Generation of Antibacterial Activity of Garlic by Heat in the Absence of Alliinase Activity against Staphylococcus aureus B33

Alliinase-Independent Inhibition of Staphylococcus aureus B33 by Heated Garlic

Kyu Hang Kyung**, Myung Hee Kim, Mee Seung Park, and Youn Soon Kim*

Department of Food Science, Sejong University, Kwangjin-ku, Seoul 143-747, Korea

*Department of Home Economy Education, Chosun University, Kwangju 501-759, Korea

Running title: Antibacterial activity of heated garlic

Choice of Journal section; Food Microbiology and Safety

**Corresponding author:

Tel: 822-3408-3225

Fax: 822-3408-3569

E-mail: kyungkh@kunja.sejong.ac.krAbstract

Heated (121C) garlic extract in which alliinase was inactivated before crushing exhibited complete bacteriostatic activity at 15% against Staphylococcus aureus. Garlic heated for 45 min showed the highest antibacterial activity and the relative peak areas of 4-heptenal, methyl allyl disulfide, diallyl disulfide, 2-vinyl-4H-1,3-dithiin, and diallyl trisulfide (DATS) were highest at 45 min of heating. Other than 4-heptenal, all these compounds are known to possess different degrees of antibacterial activity. DATS was thought to be the primary antibacterial compound in heated garlic extract. It was tentatively concluded that antibacterial compounds were formed from alliin unreacted by alliinase by marked heating. Diallyl compounds with more than three sulfur atoms and the oxidized form of allicin were not detected.

Key words: Heated garlic, Allium sativum, Staphylococcus aureus, antibacterial activity, diallyl trisulfide, alliin, allicin

Introduction

The antimicrobial activity of plant extracts, especially from Allium and Brassica, has been recognized for many years, since the antimicrobial activity of garlic (Allium sativum; Walton and others 1936) and cabbage (Brassica oleracae; Sherman and Hodge 1936) was demonstrated scientifically (Kyung and Lee 2001). The majority of the antimicrobial activity of Allium (Cavallito and Bailey 1944; Stoll and Seebeck 1951) and Brassica (Kyung and Fleming 1994) vegetables is due to volatile sulfur compounds derived from S-alk(en)yl-L-cysteine sulfoxide. The presence of S-alk(en)yl-L-cysteine sulfoxide is confined essentially to two families, the Cruciferae and the Liliaceae, and is particularly associated with the genera Allium and Brassica (Virtanen 1965).

Allicin, the principal antimicrobial compound of garlic, is only generated after the cloves are injured and the enzyme alliinase reacts with its substrate alliin (S-2-allyl-L-cysteine sulfoxide), which is an S-alk(en)yl-L-cysteine sulfoxide (Cavallito and Bailey 1944; Stoll and Seebeck 1951; Block 1992).

S-methyl-L-cysteine sulfoxide (SMCSO), a non-protein sulfur-containing amino acid found in Brassica (Synge and Wood 1956; Morris and Thompson 1956; Marks and others 1992), is structurally similar to alliin, another 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 (allyl 2-propenethiosulfinate), which are the principal antimicrobial compounds of Brassica and Allium, respectively, as a result of enzyme reaction when plant tissues are disturbed. MMTSO and allicin have similar chemical structures and are methyl- and allyl- derivatives of thiosulfinate.

There have been no reports on the antimicrobial activity of garlic generated by heating in the absence of alliinase activity. However, there are some conflicting reports on the microbial growth inhibitory activity of cabbage, which contains SMCSO, a homologue of alliin in garlic. Sherman and Hodge (1936) and Pederson and Fisher (1944) reported that heating at temperatures from 60C to boiling destroyed the antimicrobial activity of cabbage. Heating (100C) cabbage before juice extraction prevented formation of the inhibitor(s) in some cultivars, but not others (Kyung and Fleming 1994a). Yildiz and Westhoff (1981) reported that filter-sterilized cabbage juice was a better medium for Leuconostoc mesenteroides than autoclaved cabbage juice. This was confirmed by Kyung and others (1997), who maintained that the principal antimicrobial compound of autoclaved cabbage juice was methyl methanethiosulfonate (MMTSO2), one of the thermal degradation products of SMCSO. MMTSO2 is also formed by a spontaneous proportionation reaction of MMTSO (Chin and Lindsay 1994). It was surmised that relatively mild heating (60-100C) of cabbage destroys cysteine sulfoxide lyase, while severe heating (121C) not only destroys the enzyme, but also decomposes SMCSO into other compounds with antibacterial activity. In analogy with MMTSO2 formed from SMCSO in cabbage subject to severe heating, the formation of antibacterial compounds, including the oxidized form of allicin [allicin oxide (Block 1992)] from alliin, was expected in garlic subjected to severe heating.

