ANTIMICROBIAL ACTIVITIES OF SULFUR COMPOUNDS DERIVED FROM S-ALK(EN

Document Sample
ANTIMICROBIAL ACTIVITIES OF SULFUR COMPOUNDS DERIVED FROM S-ALK(EN Powered By Docstoc
					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


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


2
Dept. 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 CO 2 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.


REFERENCES
1 .Walton, L., Herbold, M. and Lindegren, C. C. (1936). Bactericidal effects of vapors from crushed garlic,

  Food Res. 1, 163-169.



2. Sherman, J. M. and Hodge, H. M. (1936). The bactericidal properties of certain plant juices, J. Bacteriol., 31,

96.


3. Cavallito, C. J. and Bailey, J. H. (1944). Allicin, the antibacterial principle of Allium sativum. I. Isolation,

physical properties and antimicrobial action, J. Amer. Chem. Soc., 66, 1950-1951.



4. Stoll, A. and Seebeck, E. (1951). Chemical investigation of alliin, the specific principle of garlic, Adv.

Enzymol., 11, 377-400.


5. Kyung, K. H. and Fleming, H. P. (1994). S-Methyl-L-cysteine sulfoxide as the precursor of methyl

methanethiosulfinate, the principal antibacterial compound in cabbage, J. Food Sci., 59(2), 350-355.


6. Virtanen, A.I. (1962). Some organic sulfur compounds in vegetables and fodder plants and their significance

in human nutrition, Angew. Chem. (Int. ed.), 6, 299-306.



7. Zsolnai, V. T. (1966). Die antimicrobielle Wirkung von Thiocyanaten und Isothiocyanaten. I. Mitteilung,

Arzneim. Forsch., 16, 870-876.


8. Delaquis, P. J. and Mazza, G. (1995). Antimicrobial properties of isothiocyanates in food preservation, Food

Technol., 49(11), 73-84.


9. Virtanen, A. J. (1965). Studies on organosulfur compounds and other labile substances in plants,

Phytochemistry, 4, 207-228.



10. Block, E., Naganathan, S., Putman, D. and Zhao, S.-H. (1992). Allium chemistry: HPLC analysis of

thiosulfinates from onion, garlic, wild garlic (Ramsons), leek, scallion, shallot, elephant (great-headed) garlic,

chive, and Chinese chive. Uniquely high allyl to methyl ratios in some garlic samples, J. Agric. Food Chem., 40,

2418-2430.


11. Pederson, C. S. and Fisher, P. (1944). The bactericidal action of cabbage and other vegetable juices, N.Y

State Agric. Exp. Sta. Bull., 273.


12. Little, J. E. and Graubaugh, K. K. (1946). Antibiotic activity of some crude plant juices, J. Bacteriol., 52,

587-591.
13. Liu, J. Y., Teraoka, T., Hosokawa, D. and Watanabe, M. (1986). Bacterial multiplication and antibacterial

activities in cabbage leaf tissue inoculated with pathogenic and nonpathogenic bacterium, Ann. Phytopath.

Soc.(Japan) 52, 669-674.



14. Dickerman, J. M. and Lieberman, S. (1952). Studies on the chemical nature of an antibiotic present in

water extract of cabbage, Food Res., 17, 438-441.


15. Synge, R. L. M. and Wood, J. C. (1956). (+)-(S-methyl-L-cysteine S-oxide) in cabbage, Biochem. J., 64,

252-259.



16. Morris, C. J. and Thompson, J. F. (1956). The identification of (+)S-methyl-L-cysteine sulfoxide in plants,

J. Am. Chem. Soc., 78, 1605-1608.


17. Marks, H. S., Hilson, J. A., Leichtweis, H. C. and Stoewsand, G. S. (1992). S-Methylcysteine sulfoxide in

Brassica vegetables and formation of methyl methanethiosulfinate from Brussels sprout, J. Agric. Food Chem.,

40, 2098-2101.


