World Journal
of Microbiology
and Biotechnology,
9, 303-307
A quantitative assessment microbial activity of garlic
of the anti(A/hum sativum)
L.P. Rees, S.F. Minney, N.T. Plummet-, J.H. Slater* and D.A. Skyrme An aqueous extract of freeze-dried garlic (Allium sativum), when incorporated into growth media, inhibited many representative bacteria, yeasts, fungi and a virus. All microorganisms tested were susceptible to garlic. Quantitative assessment of the minimum inhibitory concentrations for bacteria and yeasts showed values ranging from 0.8 to 40.0 mg garlic ml-‘. Fungal radial colony growth was inhibited by at least 25% at concentrations as low as 2.0 mg garlic ml-‘. The 50% endpoint neutralization titre for rotavirus was 2.4 to 2.8 pg ml-‘. Lactic acid bacteria were the least sensitive microorganisms to the inhibitory effects of garlic. In mixed culture studies of Lactobacillus acidophilus and Escherichia co& garlic prevented the establishment of E. co& although the final outcome of competition was not affected. Key
zoord~:
Anti-microbial
activity,
competition,
garlic, MIC values, mixed culture.
Garlic (Allium satimm) is a member of the Lilliaceae family, which includes onions, chives and leeks. Historically, garlic has been used medicinally as an anti-microbial and an anti-lipidemic agent, and more recently as a de-toxification and anti-oxidant agent, an anti-carcinogen, an anticoagulant, an anti-hypertensive agent and an immune system modulator. The anti-microbial activity of garlic exceeds that of most broad spectrum antibiotics. It exhibits anti-bacterial, anti-fungal and anti-viral properties. Susceptible bacteria include Sfupphylococctls uureus (Huddleson et al. 1944, Proteus sp., Escherichia cd, Salmonella sp. (Jezpwa et al. 1966), Bucih sp. and Hafniu sp. (Sharma et al. 1977). Anti-fungal activity has been demonstrated against dermatophytes (Amer et al. 1980), Cryptococcus sp. (Fromtling & Bulmer 1978) and, particularly, Candidu ulbicuns (Adetumbi et al. 1986). Activity in vitro against influenza B and herpes simplex viruses has been observed (Tsai et al. 1985). This study was designed to quantify the effects of an aqueous garlic suspension on a number of different bacteria, yeasts and fungi, and a virus. Furthermore, we wished to
determine whether or not differences in the effects of garlic might be used selectively to influence the outcome of competition between two different microorganisms.
Materials Microorganisms
@,I 1993 Rapid Communications
of Oxford
and their Maintenance
The microorganisms used in this study were obtained from the National Collection of Industrial and Marine Bacteria, Aberdeen, the National Collection of Type Cultures, London, and the International Mycological Institute, Egharn, Surrey (Tables 1 and 2). Proteus mirabilis strain R3.68 was kindly provided by Dr J. King (University of Wales Cardiff). Strains of S. typhimuritrm and S. enteriditis were a gift from Dr S. Bale (University of Bristol). Candida a&cans was kindly provided by Dr J. Douglas (University of Glasgow). All strains designated INT were isolated and identified by Interprise Ltd (Port Talbot). Cultures of bacteria and yeasts were grown overnight on nutrient broth (NB) or deMann, Rogosa, Sharpe broth (MRS) supplemented with glycerol to commercial preparations 10% (v v), and stored at -.2O”C. Fungi were maintained on yeast extract glucose chlorarnphenicol agar (YGC) at 4°C. Preparation
L.P. Rees, S.F. Minney and N.T. Plummer are with lnterprise Ltd, Baglan Bay Industrial Park, Port Talbot SA12 7DJ, UK. J.H. Slater and D.A. Skyrme are with the School of Pure and Applied Biology, University of Wales, Cardiff, P.O. Box 915, Cardiff CFl 3TL, UK. ‘Corresponding author.
and Methods
of Aqueous
Garlic
Extract
(AGEJ
A 20 mg ml-’ suspension of freeze-dried garlic powder (a commercial preparation manufactured and supplied by Interprise Ltd) was centrifuged twice at 3900 x g to give a clear supematant (AGE). The supematant was filter-sterilized (0.2 pm pore size). For
Ltd World Journal of Microbdogy
and Biotechnology, Vol 9, 1993
303
L.2 Rees et al. testing sensitive strains, AGE was diluted of double strength NB or MRS broth to 10 mg garlic ml-‘. For the less sensitive broth at 100 mg garlic ml-’ was prepared Determination
of Minimtim
Inhibitory
with an equal volume give stock solutions of strains, AGE in MRS in the same way.