Boiled garlic extract, in which alliinase reacted with its substrate to produce antibacterial compounds, retained antibacterial activity against Helicobacter pylori, but at a much reduced level compared with unheated fresh garlic extract (Cellini and others 1996). This report examines the generation of antibacterial compounds in heated garlic in the absence of alliinase activity. Its objectives were to show the presence of antibacterial activity in heated garlic extract in which alliinase failed to act and to identify the inhibitory compounds in heated garlic.

Materials and Methods

Materials

Garlic (Allium sativum) of unknown origin was purchased from a local market in Seoul, Korea. Cysteine and heptadecane were purchased from Sigma Chemical Co. (St. Louis, MO). Glutathione and dichloromethane were purchased from Aldrich Chemical Co. (Milwaukee, WI) and Mallinckrodt-Baker Inc. (Phillipsburg, PA), respectively.

Bacterial strain and culture conditions

Staphylococcus aureus B33 was from Dr. Henry P. Fleming, Food Fermentation Laboratories, USDA/ARS, North Carolina State University, Raleigh, NC. The bacterium was stored at -64C in tryptic soy broth (TSB; Difco Laboratories, Detroit, MI) containing 16% glycerol. For resuscitation, the frozen culture was streaked on TSA and an isolated colony picked and cultivated in TSB before using a 16-hr culture for final inoculation. A 10-L (~1.0 106 cells) 10 diluted aliquot of an overnight statically grown culture of S. aureus was inoculated into 10 mL of TSB in 16 150-mm glass culture tubes. Numbers of viable cells were estimated as colony-forming units (CFU)/mL by spiral plating (Spiral Biotech, Bethesda, MD) onto plate count agar (Difco Laboratories) and incubating for 24-48 hr. All growth experiments were performed at 30C.

Protection of S. aureus from the antibacterial activity of garlic by SH compounds

Stock solutions (500 mM each) of cysteine and glutathione were made in distilled water. Twenty, 100, or 200 L of stock solution was added to 10 mL of TSB containing garlic extract, resulting in 1, 5, and 10-mM solutions of the compounds, respectively. TSB containing 15% garlic extract with either cysteine or glutathione was inoculated similarly and the numbers of viable cells were estimated.

Preparation of heated garlic extract

Three different garlic extracts were prepared. Peeled and trimmed garlic cloves were washed with water and blanched by boiling in water for 5 min to inactivate alliinase. The blanched garlic cloves were cooled in tap water, blended (Waring Blender, Dynamics Co., New Hartford, CT) with an equal amount (w/w) of water, and centrifuged (HMR-2201V, Hanil Centrifuge Co., Inchon, Korea) for 20 min at 17,600 g. The supernatant was distributed into 8 capped glass tubes and autoclaved (121C) for between 0 and 120 min at 15 min intervals. The garlic extract prepared in this way was designated as garlic crushed after blanching and heated (Extract 1; Fig. 1). In addition, washed fresh garlic cloves were ground with a Waring blender with an equal amount (w/w) of water, and the ground garlic was stored at 37C overnight to allow the alliin to be degraded by alliinase. Garlic extract was obtained by squeezing the ground garlic stored at 37C through multiple layers of cheesecloth. The first half of the extract was distributed into 8 capped glass tubes, and autoclaved as described above. This second extract was designated as whole extract heated (Extract 2; Fig. 1). Furthermore, all volatile compounds were removed from the second half of the ground garlic extract stored at 37C by extracting it twice with dichloromethane; the product was distributed into 8 capped glass test tubes and autoclaved in the same manner. This was designated as (whole extract - volatiles) heated (Extract 3; Fig. 1).

##upload image: Fig. 1. Flow chart for the preparation of heated garlic extract and a description of the nature of the extracts.##

After autoclaving, the clear upper layer was pipetted aseptically, without disturbing the precipitated insolubles, into TSB in sterile glass tubes to make test media with appropriate concentrations of garlic.