18. Marks, H. S. (1992). Isolation, identification, and genotoxic inhibitory efficacy of naturally occurring

organosulfur compounds present in Brassica vegetables, Cornell Univ., Doctoral Diss.


19. Marks, H. S., Leichtweis, H. C. and Stoesand, G. S. (1991). Analysis of a reported organosulfur,

carcinogen inhibitor; 1,2-dithiole-3-thione in cabagge, J. Agric. Food Chem., 39, 893-895.


20. Caragay, A. B. (1992). Cancer-preventive foods and ingredients, Food Technol., 46, 65-68.


21. Alberto-Puleo, M. (1983). Physiological effects of cabbage with reference to its potential as a dietary

cancer-inhibitor and its use in ancient medicine, J. Ethnopharmacol., 9, 261-272.



22. Cheney, G. (1950). Anti-peptic ulcer dietary factor (vitamin U) in the treatment of peptic ulcer, J. Am.

Dietet. Ass., 26, 668-672.


23. Augusti, K. T. and Mathew, P. T. (1974). Lipid lowering effect of allicin (diallyl disulphide-oxide) on long

term feeding to normal rats, Experientia, 30(5), 468-470.


24. Rees, L. P., Minney, S. F., Plummer, N. T., Slator, J. H. and Skyrme, D. A. (1993). A quantitative

assessment of the antimicrobial activity of garlic (Allium sativum), World J. Microbiol. Biotechnol., 9, 303-307.



25. Block, E., Ahmed, S., Jain, M.K., Creely, R. W., Apitz-Castro, R. and Cruz, M. R. (1984). (E,Z)-Ajoene: A

potent antithrombotic agent from garlic, J. Am. Chem. Soc., 106, 8295-8296.
26. Whittle, P. J., Smith, R. H. and McIntosh, A. (1976). Estimation of S-methylcysteine sulphoxide (kale

anaemia factor) and its distribution among Brassica forage and root crops, J. Sci. Food Agric., 27, 633-642.


27. Smith, R. H. (1980). Kale poisoning: The brassica anaemia factor, Veter. Record, 107, 12-15.


28. Wills, E. D. (1956). Enzyme inhibition by allicin, the active principle of garlic, Biochem. J., 63, 514-520.


29. Adetumbi, M., Javor, G. T. and Lau, B.H.S. (1986). Allium sativum (garlic) inhibits lipid synthesis in

Candida albicans, Antimicrob. Agents Chemother., 30, 499-501.


30. Ghannoum, M. A. (1988). Studies of the antimicrobial mode of action of Allium sativum (garlic)J. Gen.

Microbiol., 134, 2917-2924.


31. Feldberg, R. S., Chang, S. C., Kotik, A.N., Nadler, M., Neuwirth, Z., Sundstrom, D. C. and Thompson, N. H.

(1988). In vitro mechanism of inhibition of bacterial cell growth by allicin, Antimicrob. Agents Chemother. 32,

1763-1768.



32. Focke, M., Feld, A. and Lichtenthaler, H. K. (1990). Allicin, a naturally occurring antibiotic from garlic,

specifically inhibits acetyl-CoA synthetase, FEBS, 261(1), 106-108.


33. Dababneh, . F. A. and Al-Delaimy, K. S. (1984). Inhibition of Staphylococcus aureus by garlic extract,

Lebensm. Wissenschaft. Technol., 17(1), 29-31.


34. Karaioannoglou, P. G., Mantis, A. J. and Panetos, A. G. (1977). The effects of garlic extract on lactic acid

bacteria (Lactobacillus plantarum) in culture media, Lebensm. Wissenschaft. Technol., 10, 148-150.


35. Mantis, A. J., Karaioannoglou, P. G., Spanos, G. P. and Panetos, A. G. (1978). The effect of garlic extract

on food poisoning bacteria in culture media. I. Staphylococcus aureus, Lebensm. Wissenschaft. Technol., 11,

26-28.


36. Conner, D. E. and Beuchat, L. R. (1984). Effects of essential oils from plants on growth of food spoilage

yeasts, J. Food Sci., 49, 429-434.