Concentration
(MrC)
From starting concentrations of 10 or 100 mg garlic ml-‘, two-fold dilutions were prepared. NB was used for all strains except the lactic acid bacteria, which required MRS broth. Overnight cultures of the test organisms were inoculated into a dilution series to give initial cell densities of about 1 x lo6 viable organisms ml-‘. Controls, which lacked AGE, were also established. The cultures were incubated aerobically with shaking at 37°C and viable cell numbers determined after 0, 6 and 24 h growth using a modified Miles and Misra plate count procedure (Miles & Misra 1938). MIC was defined as the garlic concentration at which the viable
cell
number
after
incubation
was
equal
to
the
starting
concentration of I x lo6 viable organisms ml-‘. For this study, the MIC values given are those determined after 24 h incubation. The MIC ranges given which were immediately
represent the tested garlic concentrations below and above the MIC value.
Determination of Garlic Activity Against Porcine Rofavirtts Garlic toxicity to MA104 simian epithelial cell line (European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK) was initially tested using 10 mg garlic ml-‘, and it was shown that there was no evidence of cytotoxicity. Complete tissue culture medium (CM) from the cell line supplier was supplemented to give 10 mg garlic ml-’ and a two-fold dilution series prepared and inoculated with porcine rotavirus strain OSU (ATCC VR-893; American Type Culture Collection, Rockville, MD, USA) to give I x 10’ fluorescence focus-forming units ml-‘. The cultures were incubated for I h at 37°C and inoculated into eight replicate wells of a 96-well microtitre plate previously seeded with MA104 cells. The microtitre plates were centrifuged at 2000 x g for 1 h and incubated in 5% (v v) CO2 at 37°C for 7 days. The cells were
fixed, stained and examined
by indirect
immunofluorescence
microscopy for evidence of rotavirus infection (Brydan et al. 1977). The neutralization titre (50% endpoint) was defined as the garlic concentration at which 50% of the wells showed no evidence of rotavirus infection. The 50% endpoint value was determined graphically from plots of the percentage of wells containing rotavirus infected cells versus garlic concentration.
Determination
of Garlic
Acfivity
Against
Fungal
Radial
Results
and Discussion
MlC determinations for Bacteria and Yeasts The growth response profiles after 6 or 24 h growth for S. dtrblinii were typical of the MIC data for bacteria and yeasts (Figure I). After 6 h growth the MIC was between 0.3 and 1.3 mg garlic ml-‘. However, viable cell numbers in 24-h-old cultures containing up to 1.3 mg garlic ml-’ reached similar levels to those in the control cultures of the same age, i.e. garlic at these concentrations had little effect on the size of the final bacterial population. These results demonstrated a threshold level for the anti-microbial activity of garlic; after 6 h incubation of S. d&linii at garlic concentrations up to 1.3 mg ml-‘, growth was inhibited, but during a further 18 h incubation period the cultures recovered and then appeared unaffected by the presence of garlic. At concentrations of garlic above 2.5 mg ml-‘, viable cell numbers were found to have decreased after both 6 and 24 h incubation. Furthermore, the viable cell numbers after 24 h incubation were substantially lower than those in 6 h incubations at the two higher garlic concentrations of 5 and 10 mg ml-’ (Figure I); this indicated that garlic was acting bactericidally and not bacteristatically. The bacterial and yeast strains evaluated in this study were inhibited by garlic to varying degrees after 24 h incubation (Table I). There were considerable variations in the MIC values determined. The majority of the enteric microorganisms tested were sensitive to garlic between 0.8 and 3.3 mg ml-‘. However, one group of enteric microorganisms, the lactic acid bacteria, showed MIC values significantly higher, at 12.5 to 40 mg ml-‘. The sensitive strains included Gram-positive and Gram-negative microorganisms, some of which were pathogenic. It appeared that
Growth
Inhibition of fungal growth was determined by examining radial growth on YGC agar supplemented with garlic at concentrations ranging from 0 to 10 mg ml-‘. Supplemented and control plates were inoculated with a 2.0 mm’ agar block of the appropriate fungus and incubated at 20°C. The diameter of the fungal colonies were measured after 3 and 8 days growth.