Identification of volatile sulfur compounds

Mass spectra of the isolated compounds were obtained by combination GC/MS (Platform II mass spectrometer and Chemstation with MassLynx 3.2 Buildoo4 Software; Micromass Ltd., Manchester, UK). Aliquots of 10 mL of heated garlic extract (garlic crushed after heating) were extracted with 5 mL of dichloromethane, and 2-L portions of the dichloromethane extract were injected for GC/MS analysis. Heptadecane was used as the internal standard. The dichloromethane extract was held at 64C, when held before analysis. The GC column (30m capillary, J & W Scientific Inc., Folsom, CA) was coated with DB-5 (0.25 thickness), and the oven temperature was programmed to increase from 28 to 150C at 6C /min, holding at the initial and final temperatures for 2 min. The carrier gas was He (1.5 mL/min). Samples were injected using the splitless mode. The injector port and detector temperatures were 250C. Electron impact ionization (potential 70eV) was used and the mass range scanned was 33-350 Daltons.

Results and Discussion

Antibacterial activity of heated garlic extract

The antibacterial activity of heated garlic against S. aureus was tested in TSB containing different concentrations of garlic extract heated (Extract 1) at 121C for different periods of time (0 to 120 min) at 15 min intervals (Fig. 2). The antibacterial activity of heated garlic progressively increased towards 45 min of heating. Garlic extract at a concentration of 15% heated for 45 min was completely bacteriostatic for S. aureus for 24 hr. However, the activity progressively decreased as heating time was increased further. Since the extract of blanched garlic (0 min heating) showed no inhibition (Fig. 2), the compounds with antibacterial activity must have been newly formed from garlic constituent(s) by heating and accumulated up until 45 min of heating. From then on, the generation of the antibacterial compound(s) was outpaced by their destruction. The extract heated for 120 min retained some antibacterial activity and the final cell number was smaller than that of the extract of blanched garlic. Alliin is reported not to be antimicrobial unless reacted by the enzyme alliinase (Brown and others 1954; Stoll and Seebeck 1951). The boiled garlic extract of Cellini and others (1996) was antimicrobial because they boiled the garlic extract after the garlic was crushed, so that the alliinase enzyme had an opportunity to react with its substrate. Therefore, the antimicrobial activity of boiled garlic extract must be the residual antimicrobial activity after boiling destroys most of the activity.

##upload image: Fig. 2. Growth of S. aureus B 33 at 30C for 24 hours in TSB containing 15% garlic extract heated at 121C for different periods of time.##

##upload image: Table 1. Growth of S. aureus in TSB with different concentrations of heated garlic extracts prepared in three different waysa.##

The antimicrobial activity of the extract (Extract 1) of heated whole garlic was compared with that of garlic extracts (Extract 2) heated after they were maintained at 37C overnight to exhaust alliin (Table 1). This whole extract showed some antibacterial activity, inhibiting bacterial growth completely at 45%. This activity is similar to that found by Cellini and others (1996), in the sense that it was the residual activity after most of the activity was destroyed on boiling. With this manner of extract preparation (Fig. 1), the antibacterial compounds (e.g., allicin) in crushed fresh garlic were thermally decomposed to generate compounds with relatively weaker activities on heating. The extract (Extract 3) heated after the garlic extract was made and the volatile compounds removed with dichloromethane exhibited weak activity, inhibiting the growth of S. aureus slightly, even at 50%. In the extract (Extract 1) without alliinase activity, it was presumed that heating formed antibacterial compounds from alliin unreacted by alliinase. The antimicrobial activity of heated garlic extract (Extract 1) was apparent (Fig. 2), but much weaker than that of fresh extract. Fresh garlic extract inhibited S. aureus at 1-2% (Dababneh and Al-Delaimy 1984).

##upload image: Table 2. Effect of adjusting pH before heating whole garlic cloves on the antibacterial activity of garlic.##

When the pH of garlic extract was adjusted before heating (after blanching followed by blending; Table 2), the antibacterial activity was higher when the pH was acidic (pH 4.0-6.0) than when it was alkaline (pH 7.2-9.0). It was postulated that, at lower pH, the alliin in garlic was decomposed into compounds with stronger antibacterial activity, or that more antibacterial compounds were produced. It was reported that the formation of 3-vinyl-4H-1,2-dithiin and 2-vinyl-4H-1,3-dithiin, which are derivatives of allicin, was favored by heating at a pH around 5.5, and the formation of diallyl trisulfide (DATS) and methyl allyl trisulfide was favored in neutral or weak acidic conditions (Yu and others 1989).

Reversal of the antibacterial activity of heated garlic extract by thiol compounds

The antimicrobial activity of thiosulfinates (Cavallito and others 1944; Small and others 1947, 1949), cabbage (Kyung and Fleming 1994), and garlic (Cavallito and others 1944) is inactivated by cysteine. Ten mM of either cysteine or glutathione completely abolished the antibacterial activity of heated garlic extract (Table 3). Therefore, the antibacterial compounds from heated garlic seem to work in the same manner as thiosulfinates, represented by allicin from fresh garlic extract, as shown in the following section.