37. Johnson, M. G. and Vaughn, R. H. (1969). Death of Salmonella typhimurium and Escherichia coli in the

presence of freshly reconstituted dehydrated garlic and onion, Appl. Microbiol., 17(6), 903-905.



38. Saleem, Z. M. and Al-Delaimy, K. S. (1982). Inhibition of Bacillus cereus by garlic extracts, J. Food Prot.,

45(11), 1007-1009.
39. DeWit, J. C., Notermans, S., Gorin, N. and Kampelmacher, E. H. (1979). Effects of garlic oil or onion oil on

toxin production by Clostridium botulinum in meat slurry, J. Food Prot., 42, 222-224.



40. Mantis, A. J., Koidis, P. A., Karaioannoglou, P. G. and Panetos, A. G. (1979). Effect of garlic extract on

food poisoning bacteria, Lebensm. Wissenschaft. Technol., 12, 230-232.


41. Barone, F. E. and Tansey, M. R. (1977). Isolation, purification, identification, synthesis, and kinetics of

activity of the anticandidal component of Allium sativum, and a hypothesis for its mode of action, Mycologia,

69, 793-825.



42. Al-Delaimy, K. S. and Ali, S. H. (1970). Antibacterial action of vegetable extracts on the growth of

pathogenic bacteria, J. Sci. Food Agric., 21, 110-112


43. Srivastava, K. C., Perera, A. D. and Saridakis, H. O. (1982). Bacteriostatic effects of garlic sap on Gram

negative pathogenic bacteria - an in vitro study, Lebensm. Wissenschaft. Technol., 15(2), 74-76.


44. Moore, G. S. and Atkins, R. D. (1977). The fungicidal and fungistatic effects of an aqueous garlic extract

on medically important yeast-like fungi, Mycologia, 69, 341-348.


45. Tansey, M. R. and Appleton, J. A. (1975). Inhibition of fungal growth by garlic extract, Mycologia, 67,

409-413.


46. Singh, K. V. and Shukula, N. P. (1984). Activity of multiple resistant bacteria of garlic (Allium sativum)

extract, Fitoterapia, 15(5), 313-315.


47. Shin, D.B., Kim, Y.S. and Lee, Y.C. (1999). Effect of dehydration methods on the antimicrobial activity of

garlic juice powder, 1999 IFT Annual Meeting, 98.



48. Kyung, K. H. and Fleming, H. P. (1994). Antibacterial activity of cabbage juice against lactic acid bacteria,

J. Food Sci., 59(1), 125-129.


49. Yildiz, F. and Westhoff, D. (1981). Associative growth of lactic acid bacteria in cabbage juice, J. Food Sci.,

46, 962-963.


50. Hamamoto, A. and Mazelis, M. (1986). The C-S lyases of higher plants. Isolation and properties of

homogeneous cystine lyase from broccoli, Plant Physiol., 80, 702-706.



51. Mae, T., Ohira, K. and Fujiwara, A. (1971). Fate of (+)S-methyl-L-cysteine sulfoxide in Chinese cabbage,

Brassica pekinensis RUPR, Plant Cell Physiol., 12, 1-11.
52. Rundqvist, C. (1909). Pharmacological incestigation of Allium bulbs, Pharmaceutiskt Notisblad, 18, 323-

333.



53. Stoll, A. and Seebeck, E. (1948). Uber Alliin, die genuine Muttersubstanz des Knoblauchols. 1. Mitteilung

uber Allium Substanzen, Helv. Chim Acta, 31, 189-210.


54. Stoll, A. and Seebeck, E. (1951). Die Synthese des naturlichen Alliins und seiner drei optisch aktiven

Isomeren. 5. Mitteilung uber Allium-Substanzen, Helv. Chim. Acta, 34, 481-487.



55. Ziegler, S. J. and Sticher, O. (1989). HPLC of S-alk(en)yl-L-cysteine derivatives in garlic including

quantitative determination of (+)S-allyl-L-cysteine sulfoxide (alliin), Planta Medica, 55, 372-377.


