulfoxides is confined essentially to two

families; the Cruciferae and the Lilliaceae where they are particularly associated with Allium and Brassica

(9).

The antimicrobial activity of garlic has been the most studied area for natural antimicrobials. Cavallito

and Bailey (3) succeeded in isolating allicin (allyl 2-propenethiosulfinate) which is absent in intact garlic,

but generated from its precursor, alliin (S-allyl-L-cysteine sulfoxide), through enzymatic hydrolysis when

the tissue of garlic is disrupted (3, 4, 10). Although relatively fewer research has been conducted on the

antimicrobial activity of cabbage compared with garlic, the presence of antimicrobial activity of Brassica

including cabbage, has been confirmed (5, 11-14) following the initial demonstration of the activity in

1936 by Sherman and Hodge (2). S-Methyl-L-cysteine sulfoxide (SMCSO), a non-protein sulfur-containing

amino acid in Brassica (15-17), is structurally similar to alliin, another example of non-protein amino acid

commonly found in Allium including garlic. SMCSO and alliin are methyl and allyl derivatives of L-cysteine

sulfoxide, respectively. The two compounds generate methyl methanethiosulfinate (MMTSO) and allicin,

which are principal antimicrobial compounds of Brassica and Allium, respectively, as a result of enzyme

reaction when the tissue of vegetables is disrupted. MMTSO and allicin have similar chemical structure,

and are methyl and allyl derivatives of thiosulfinate. Some other genera among Cruciferae, Synapis (S.

alba L.; mustard), Raphanus (R. sativus; radish), Cherianthus (C. cheiri; wallflower), and Capsell (C.

bursa-pastoris L.; shepherd's purse) also contain SMCSO (15). Although Allium and Brassica vegetables

have been reported to have other biological activities, such as cancer-preventive (17, 18, 20, 21), anti-

ulcerative (22), serum lipid-lowering (23), antiviral (24), antithrombotic (25), hemolytic anaemia-causing

(26, 27) and enzyme inhibitory (28-32) activities, this article reviews the chemistry of S-alk(en)yl-L-

cysteine-sulfoxides of Allium and Brassica and the antimicrobial activity of sulfur compounds derived

from them.

ANTIMICROBIAL ACTIVITY OF EXTRACTS OF ALLIUM AND BRASSICA VEGETABLES

Antimicrobial activity of garlic extract has been recognized for many years. It was reported that 1-2%

garlic extract inhibited microbial growth, and higher concentrations were germicidal. Dababneh and Al-

Delaimy (33) reported that 1% garlic extract inhibited Staphylococcus aureus. Karaioannoglou et al. (34)

indicated that garlic extract >1% in culture media was inhibitory to Lactobacillus plantarum, and >2%

was bactericidal. Mantis et al. (35) showed that 2% garlic extract in culture media had growth inhibitory

effects against S. aureus. Extracts of <1% were non-inhibitory and those of >5% were germicidal. Garlic

extract at 1% was strongly inhibitory against yeasts (36). Salmonella typhimurium (37), Bacillus cereus

(38), Clostridium botulinum (39), C. perfringens (40), Candida utilis (24, 41) and many other bacteria (24,

42, 43) and fungi (24, 44, 45) have been inhibited by garlic extract. S. aureus, Escherichia coli, Proteus

mirabilis and Pseudomonas aeruginosa which are multiple resistant to antibiotics including penicillin,

streptomycin, doxycilline and cephalexin were inhibited by garlic extract (46). A multiple resistant

Klebsiella sp. was not inhibited by garlic extract. hin et al. (47) evaluated the antimicrobial activity of

garlic juice powder dehydrated by different methods. Freeze-dried garlic juice powder showed high

inhibitory effect on Gram positive bacteria (Bacillus subtilis, S. aureus and Streptococcus mutans) having

0.3 2.0% minimum inhibitory concentrations (MIC). But spray-dried garlic juice powder did not have

inhibitory effect on Gram positive bacteria, except B. subtilis. Garlic juice powder processed with both

hydration methods did not show any difference in inhibitory effects on E. coli and E. coli O 157:H7 with

1.0 2.0%. The antimicrobial activity of onion is relatively weaker than that of garlic and an area of fewer

research. While 1-4% garlic extract completely inhibited E. coli, S. typhosa, Shigella dysenterie and S.

aureus, 4% onion extract completely inhibited the growth of only S. dysenterie and S. aureus. (42).