##upload image: Table 3. Effect of SH compounds on the antibacterial activity of heated garlic extract tested against S. aureus a.##

Volatile sulfur compounds in heated garlic extract

GC chromatograms of heated garlic volatiles are shown in Fig. 3 and the volatile compounds separated are listed in Table 4. Many of the sulfur compounds identified (Table 4), including methyl allyl sulfide, diallyl (mono)sulfide (DAMS), methyl allyl disulfide, allyl propyl sulfide, diallyl disulfide (DADS), 2-vinyl-4H-1,3-dithiin, and DATS are degradation products of S-alk(en)yl-L-cysteine sulfoxides (Yu and others 1993, 1994) and are expected to have varying degrees of antibacterial activity. Five of the 12 separated volatile compounds, 4-heptenal, methyl allyl disulfide, diallyl disulfide, 2-vinyl-4H-1,3-dithiin, and DATS, had the highest relative peak areas when garlic was heated for 45 min (Fig. 4). The major antibacterial compound(s) in heated garlic are expected to be in this group. The relative peak areas of the other compounds showed either decreasing or increasing trends throughout, or peaked at heating times other than 45 min (Fig. 5). Therefore, this second group does not include any major antibacterial compound found in heated garlic extract.

##upload image: Fig. 3. GC chromatograms of volatile compounds from garlic extract heated at 121C. A: blanched extract heated for 0 min, B: heated for 45 min, C: heated for 120 min.##

##upload image: Table 4. Volatile compounds found in heated garlic extract .##

##upload image: Fig. 4. Volatile compounds in garlic extract heated for 45 min with the highest relative peak areas. : 4-heptenal, : methyl allyl disulfide, : diallyl disulfide, : 2-vinyl-4H-1,3-dithiin, : diallyl trisulfide ##

The diallyl sulfides of garlic materials have significant activity against H. pylori (OGara and others 2000). The activity of diallyl sulfides increases with additional sulfur atoms. The MIC ranges of DADS, DATS, and diallyl tetrasulfide (DATTS) were 100 to 200, 13 to 25, and 3 to 6 /ml, respectively. DAMS was only slightly inhibitory (MIC range: 4148 to 2074 /ml). Allicin was more inhibitory (MIC range: 6 to 12 /ml) than any diallyl sulfur compounds except DATTS. Diallyl sulfides with more than two sulfur atoms, which are reported to be present in garlic oil (OGara and others 2000), were not detected in this work.

##upload image: Fig. 5. Volatile compounds with lower relative peak areas in garlic extract heated for 45 min. : methyl allyl sulfide, : unknown (2), : diallyl monosulfide, : allyl propyl sulfide, : unknown (8), : 3,5-dimethyl-1,2,4-trithiolane, : unknown (12) ##

Since DATS is reported to be about 10 times more inhibitory than DADS against H. pylori (OGara and others 2000) and since Fig. 4 shows that twice as much DADS was present in heated garlic extract than DATS, it was deduced that DATS is the primary antibacterial compound in heated garlic extract. The antibacterial activity of other compounds, including DADS and 2-vinyl-4H-1,3-dithiin (or allicin), was thought to be of secondary importance to that of DATS. However, it should be kept in mind that there likely will be variations in sensitivity to those compounds depending on types of bacteria. In addition there is the possibility that DATS, DADS, and other compounds might have acted synergistically.

Allicin oxide (allyl 2-propenethiosulfonate, pseudoallicin; Block 1992), which is homologous to MMTSO2 from heated cabbage (Kyung and others 1997), was not detected, even after efforts were made. Block (1992) was also unable to find it. Allicin oxide is reported to have only 50% of the antimicrobial activity of allicin. MMTSO2 is formed from SMCSO when cabbage juice is heated (Kyung and others 1997). MMTSO2 is slightly more inhibitory to various types of bacteria than MMTSO, but is less inhibitory to yeasts (Kyung and Fleming 1997). Small and others (1949) reported that MMTSO2 was equally or more antimicrobial than MMTSO against various bacteria.