56. Freeman, G. G. and Whenham, R. J. (1975). The use of synthetic (+_)-S-1-propyl-L-cysteine sulphoxide

and of alliinase preparations in studies of flavour changes resulting from processing of onion ( Allium cepa L.),

J. Sci. Food Agric., 26, 1333-1346.


57. Fujiwara, M., Yoshimura, M., Tsuno, S. and Murakami, F. (1958). Allithiamine, a newly found derivative of

vitamin B1. IV. On the allicin homologues in the vegetables, J. Biochem. (Japan), 45, 141-149.


58. Horhammer, L., Wagner, H., Seitz, M. and Vejdelek, Z. J. (1968). Onion flavors and their analysis by gas

chromatography-mass spectrometry, Pharmazie, 23, 462-466.


59. Sinha, N. K., Guyer, D. E., Gage, D. A. and Lira, C. T. (1992). Supercritical carbon dioxide extraction of

onion flavors and their analysis by gas chromatography-mass spectrometry, J. Agric. Food Chem., 40, 842-

845.


60. Kunelius, H. T., Sanderson, J. B. and Narashimhalu, P. R. (1987). Effect of seeding date on yields and

quality of green forage crops, Can. J. Plant Sci., 67, 1045-1050.


61. Bradshaw, J. E. and Borzucki, R. (1982). Digestability, S-methyl-L-cysteine sulfoxide content and

thiocyante ion content of cabbage of stockfeeding, J. Sci. Food Agric., 33, 1-5.


62. Arnold, W. N. and Thompson, J. F. (1962). The formation of (+)S-methyl-L-cysteine sulfoxide from S-

methyl-L-cysteine in crucifers, Biochim. Biophys. Acta, 57, 604-606.


63. Lancaster, J. E. and Collin, H. A. (1981). Presence of alliinase in isolated vacuoles and of alkyl cysteine

sulfoxides in cytoplasm of bulbs of onion (Allium cepa), Plant Sci. Lett., 22, 169-176.
64. Anderson, N. W. and Thompson, J. F. (1979). Cystine lyase: beta-cystathionase from turnip roots,

Phytochemistry, 18, 1953-1958.



65. Nock, L. P. and Mazelis, M. (1985). Some new observations on the properties of garlic alliinase, Plant

Physiol., 77, S-115


66. Hall, D. I. and Smith, I. K. (1983). Partial purification and characterization of cystine lyase from cabbage

(Brassica oleraceae var capitata), Plant Physiol., 72, 654-658.



67. Schwimmer, S. (1971). Enzymatic conversion of gamma-L-glutamyl cysteine peptides to pyruvic acid, a

coupled reaction for enhancement of onion flavor, J. Agric. Food Chem., 19, 980-983.


68. Schwimmer, S. and Austin, S. J. (1971). Enhancement of pyruvic acid release and flavor in dehydrated

Allium powders by gamma glutamyl transpeptidases, J. Food Sci., 36, 1081-1085.


69. Mazelis, M. (1963). Demonstration and characterization of cysteine sulfoxide lyase in cruciferae,

Phytochemistry, 2, 15-22.



70. Freeman, G. G. (1975). Distribution of flavor components in onion (Allium cepa L.), leek (Allium porrum)

and garlic (Allium sativum), J. Sci. Food Agric., 26, 471-481.


71. Nomura, J., Nishizuka, Y. and Hayaishi, O. (1963). S-Alkylcysteinase : Enzymatic cleavage of S-methyl-

L-cysteine and its sulfoxide, J. Biol. Chem., 238(4), 1441- 1446.


72. Murakami, F. (1960). Studies on the nutritional value of Allium plants (XXXVI) Decomposition of alliin

homologues by microorganism and formation of substance with thiamine masking activity, Vitamins (Tokyo),

20, 126.



73. Cavallito, C. J., Buck, J. S. and Suter, C. M. (1944). Allicin, the antibacterial principles of Allium sativim. II.

Determination of the chemical structure, J. Am. Chem. Soc., 66, 1952-1954.