Determination of the Effect of Garlic on the Mixed Culture Growth of E. coli and L. acidophilus Duplicate conical flasks (250-ml), each containing 100 ml MRS broth supplemented with 10.0 mg garlic ml-’ were inoculated with overnight cultures of L. ucidophilrcs (L9) and E. coli (Mll) to an initial density of 6.5 x lo6 and 1 x lo6 viable organisms ml-‘, respectively. Duplicate control cultures without added garlic were established in the same way. The cultures were incubated without shaking at 37°C and viable cell numbers for each population determined regularly for 30 h on MRS and McConkey agars.
01 0
'
2
'
'
4
'
Garlic concentration Figure viable growth.
'
6
'
a
-
'
10
(mg ml-‘)
1. The effect of different concentrations of garlic on the cell numbers of S. dub/M after 6 h (0) or 24 h (0) The horizontal line indicates the MIC value.
Anti-microbial Table
1. Strains
of microorganisms
evaluated,
Strain
Staphylococcus
Strain
aureus
INT C6 INT Ml8
Figure viable growth.
MIC values MC (mg
range ml-‘)
and
MIC ranges. MIC at 24 h (mg ml-‘)
0.6 to 1.3 0.6 to 1.3 0.6 to 1.3
0.8 1.0 1.0
1.3 to 2.5
1.4 1.7
1.3 to 2.5 1.3 to 2.5
1.8
NCIMB 8993 INT M29
1.3 to 2.5 1.3 to 2.5
1.8 1.9
R3.68 INT M40
1.3 to 2.5 1.3 to 2.5
1.9 2.2
NCTC 11994 INT M41 P227165
1.3 to 2.5 1.3 to 2.5 1.3 to 2.5
2.3 2.3 2.4
P227221 NCTC 8172
2.5 to 5.0 2.5 to 5.0
2.8 3.3
NCTC 8626 INT Lil
5.0 to 10.0 10.0 to 15.0
9.1 12.5
INT L9 NCIMB
10.0 to 25.0
16.7 17.5
NCTC
2599
11970
INT AP7
15.0 to 20.0 10.0 to 25.0
INT AP8 INT AP6
25.0 to 37.5 37.5 to 50.0
10
40 60 80 concentration (mg ml-‘)
11857
INT21 INT Ml1
the garlic-sensitive strains were catalase positive whilst the less sensitive lactic acid bacteria were catalase negative. The growth profile responses for the less sensitive microorganisms, such as Pediococcus pentusacetis, were slightly different (Figure 2). No growth occurred after 6 h incubation with 25 and 100 mg garlic ml-’ and the viable cell numbers were the same as in the initial inoculum. That is, the cultures showed a bacteriostatic response. However, after 24 h incubation with 25 mg garlic ml-‘, the population had recovered and grown. At 50 mg garlic ml-‘, the
Garlic
no.