However, in a test with non-growing S. typhimurium, freshly reconstituted dehydrated onion showed a

stronger bactericidal activity compared with freshly reconstituted dehydrated garlic (37). The presence

of the antimicrobial activity in cabbage is definite (5, 11-13, 48, 49), but much less potent compared with

those of garlic and onion. The antimicrobial activity of cabbage was reported to be destroyed by heating

(2, 11) as is the case with garlic. Yildiz and Westhoff (49), however, reported that heating caused

cabbage extract to become inhibitory. Kyung and Fleming (5) demonstrated inhibitory activity in fresh,

unheated juice of several cultivars of cabbage. Heating the cabbage before juice extraction prevented

formation of the inhibitor(s) in some cultivars, but not others. The growth inhibitory substance of fresh

cabbage was suggested to be carbohydrate in nature and of low molecular weight (13, 14). The identity

of the inhibitory compound has recently been elucidated as MMTSO generated from SMCSO in cabbage

(48).

S-ALK(EN)YL-L-CYSTEINE SULFOXIDES; THE PRECURSORS

Brassica is taxonomically far apart from the genus Allium which includes garlic and onion. However, they

have in common, that S-alk(en)yl-L-cysteine sulfoxides as major non-protein amino acids (50). S-

Alk(en)yl-L-cysteine sulfoxides were suggested to be important in sulfur metabolism, acting as a soluble

pool for organic sulfur (51). The amounts vary widely depending on plant species and on different parts

of the plants (16, 48). The general structure of the S-alk(en)yl-L-cysteine sulfoxides is shown in Fig. 1.

Five S-alk(en)yl-L-cysteine sulfoxides differing in R-side group have been described; four in Allium and

one in Brassica. Variety of R-side group of S-alk(en)yl-L-cysteine sulfoxides together with some source

plants and their amounts are given in Table 1.

Figure 1. Enzymatic cleavage of S-alk(en)yl-L-cysteine sulfoxides

Rundqvist (52) made the first effort to isolate the basic principle from which volatile sulfur compounds

are generated in garlic. Owing to the fact that the precipitation he obtained contained considerable

quantities of carbohydrate, he thought that the compound he was seeking was a glucoside which he

named "alliin". The term alliin has since been used (4). Later, pure alliin has been isolated by Stoll and

Seebeck (53) and identified as an amino acid. Alliin was chemically synthesized in 1951 by the same

group(54). SMCSO was initially isolated from cruciferous vegetables in two separate laboratories at the

approximately same time (15, 16). They analyzed SMCSO in cruciferous vegetables among which

Brassica contained it invariably, while other genera in Cruciferae showed mixed results.

Table 1. Kinds and amounts of S-alk(en)yl-L-cysteine sulfoxides in Allium and Brassica*.

Genus Alk(en)yl group Plant sources and amounts of S-alk(en)yl-L-cysteine sulfoxides(mg/kg) in parentheses

Allium methyl garlic, elephant garlic, wild garlic, onion [200 (9)], leek, scallion, shallot, Chinese chive

propyl onion [50 (9)], leek, scallion, shallot, chive

propenyl garlic, elephant garlic, wild garlic, onion [40* (9), leek, scallion, shallot, Chinese chive, chive

allyl garlic [900-11500 (54)], elephant garlic, wild garlic, Chinese chive

Brassica methyl

cabbage [185-2218 (15-17, 47, 60)], kale [1310-1380 (26)], turnip[43-202 (16)], swede, Chinese

cabbage [396-786 (16, 50)], cauli-flower [2380 (16)], kohlrabi [558-1069 (16)], broccoli [343-

2406(16, 61)]

From Block et al. (10).

When S-alk(en)yl-L-cysteine sulfoxides of garlic were analyzed by reverse-phase HPLC, S-methyl-

and S-allyl-L-cysteine sulfoxides were the only compounds which were identified with certainty (55).