74. Block, E. (1993). Flavor artifacts, J. Agric. Food Chem., 41, 692


75. Brodnitz, M. H., Pascale, J. V. and von Derslice, L. (1971). Flavor components of garlic extract, J. Agric.

Food Chem., 19, 273-275.


76. Yu, T.-H. and Wu, C.-M. (1989). Stability of allicin in garlic juice, J. Food Sci., 54(4), 977-981.
77. Small, L. D., Bailey, J. H. and Cavallito, C. J. (1947). Alkyl thiosulfinates, J. Am. Chem. Soc., 69, 1710-

1713.



78. Moore, T. L. and O'connor, D. E. (1966). The reaction of methanesulfenyl chloride with alkoxides and

alcohols. Preparation of aliphatic sulfenate and sulfinate esters, J. Org. Chem., 31, 3587-3592.


79. Ostermayer, F. and Tarbell, D. S. (1960). Products of acidic hydrolysis of S-methyl-L-cysteine sulfoxide;

isolation of methyl methanethiosulfinate and mechanism of the hydrolysis, J. Am. Chem. Soc., 82, 3752-3755.



80. Ingersoll, R. L., Volirath, R. E., Scott, B. and Lindegren, C. C. (1938). Bactericidal activity of

crotonaldehyde, Food Res., 3, 389-392.


81. Small, L. D., Bailey, J. H. and Cavallito, C. J. (1949) Comparison of some properties of thiosulfonates and

thiosulfinates, J. Am. Chem. Soc., 71, 3565-3566.


82. Yoshida, S., Kasuga, S., Hayashi, N., Ushiroguchi, T., Matsuura, H. and Nakagawa, S. (1987). Antifungal

activity of ajoene derived from garlic, Appl. Environ. Microbiol., 53(3), 615-617.



83. Naganawa, R., Iwata, N., Ishikawa, K., Fukyda, H., Fujino, T. and Suzuki, A. (1996). Inhibition of microbial

growth by ajoene, a sulfur-containing compound derived from garlic, Appl. Environ. Microbiol., 62(11), 4238-

4242.


84. Brown, H. D., Matzuk, A. R., Becker, H. J., Conbere, J. P., Constantin, J. M., Solotorovsky, M., Winsten, S.,

Ironson, E. and Quastel, J. H. (1954). The antituberculosis activity of some ethylmercapto compounds, J. Am.

Chem. Soc., 76, 3860


85. Chin, H.-W. and Linsay, R. C. (1994). Mechanisms of formation of volatile sulfur compounds following the

action of cysteine sulfoxide lyases, J. Agric. Food Chem., 42, 1529-1536.


86. Kyung, K. H. and Fleming, H. P. (1997). Antimicrobial activity of sulfur compounds derived from cabbage,

J. Food Prot., 60(1), 67-71.


87. Kyung, K. H., Han, D. C. and Fleming, H. P. (1997). Antimicrobial activity of heated cabbage juice, S-

methyl-L-cysteine sulfoxide and methyl methanethiosulfonate, J. Food Sci., 62(2), 406-409.


88. Kubelik, J. (1970). Antimicrobielle Eigenschaften des Knoblauchs, Pharmazie, 25, 266-269.



89. Lubeck, V. L. (1956). Verfahren zum Stabilisieren von antibiotisch wirksamen Inhaltsstoffen von Allium

sativum, Deutch. Pat. 943250.
90. Al-Delaimy, K. S. and Barakat, M. M. F. (1971). Antimicrobial and preservative activity of garlic on fresh

ground camel meat. I. Effect of fresh ground garlic segments, J. Sci. Food Agric., 22, 96-98.




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

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:13
posted:2/21/2010
language:English
pages:19
Lingjuan Ma Lingjuan Ma MS
About work for China Compulsory Certification. Some of the documents come from Internet, if you hold the copyright please contact me by huangcaijin@sohu.com