NUMB
Staphylococcus aureus Escherichia co/i Candida albicans Escherichia coli Bacillus cereus Bacillus subtilis Saccharomyces cerevisiae Proteus mirabilis Escherichia co/i Listeria monocytogenes Salmonella dublinii Salmonella enteriditis Salmonella typhimurium Klebsiella aerogenes Pseudomonas aeruginosa Lactobacillus plantarum Lactobacillus acidophilus Lactobacillus casei Enterococcus faecium Pediococcus pentasaceus Pediococcus pentasaceus
their
100
2. The effect of different concentrations of garlic on the cell numbers of L. acidophilus after 6 h (0) or 24 h (a) The horizontal line indicates the MIC value.
activity of garlic
18.5 34.5 39.7
population size was 50% below the MIC level, suggesting that some killing of the population occurred. Interestingly, however, at 75 and 100 mg garlic ml-‘, the viable cell numbers after 24 h were comparable with the MIC level and with the population sizes after 6 h incubation at these garlic concentrations. This indicated that garlic was not exerting a bactericidal effect, in contrast to the response profile for sensitive strains such as 5. dublinii (Figure 1). A similar effect was reported for the /3-lactam antibiotics (Eagle & Musselman 1948); a concentration of antibiotic was identified which resulted in optimum bacteriocidal action but above which the organism death rate was less marked. The MIC values reported in this paper refer to the concentration of freeze-dried garlic powder used to prepare AGE. Garlic is a complex mixture and the active anti-microbial compounds are only a proportion of the total weight of powder used in the assays. Allicin is normally considered to be the main anti-microbial compound in garlic; it represents approximately 0.4% (w/w) of garlic fresh weight (Iberl et al. 1990) and is known to inhibit a variety of enzymes (Wills 1956; Ghannoum 1988). Assuming that the dry matter content of garlic is 33% (N. T. Plummer, unpublished observations) then the allicin content of the garlic powder used in this study would be 1.2% (w/w). Thus the MIC values expressed as concentrations of allicin rather than garlic powder would be about 10 to 480 pg ml-‘. Effect of Garlic on Fungal Colony Radial Growth It was shown that the radial growth rates of four fungi were strongly inhibited by garlic (Table 2). Colony growth was
L.P. Rees et al. Table
2. lnhlbition
of fungal
growth
Strain
by garlic. Diameter
of fungal garlic
colony (mm) concentrations
0
2
4
6
6
10
6 19 23 a7 17 59 ia ND
of Microbiology
and Biotechnology,
9, 303-307
A quantitative assessment microbial activity of garlic
of the anti(A/hum sativum)
L.P. Rees, S.F. Minney, N.T. Plummet-, J.H. Slater* and D.A. Skyrme An aqueous extract of freeze-dried garlic (Allium sativum), when incorporated into growth media, inhibited many representative bacteria, yeasts, fungi and a virus. All microorganisms tested were susceptible to garlic. Quantitative assessment of the minimum inhibitory concentrations for bacteria and yeasts showed values ranging from 0.8 to 40.0 mg garlic ml-‘. Fungal radial colony growth was inhibited by at least 25% at concentrations as low as 2.0 mg garlic ml-‘. The 50% endpoint neutralization titre for rotavirus was 2.4 to 2.8 pg ml-‘. Lactic acid bacteria were the least sensitive microorganisms to the inhibitory effects of garlic. In mixed culture studies of Lactobacillus acidophilus and Escherichia co& garlic prevented the establishment of E. co& although the final outcome of competition was not affected. Key
zoord~:
Anti-microbial
activity,
competition,
garlic, MIC values, mixed culture.
Garlic (Allium satimm) is a member of the Lilliaceae family, which includes onions, chives and leeks. Historically, garlic has been used medicinally as an anti-microbial and an anti-lipidemic agent, and more recently as a de-toxification and anti-oxidant agent, an anti-carcinogen, an anticoagulant, an anti-hypertensive agent and an immune system modulator. The anti-microbial activity of garlic exceeds that of most broad spectrum antibiotics. It exhibits anti-bacterial, anti-fungal and anti-viral properties. Susceptible bacteria include Sfupphylococctls uureus (Huddleson et al. 1944, Proteus sp., Escherichia cd, Salmonella sp. (Jezpwa et al. 1966), Bucih sp. and Hafniu sp. (Sharma et al. 1977). Anti-fungal activity has been demonstrated against dermatophytes (Amer et al. 1980), Cryptococcus sp. (Fromtling & Bulmer 1978) and, particularly, Candidu ulbicuns (Adetumbi et al. 1986). Activity in vitro against influenza B and herpes simplex viruses has been observed (Tsai et al. 1985). This study was designed to quantify the effects of an aqueous garlic suspension on a number of different bacteria, yeasts and fungi, and a virus. Furthermore, we wished to
determine whether or not differences in the effects of garlic might be used selectively to influence the outcome of competition between two different microorganisms.