Other sulfur amino acids were not found. Ziegler & Sticher (55) opined that the minor derivatives of

L-cysteine sulfoxide were absent or were below detection limits under the chromatographic

conditions. From the GC analytical results of thiosulfinates of crushed garlic, Freeman and

Whenham (56) showed that the L-cysteine sulfoxide fraction of garlic consists of 85% alliin along

with 2% S-propyl cysteine sulfoxide and 13% SMCSO. Therefore it can be safely assumed that the

concentration of minor L-cysteine sulfoxides could be too low to be detected by HPLC by Ziegler and

Sticher (55). Block et al. (10) reported HPLC analytical results of thiosulfinates of garlic and showed

that their ratios of allyl/methyl were similar to that of Freeman and Whenham (56). The allyl/methyl

ratio of garlic (10) ranged from 94:2 (New York grown) to 80:16 (store bought garlic) to 74:24

(Indian garlic grown at 32℃). Occurrence of ethyl (57, 58), and propyl (56) and butyl (58)

derivatives in garlic and allyl derivatives in onion (59) was suggested, but has never been positively

confirmed since (55). Allyl groups are absent in onion, scallion, shallot, leek, and chive and propyl

groups are absent in garlic, elephant garlic, wild garlic and Chinese chive (10; Table 1), judging from

various thiosulfinates isolated from Allium plants (Table 2).

Table 2. Thiosulfinates from the extracts of Allium and Brassica

Thiosulfinates Allium and Brassica vegetables

AllSS(O)propenyl-(E): garlic, elephant garlic

AllS(O)Spropenyl-(Z,E): garlic, elephant garlic, wild garlic

AllS(O)Sall: garlic, elephant garlic, wild garlic

n-pro-SS(O)propenyl-(E): onion, shallot, scallion, leek, chive

n-pro-S(O)S-propenyl-(Z,E): onion, shallot, scallion, leek, chive

n-pro-S(O)S-pro-n: onion, shallot, scallion, leek, chive

Me-S(O)S-Me: onion, shallot, scallion, leek, garlic, all Brassica

All-S(O)S-Me: garlic, elephant garlic, wild garlic, Chinese chive

Me-S(O)S-propenyl-(Z,E): onion, shallot, scallion, leek, garlic, elephant garlic, wild garlic, Chinese chive, chive

Me-SS(O)-pr: onion, shallot, scallion, leek, chive

Me-S(O)S-pr: onion, shallot, scallion, leek, chive

All-SS(O)-Me: garlic, elephant garlic, wild garlic, Chinese chive

Me-S(O)S-Me: onion, shallot, scallion, leek, garlic, elephant garlic, wild garlic, Chinese chive, chive

From Block et al. (1992). For Brassica, Marks et al. 1992

The concentration of the minor unsymmetrical thiosulfinates, which possess 1-propenyl group, varied

with the age and storage condition of garlic following harvesting. 1-Propenyl levels increased upon

refrigeration (10). All the Brassica vegetables including kale, swede, turnip, cabbage, Chinese cabbage,

cauliflower, kohlrabi and broccoli contains only S-methyl- derivative of L-cysteine sulfoxide (16; Table 1).

Onion contained γ-L-glutamyl-(+)-S-propenyl-L-cysteine sulfoxide and cycloalliin in addition to propyl,

methyl, propenyl derivatives of L-cysteine sulfoxide. Cycloalliin could be an artefact during elution from

cationic exchange resin, since S-propenyl-L-cysteine sulfoxide spontaneously cyclizes at pH> 7 (9).

Growth condition is known to modify the profile of S-alkenyl-L-cysteine sulfoxides of Allium and Brassica.

Some garlic varieties grown in cooler climates show a higher allyl to methyl ratio than garlic grown in

warmer climates (10). For example, NY grown garlic revealed low levels of (+)S-methyl-L-cysteine

sulfoxide (0.08-0.25mg/g of garlic compared to 1-1.6mg/g in California garlic) with normal levels of other

derivatives (10). Block et al.(10) suggested that garlic grown in colder climates are subject to stress and

that this stress causes reduced synthesis of SMCSO. Cruciferous vegetable species like kale, however,

has been known to accumulate more SMCSO when grown during periods of frost (60, 61).

ENZYMES AND THEIR REACTION PRODUCTS

Enzymes

In Allium plants, Alliin and alliinase are located in different compartments (63); the substrates in the

cytoplasm and enzyme in the vacuole. The reaction of alliinase (Fig. 1) takes place extremely rapidly, a

fact which is in agreement with the instantaneous appearance of the typical odor on crushing garlic.