The antimicrobial activity of dimethyl sulfides, formed as degradation products of SMCSO, has been studied. Dimethyl monosulfide was not antimicrobial up to 500ppm (Kyung and Fleming 1997). Dimethyl disulfide (DMDS), which slightly inhibited the growth of certain microorganisms at 500 ppm, delaying the onset of growth, did not completely inhibit the growth of any microorganisms tested (Kyung and Fleming 1997). Disulfides, such as DADS and DMDS, have been shown to be antimicrobial (Brown and others 1954; Virtanen 1962). However, the activity is reported to be weaker than that of the corresponding thiosulfinates (Virtanen 1962). Sulfides of diallyl and dimethyl derivatives are also formed in unheated extract of garlic (Cellini and others 1996) and cabbage (Chin and Lindsay 1994; Marks and others 1992).

Theories as to how DMDS affects cellular activity have been proposed. Steven and others (1981) suggested that DMDS inactivates active papain by forming an inactive papain-methyl sulfide complex (papain-SH + DMDS papain-S-S-CH3 + CH3SH). Smith (1980), in reporting kale poisoning of ruminant animals feeding on cruciferous vegetables, explained that DMDS may harm red blood cells by oxidizing reduced glutathione to oxidized glutathione (2G-SH + CH3-S-S-CH3 G-S-S-G + 2CH3SH). The basic principles of the two theories are not very different, and are similar to the way in which thiosulfinates, represented by allicin and MMTSO, inhibit microorganisms (cellular protein-SH + R-S-S(O)-R' cellular protein-S-S-R + R'SOH; Small and others 1947).

Garlic and omeprazole work synergistically against H. pylori (Jonkers and others 1999; Cellini and others 1996). The active components of both garlic and omeprazole are known to inhibit bacteria by binding the -SH groups of bacterial enzymes.Conclusions

Garlic antibacterial activity was generated by heat in the absence of alliinase activity. Alliin is thought to be thermally decomposed into compounds with different degrees of antibacterial activity. Diallyl trisulfide seemed to be the primary antibacterial compound and other compounds, such as diallyl disulfide and 2-vinyl-4H-1,3-dithiin (or allicin) are of secondary importance.

Acknowledgments

This work was supported by a research grant (HMP-99-F-06-0001) from the Ministry of Health and Welfare, Republic of Korea. We thank C.T. Kim (Nong Shim Foods Co.) for MS analyses and help with data interpretation.

References

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Table 1. Growth of S. aureus in TSB with different concentrations of heated garlic extracts prepared in three different waysa

Font(%)

Garlic crushed before heating

Extract 1c

Extract 2c

Extract 3c

0

9.11

9.28

9.08

5

9.04

9.08

7.30

10

9.08

9.11

6.85

15

8.76

8.89

5.15

20

8.70

8.83

- b

25

7.89

8.77

-

30

7.04

8.76

-

35

6.68

8.60

-

40

6.26

8.48

-

45

4.92

8.30

-

50

4.56

7.63

-

a ; Log CFU/mL after incubation at 30C for 24h with an inoculum to make initial number of 1.5105cells/mL

b ; < 2.6

c ; see Fig.1.

Table 2. Effect of adjusting pH before heating whole garlic cloves on the antibacterial activity of garlic

pH

Extract 1 (15%)

4.0

4.68a

5.0

4.79

5.9

4.83

6.2 (pH unadjusted)

5.04

7.2

7.53

8.0

7.60

9.0

7.85

a ; Log CFU/mL after incubation at 30C for 24h with an inoculum to make initial number of 1.5105cells/mL. Basal medium was TSB containing 15% heated garlic extract.

Table 3. Effect of SH compounds on the antibacterial activity of heated garlic extract tested against S. aureus a

SH compounds

Concentration (mM)

0

1

5

10

Cysteine

5.08

7.17

7.43

8.54

Glutathione

5.08

7.52

7.85

8.92

a ; Log CFU/mL after incubation at 30C for 24h with an inoculum to make initial number of 1.5105cells/mL. Basal medium was TSB containing 15% heated garlic extract.

Table 4. Volatile compounds found in heated garlic extract

No.

R.T.

Compounds

M.W.

1

3.98

methyl allyl sulfide

88

2

6.15

Unknown

[54(100), 80(43), 55(41), 69(13)]

(98)

3

7.77

4-heptenal

112

4

8.12

diallyl monosulfide

114

5

9.76

methyl allyl disulfide

120

6

9.94

allyl propyl sulfide

116

7

14.36

diallyl disulfide

146

8

16.25

Unknown

[73(100), 74(97), 138(93), 45(68), 64(50)]

(138)

9

17.79

2-vinyl-4H-1,3-dithiin

144

10

19.92

diallyl trisulfide

178

11

21.4

3,5-dimethyl-1,2,4-trithiolane

180

12

27.21

Unknown

[138(100), 58(92), 73(90), 74(85), 45(43)]

(230)