Materials Microorganisms
@,I 1993 Rapid Communications
of Oxford
and their Maintenance
The microorganisms used in this study were obtained from the National Collection of Industrial and Marine Bacteria, Aberdeen, the National Collection of Type Cultures, London, and the International Mycological Institute, Egharn, Surrey (Tables 1 and 2). Proteus mirabilis strain R3.68 was kindly provided by Dr J. King (University of Wales Cardiff). Strains of S. typhimuritrm and S. enteriditis were a gift from Dr S. Bale (University of Bristol). Candida a&cans was kindly provided by Dr J. Douglas (University of Glasgow). All strains designated INT were isolated and identified by Interprise Ltd (Port Talbot). Cultures of bacteria and yeasts were grown overnight on nutrient broth (NB) or deMann, Rogosa, Sharpe broth (MRS) supplemented with glycerol to commercial preparations 10% (v v), and stored at -.2O”C. Fungi were maintained on yeast extract glucose chlorarnphenicol agar (YGC) at 4°C. Preparation
L.P. Rees, S.F. Minney and N.T. Plummer are with lnterprise Ltd, Baglan Bay Industrial Park, Port Talbot SA12 7DJ, UK. J.H. Slater and D.A. Skyrme are with the School of Pure and Applied Biology, University of Wales, Cardiff, P.O. Box 915, Cardiff CFl 3TL, UK. ‘Corresponding author.
and Methods
of Aqueous
Garlic
Extract
(AGEJ
A 20 mg ml-’ suspension of freeze-dried garlic powder (a commercial preparation manufactured and supplied by Interprise Ltd) was centrifuged twice at 3900 x g to give a clear supematant (AGE). The supematant was filter-sterilized (0.2 pm pore size). For
Ltd World Journal of Microbdogy
and Biotechnology, Vol 9, 1993
303
L.2 Rees et al. testing sensitive strains, AGE was diluted of double strength NB or MRS broth to 10 mg garlic ml-‘. For the less sensitive broth at 100 mg garlic ml-’ was prepared Determination
of Minimtim
Inhibitory
with an equal volume give stock solutions of strains, AGE in MRS in the same way.
Concentration
(MrC)
From starting concentrations of 10 or 100 mg garlic ml-‘, two-fold dilutions were prepared. NB was used for all strains except the lactic acid bacteria, which required MRS broth. Overnight cultures of the test organisms were inoculated into a dilution series to give initial cell densities of about 1 x lo6 viable organisms ml-‘. Controls, which lacked AGE, were also established. The cultures were incubated aerobically with shaking at 37°C and viable cell numbers determined after 0, 6 and 24 h growth using a modified Miles and Misra plate count procedure (Miles & Misra 1938). MIC was defined as the garlic concentration at which the viable
cell
number
after
incubation
was
equal
to
the
starting
concentration of I x lo6 viable organisms ml-‘. For this study, the MIC values given are those determined after 24 h incubation. The MIC ranges given which were immediately
represent the tested garlic concentrations below and above the MIC value.