More than 80% of the alliin is split by the enzyme within 2 minutes (4). The molecular weight of the

enzymes in onion and broccoli are quite similar (50, 64) and the enzyme appears to consist of a trimer

with a subunit molecular weight of approximately 50,000. All of these enzymes are glycoproteins

consisting of 5.8-6.0% carbohydrate by weight (50, 65). Cabbage leaves (66) and broccoli (50) have two

cystine lyases with somewhat different specificities. Alliinase of Allium and cystine lyase of Brassica act

on common substrate, S-alk(en)yl-L-cysteine sulfoxide (50) and Hamamoto & Mazelis (50) proposed a

new name L-cysteine sulfoxide lyase. Optimum pH range of onion and broccoli enzymes is 8.0-8.6 (50,

69), and of garlic enzyme is 5-8 (4, 50). Action of alliinase on the mixture of sulfoxides forms

allyl/methane, methyl/methane and other mixed thiosulfinates in addition to allicin (56). When studied

with synthetic alliin, (-)-S-allyl-L-cysteine sulfoxide was disintegrated more slowly compared to its (+)

isomer (54). In addition to methyl and propenyl derivatives of L-cysteine sulfoxide, as flavor precursors of

onion, γ-L-glutamyl-L-cysteine sulfoxide is present and is thus insusceptable to the action of the C-S lyase

(68). Since γ-L-glutamyl transpeptidase catalyzes the hydrolysis (Fig. 2) as well as glutamyl transfer, the

addition of this enzyme to onion liberate the flavor precursor, which in turn destroyed by C-S lyase (67,

68). γ-L-Glutamyl transpeptidase is found in sprouted onion. In addition to already-described alliinase

reaction common to Allium, alliinase of onion and leek, but not of garlic, has been reported to have an

activity of generating lachrymatory compound, thiopropanal S-oxide (70). Marks,et al. (17) reported the

formation of MMTSO, methyl methanethiosulfonate (MMTSO2) and dimethyl trisulfide (DMTS) in a model

system composed of SMCSO and partially purified cabbage C-S lyase. Bacteria are also known to have

enzyme(s) that catalyzes the hydrolysis of S-alk(en)yl-L-cysteine sulfoxides. Stoll and Seebeck (4)

reported the development of an odor of garlic, when E. coli was cultured in synthetic nutrient medium

with 0.2% alliin. Bacterial enzymes of Pseudomonas cruciviae (71) and Bacillus subtilis (72) catalyzed the

stoichiometric conversion of SMCSO to MMTSO, pyruvate and ammonia and required pyridoxal phosphate

as a coenzyme (71), as other alliinases and C-S lyases did. SMCSO is converted to dimethyl disulfide

(DMDS) by unspecified rumen microorganisms and cause hemolytic anaemia in cattle and sheep, known

as kale poisoning (27).

Figure 2. Liberation of L-cysteine sulfoxide from γ-glutamylpeptide in onion

Products and Their Chemistry

Cavallito and Bailey (3) succeeded in isolating a water-soluble antimicrobial substance from an aqueous

ethanolic extract of garlic by steam distillation under reduced pressure and named it "allicin", and the

Cavallito group (73) correctly assigned the structure as allyl-S(O)-S-allyl. Block et al. (10) made an HPLC

analysis of thiosulfinates in garlic and reported that the major thiosulfinates from garlic and elephant

garlic was allicin. Other kinds of thiosulfinates identified from the extracts of Allium species are as in

Table 2. Sinha et al. (59) reported a finding of allicin from the supercritical CO2 extract of onion. The

discrepancy was explained by Block et al. (10) as an artifact due to very high injection port temperature

employed by Sinha et al. (59). GC analysis employing high temperature may induce chemical

modification of less stable compounds during the procedure of chromatography. Block (74) urges to use

nonthermal methods of analysis like HPLC when volatile compounds of garlic are of interest. Allicin is not

stable, even at 3℃, and it loses its activity within 14 days (4). Brodnitz et al. (75) observed that allicin

underwent complete decomposition at 20℃ after 20hr resulting in DADS, diallyl trisulfide (DATS), diallyl

sulfide and sulfur dioxide. But allicin in garlic juice underwent complete decomposition at 40℃ after

144hr (76). The authors postulated that allicin was more stable in garlic juice than in pure state. The

particular instability of the allyl compound appears to be associated with the double bond (77).

Thiosulfinates are unstable toward alkalies, but are stable in dilute acids. The generation of MMTSO was

confirmed in a water extract of macerated Brussels sprouts which was the first evidence of MMTSO

generated enzymatically under natural conditions (17). Unless stored at dry ice temperature, MMTSO,

the primary breakdown product of SMCSO, has been shown to be degraded into volatile sulfur

compounds, including methyl methanethiosulfonate (MMTSO2) and dimethyl disulfide (DMDS) (78, 79;

Fig. 3).

Figure 3. Spontaneous disproportionation of methyl methanethiosulfinate

ANTIMICROBIAL COMPOUNDS DERIVED FROM S-ALK(EN)YL-L-CYSTEINE SULFOXIDES

Before the nature of the antimicrobial substance of garlic is known as allicin by Cavallito and Bailey (3),

some workers ascribed the garlic antimicrobial activity to other compounds including acrolein and

related aldehydes (80). However, antimicrobial activity is not confined to allicin alone, but is a general

property of alk(en)yl esters of alka(e)nethiosulfinate (4). The action of allicin, the first known natural

thiosulfinate is considerably more bacteriostatic than bactericidal (3). It is about equally effective against

Gram-positive and Gram-negative bacteria. A few observations were made by Small et al. (77) relative to

the chemical structure and antimicrobial activity of the thiosulfinates. In general, it requires about the

same quantities of the lower molecular weight thiosulfinates to inhibit Gram-positive as compared with

Gram-negative bacteria, but as the carbon chain length increases, activity against Gram-negative

organisms decreases, while that against Gram positive bacteria increases. Branching results in lowered

activity. Small et al. (81) compared the antimicrobial activity of thiosulfinate and thiosulfonate (synthetic

ethyl ethanethiosulfinate and ethyl ethanethiosulfonate, not found naturally). The two thiol esters were

of comparable antimicrobial activity, with thiosulfonate being slightly more effective against S. aureus

and Klebsiella pneumoniae.

Figure 4. Formation of ajoene from allicin (Block et al., 1984)

Ajoene, a derivative of allicin (Fig. 4), originally reported for its potent antithrombotic activity (25)

exhibited a strong antifungal activity toward Aspergillus niger and C. albicans at <20μg/ml (82). Yoshida

et al.(82) concluded that ajoene had stronger antifungal activity than allicin and that it damages the cell

wall of fungi and thus maintained that growth inhibitory activity of ajoene toward bacteria was not

expected except for a specific strain. Later Naganawa et al. (83) showed a different result concerning

antimicrobial activity of ajoene. They reported that ajoene was strongly inhibitory against Gram-positive

bacteria and yeasts and had various degrees of inhibition against Gram-negative bacteria like E. coli and

P. aeruginosa. DADS, one of degradation products of allicin, was shown to possess antituberculosis

activity (84). MMTSO, the primary breakdown product of SMCSO in Brassica (17, 48), has been shown to

be antibacterial (48). MMTSO is not as potent as allicin. MMTSO decomposes on standing to give

principally MMTSO2, DMDS and dimethyl trisulfide (DMTS) (17, 85), thus decreasing antimicrobial activity

by approximately half, because MMTSO2 has comparable antimicrobial activity with MMTSO, but DMDS

has only slight growth inhibitory activity. Kyung and Fleming (86) tested antimicrobial activity of SMCSO

and its derivatives against 15 bacteria and 4 yeasts. SMCSO itself was uninhibitory while DMDS was only

slightly inhibitory, and DMTS was weakly inhibitory. MMTSO2 was as inhibitory as MMTSO but with

different inhibitory patterns. MMTSOO is also generated in autoclaved cabbage and SMCSO solution and

was shown to be antibacterial (87).

Cysteine inhibits antimicrobial activity of allicin, which may be reactivated by hydrogen peroxide (88).

Antimicrobial activity of garlic was known to be stabilized by hydrogen peroxide (89). However, it is not

in agreement with the report (24) that catalase positive bacteria were sensitive to garlic while less

sensitive bacteria, e.g., lactic acid bacteria, were catalase negative.

MODE OF ACTION

The principal antimicrobial compounds of Allium and Brassica are those belonging to a group known as

thiosulfinate. The antimicrobial activity of thiosulfinates has been explained as a general reaction

between thiosulfinates and -SH groups of essential cellular proteins (41, 73, 77, 81). Small et al. (77)

mentioned that -S(O)S- was responsible for the antimicrobial activity and that reacted readily with

cysteine to yield mixed disulfides. Fujiwara et al. (57) showed essentially the same reaction between

allicin and thiamine. The general reaction (Fig. 5), as proposed by Small et al. (77) can apply to where

thiosulfinates are involved and the reaction is believed to be the common mechanism of antimicrobial

activity of thiosulfinates. Since the proposal of the general inhibitory mechanism of thiosulfinates, some

workers (28-32) have reported specific target processes or enzymes of thiosulfinates. Ghannoum (30)