Determination of Garlic Activity Against Porcine Rofavirtts Garlic toxicity to MA104 simian epithelial cell line (European Collection of Animal Cell Cultures, Porton Down, Salisbury, UK) was initially tested using 10 mg garlic ml-‘, and it was shown that there was no evidence of cytotoxicity. Complete tissue culture medium (CM) from the cell line supplier was supplemented to give 10 mg garlic ml-’ and a two-fold dilution series prepared and inoculated with porcine rotavirus strain OSU (ATCC VR-893; American Type Culture Collection, Rockville, MD, USA) to give I x 10’ fluorescence focus-forming units ml-‘. The cultures were incubated for I h at 37°C and inoculated into eight replicate wells of a 96-well microtitre plate previously seeded with MA104 cells. The microtitre plates were centrifuged at 2000 x g for 1 h and incubated in 5% (v v) CO2 at 37°C for 7 days. The cells were
fixed, stained and examined
by indirect
immunofluorescence
microscopy for evidence of rotavirus infection (Brydan et al. 1977). The neutralization titre (50% endpoint) was defined as the garlic concentration at which 50% of the wells showed no evidence of rotavirus infection. The 50% endpoint value was determined graphically from plots of the percentage of wells containing rotavirus infected cells versus garlic concentration.
Determination
of Garlic
Acfivity
Against
Fungal
Radial
Results
and Discussion
MlC determinations for Bacteria and Yeasts The growth response profiles after 6 or 24 h growth for S. dtrblinii were typical of the MIC data for bacteria and yeasts (Figure I). After 6 h growth the MIC was between 0.3 and 1.3 mg garlic ml-‘. However, viable cell numbers in 24-h-old cultures containing up to 1.3 mg garlic ml-’ reached similar levels to those in the control cultures of the same age, i.e. garlic at these concentrations had little effect on the size of the final bacterial population. These results demonstrated a threshold level for the anti-microbial activity of garlic; after 6 h incubation of S. d&linii at garlic concentrations up to 1.3 mg ml-‘, growth was inhibited, but during a further 18 h incubation period the cultures recovered and then appeared unaffected by the presence of garlic. At concentrations of garlic above 2.5 mg ml-‘, viable cell numbers were found to have decreased after both 6 and 24 h incubation. Furthermore, the viable cell numbers after 24 h incubation were substantially lower than those in 6 h incubations at the two higher garlic concentrations of 5 and 10 mg ml-’ (Figure I); this indicated that garlic was acting bactericidally and not bacteristatically. The bacterial and yeast strains evaluated in this study were inhibited by garlic to varying degrees after 24 h incubation (Table I). There were considerable variations in the MIC values determined. The majority of the enteric microorganisms tested were sensitive to garlic between 0.8 and 3.3 mg ml-‘. However, one group of enteric microorganisms, the lactic acid bacteria, showed MIC values significantly higher, at 12.5 to 40 mg ml-‘. The sensitive strains included Gram-positive and Gram-negative microorganisms, some of which were pathogenic. It appeared that
Growth
Inhibition of fungal growth was determined by examining radial growth on YGC agar supplemented with garlic at concentrations ranging from 0 to 10 mg ml-‘. Supplemented and control plates were inoculated with a 2.0 mm’ agar block of the appropriate fungus and incubated at 20°C. The diameter of the fungal colonies were measured after 3 and 8 days growth.
Determination of the Effect of Garlic on the Mixed Culture Growth of E. coli and L. acidophilus Duplicate conical flasks (250-ml), each containing 100 ml MRS broth supplemented with 10.0 mg garlic ml-’ were inoculated with overnight cultures of L. ucidophilrcs (L9) and E. coli (Mll) to an initial density of 6.5 x lo6 and 1 x lo6 viable organisms ml-‘, respectively. Duplicate control cultures without added garlic were established in the same way. The cultures were incubated without shaking at 37°C and viable cell numbers for each population determined regularly for 30 h on MRS and McConkey agars.
01 0
'
2
'
'
4
'
Garlic concentration Figure viable growth.
'
6
'
a
-
'
10
(mg ml-‘)
1. The effect of different concentrations of garlic on the cell numbers of S. dub/M after 6 h (0) or 24 h (0) The horizontal line indicates the MIC value.
Anti-microbial Table
1. Strains
of microorganisms
evaluated,
Strain
Staphylococcus
Strain
aureus
INT C6 INT Ml8
Figure viable growth.