and Neuwirth et al., (31) reported that allicin inhibited lipid biosynthesis and RNA synthesis, respectively,

without pointing out target enzymes. Wills (28) reported that allicin inhibited the acticity of many -SH

enzymes. Among them most strongly inhibited were xanthine oxidase, succinic dehydrogenase and

triose phosphate dehydrogenase. He confirmed the results of Small et al. (77) that -S(O)-S- group was

essential for the inhibition of -SH enzymes, while -S-S-, -S- and -SO- groups were not effective. Focke et

al. (32) found that allicin inhibited the incorporation of acetate, but not of acetyl CoA or malonate, into

fatty acids and concluded that only acetyl CoA synthetase for the fatty acid synthesis was inhibited by

allicin. They explained that the inhibition of acetyl CoA synthetase by allicin was specific and non-

sulfhydryl effect.

Figure 5. Proposed reaction between thiosulfinates and SH group of cellular proteins

Ajoene is reported to be another potent antimcirobial compound. Yoshida et al. (82), maintaining that

ajoene was even more potent antifungal agent than allicin, assumed that ajoene may damage the cell

walls of fungi, thus not expecting significant antibacterial activity, except for Staphylococcus aureus.

Later, however, Naganawa et al. (83) showed a different result concerning antimicrobial activity of

ajoene. They reported that ajoene was strongly inhibitory against Gram-positive bacteria and yeasts and

had various degrees of inhibition of Gram-negative bacteria like E. coli and P. aeruginosa. Naganawa et

al. (83) postulated that the disulfide group in ajoene appears to be necessary for the antibacterial

activity, since reduction by cysteine abolished its antimicrobial activity. Ajoene does not possess

thiosulfinyl group (Fig. 4).

USES IN FOODS AS A PRESERVATIVE

Although antimicrobial activity of Allium and Brassica vegetables represents a promising area of

research, use of the vegetables as natural food preservatives has not been common. There is only one

known example of using garlic as a food preservative. When Al-Delaimy and Barakat (90) treated ground

camel meat with 5% or more of ground garlic, they could extend storage shelf-life of camel meat at any

given storage temperature. Fifteen percent or more of garlic were found to act as strong bactericides

since the initial microbial population of ground camel meat was completely destroyed and no further

growth of any type of microorganisms was observed. Such a high level of garlic added to foods would be

fine with some segments of population of the world, but not with the rest. A way to reduce the level of

garlic added to foods for extending the shelf-life could be low temperature storage of them. For example,

meat products with added garlic could be stored at the refrigerated temperature with extended shelf-life.

However, there are difficult problems to be solved before garlic products are used as natural food

additives, such as the strong garlic flavor and instability of functional compounds in garlic.

CONCLUSION

The antimicrobial activity of Allium and Brassica used as flavoring agents or food materials have been

recognized for many years. Among the vegetables, garlic has been studied most extensively. The

antimicrobial activity of Allium and Brassica is believed to be due to thiosulfinates enzymatically

generated from S-alk(en)yl-L-cysteine sulfoxides. The antimicrobial action of thiosulfinates and their

derivatives are effective due to -S(O)-S- group in the molecules, which readily react with -SH group of

essential proteins of microorganisms. Ajoene and MMTSO2 which are generated from allicin and MMTSO,

respectively, are also known to possess antimicrobial activity. Levels of garlic normally used as food-

flavoring materials may not be sufficient to obtain the desired preservative effects. Garlic showed an

acceptable preservative effect only when substantial levels were added to food, which may not be

acceptable by many people because of the strong flavor. The relative instability of the activity further

discourage the use of them as food preservatives. We have to acquire ways to enhance stability of

antimicrobial compounds and to decrease objectionable sulfurous odor before finding more use of those

vegetables possessing potent natural antimicrobial activities as a food preservative.

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Legends for Tables

Table 1. Kinds and amounts of S-alk(en)yl-L-cysteine sulfoxides in Allium and Brassica

Table 2. Thiosulfinates from the extracts of Allium and Brassica

Legends for Figures

Figure 1. Enzymatic cleavage of S-alk(en)yl-L-cysteine sulfoxides

Figure 2. Liberation of L-cysteine sulfoxide from gamma-glutamylpeptide in onion

Figure 3. Spontaneous disproportionation of methyl methanethiosulfinate

Figure 4. Formation of ajoene from allicin

Figure 5. Proposed reaction between thiosulfinates and SH group of cellular proteins