MIC values MC (mg
range ml-‘)
and
MIC ranges. MIC at 24 h (mg ml-‘)
0.6 to 1.3 0.6 to 1.3 0.6 to 1.3
0.8 1.0 1.0
1.3 to 2.5
1.4 1.7
1.3 to 2.5 1.3 to 2.5
1.8
NCIMB 8993 INT M29
1.3 to 2.5 1.3 to 2.5
1.8 1.9
R3.68 INT M40
1.3 to 2.5 1.3 to 2.5
1.9 2.2
NCTC 11994 INT M41 P227165
1.3 to 2.5 1.3 to 2.5 1.3 to 2.5
2.3 2.3 2.4
P227221 NCTC 8172
2.5 to 5.0 2.5 to 5.0
2.8 3.3
NCTC 8626 INT Lil
5.0 to 10.0 10.0 to 15.0
9.1 12.5
INT L9 NCIMB
10.0 to 25.0
16.7 17.5
NCTC
2599
11970
INT AP7
15.0 to 20.0 10.0 to 25.0
INT AP8 INT AP6
25.0 to 37.5 37.5 to 50.0
10
40 60 80 concentration (mg ml-‘)
11857
INT21 INT Ml1
the garlic-sensitive strains were catalase positive whilst the less sensitive lactic acid bacteria were catalase negative. The growth profile responses for the less sensitive microorganisms, such as Pediococcus pentusacetis, were slightly different (Figure 2). No growth occurred after 6 h incubation with 25 and 100 mg garlic ml-’ and the viable cell numbers were the same as in the initial inoculum. That is, the cultures showed a bacteriostatic response. However, after 24 h incubation with 25 mg garlic ml-‘, the population had recovered and grown. At 50 mg garlic ml-‘, the
Garlic
no.
NUMB
Staphylococcus aureus Escherichia co/i Candida albicans Escherichia coli Bacillus cereus Bacillus subtilis Saccharomyces cerevisiae Proteus mirabilis Escherichia co/i Listeria monocytogenes Salmonella dublinii Salmonella enteriditis Salmonella typhimurium Klebsiella aerogenes Pseudomonas aeruginosa Lactobacillus plantarum Lactobacillus acidophilus Lactobacillus casei Enterococcus faecium Pediococcus pentasaceus Pediococcus pentasaceus
their
100
2. The effect of different concentrations of garlic on the cell numbers of L. acidophilus after 6 h (0) or 24 h (a) The horizontal line indicates the MIC value.
activity of garlic
18.5 34.5 39.7
population size was 50% below the MIC level, suggesting that some killing of the population occurred. Interestingly, however, at 75 and 100 mg garlic ml-‘, the viable cell numbers after 24 h were comparable with the MIC level and with the population sizes after 6 h incubation at these garlic concentrations. This indicated that garlic was not exerting a bactericidal effect, in contrast to the response profile for sensitive strains such as 5. dublinii (Figure 1). A similar effect was reported for the /3-lactam antibiotics (Eagle & Musselman 1948); a concentration of antibiotic was identified which resulted in optimum bacteriocidal action but above which the organism death rate was less marked. The MIC values reported in this paper refer to the concentration of freeze-dried garlic powder used to prepare AGE. Garlic is a complex mixture and the active anti-microbial compounds are only a proportion of the total weight of powder used in the assays. Allicin is normally considered to be the main anti-microbial compound in garlic; it represents approximately 0.4% (w/w) of garlic fresh weight (Iberl et al. 1990) and is known to inhibit a variety of enzymes (Wills 1956; Ghannoum 1988). Assuming that the dry matter content of garlic is 33% (N. T. Plummer, unpublished observations) then the allicin content of the garlic powder used in this study would be 1.2% (w/w). Thus the MIC values expressed as concentrations of allicin rather than garlic powder would be about 10 to 480 pg ml-‘. Effect of Garlic on Fungal Colony Radial Growth It was shown that the radial growth rates of four fungi were strongly inhibited by garlic (Table 2). Colony growth was
L.P. Rees et al. Table
2. lnhlbition
of fungal
growth
Strain
by garlic. Diameter
of fungal garlic
colony (mm) concentrations
0
2
4
6
6
10
6 19 23 a7 17 59 ia ND
