Abstract: In the present study, antimicrobial activity of various extracts of Zingiber officinale, Curcuma longa and Alpinia galanga were screened against the common food borne bacteria such as Escherichia coli, Salmonella enteriditis, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Bacillus cereus and fungi such as Saccharomyces cerevisiae, Hansenula anomala, Mucor mucedo, Candida albicans using disc diffusion method. All the extracts showed significant antibacterial and antifungal properties. The methanol extracts (100 μg mL-1) revealed maximum zone of inhibition (p<0.001). Zingiber officinale and Curcuma longa possessed considerably greater activity than Alpinia galanga. These findings established the potential of the selected rhizomes of Zingiberaceae family as effective natural food preservatives.
INTRODUCTION
Food is the most important basic need of man and hence preserving them is of utmost importance and is one of the oldest technologies. Food borne illness consequential from the consumption of contaminated food has been of great concern for consumers despite the use of various preservatives (Shan et al., 2007). Safety researchers and regulatory bodies in food industries are constantly bothered with increase in the number of food poisoning and spoilage caused by pathogenic food borne microorganisms (Meng et al., 1998). The emergence of antibiotic resistant pathogens that can amplify food borne infections is also another concern (Perrenten et al., 1998; Stermitz et al., 2000). The demand for food with extended shelf-life, lower level of harmful chemical preservatives and absence of risk causing food borne infections have made food processors to focus on exploring naturally occurring preservatives.
Herbs have been used in foods since ancient times, not only as folk medicine, but also as flavoring agents and food preservatives (Dillon, 1994; Cutler, 1995; Charalambous, 1994) due to their antimicrobial activity against certain pathogens (Tepe et al., 2004; Erasto et al., 2004; Fukai et al., 2002; Puupponen et al., 2001; Salie et al., 1996; Xu et al., 1998; Rauha et al., 2000; Ahmad and Beg, 2001), antioxidative property (Beuchat and Golden, 1989; Shobana and Naidu, 2000) and wide array of medicinal values (Wood et al., 2001). Herbs and spices are traditionally used as one of the safest and effective remedies in curing various ailments and even long-term consumption is not known to have produced any side effects (Nielsen and Rios, 2000). They do not exhibit toxicity at levels consumed and are considered as GRAS (Generally Recognized As Safe) substance (Souza et al., 2005; Pandit and Shelf, 1994).
In Ayurveda, Zingiber officinale, Curcuma longa and Alpinia galanga belonging to the family Zingiberaceae are most commonly used rhizomes for their medicinal value. All these plants are distributed in warm tropical climates, predominantly found in Southeastern Asia and widely cultivated in India, China, Africa, Jamaica, Mexico and Hawaii (Evans, 1996).
Zingiber officinale is an aromatic plant and the rhizomes are scaly structures consisting of aromatic components, essential oil and oleoresins due to which it is used commonly in food products and beverages. Curcuma longa has been extensively utilized as a common household remedy and as a spice. It contains essential oil, yellow pigments, starch and oleoresin (Leung and Foster, 2003). Rhizomes of C. longa are utilized as carminative, anti-inflammatory and anthelmintic agents. Traditionally, the pastes from its rhizomes are applied as a remedy for wounds, bruises, inflamed joints and sprains (Eigner and Scholz, 1999). Alpinia galanga is also a traditionally used spice which has been comprehensively used as flavoring agent, carminative, treating diarrhea and for stomachache. For its distinctive aroma as well as strong flavor, the rhizomes of A. galanga are extensively used in foods of Thailand and Malaysia (Yang and Eilerman, 1999). Essential oils obtained from the rhizomes of A. galanga have been claimed to possess antimicrobial activities against bacteria, fungus, yeast and parasites (Farnsworth and Bunyapraphatson, 1992). Instead of these substantial findings showing wide range of potential therapeutic actions, these spices are still branded almost purely as flavoring agents.
The present study was undertaken to determine and compare the potential of various extracts of Z. officinale, C. longa and A. galanga as an antimicrobial agent against food borne micro organisms such as Escherichia coli, Salmonella enteriditis, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Bacillus cereus, Saccharomyces cerevisiae, Hansenula anomala, Mucor mucedo and Candida albicans as natural food preservatives.
MATERIALS AND METHODS
Preparation of Extracts
Fresh rhizomes of Z. officinale, C. longa and A. galanga
were purchased in the month of April 2008 from a local market in Cheras, Selangor,
Malaysia and were authenticated by Dr. J. Anbu Jeba Sunilson, Pharmacognosist,
Masterskill University College of Health Sciences, Malaysia. The rhizomes were
washed under tap water to remove the dirt and soil and sliced into smaller pieces.
They were dried separately in a vaccum drier at 25°C for 24 h and size reduced
to coarse powder using a cutter mill. Powders of each rhizome (500 g) were extracted
separately with petroleum ether, chloroform, methanol and water by Soxhlet extraction
technique successively (Sunilson et al., 2009).
All the extracts were concentrated using rotary vaccum evaporator and kept in
dessicator until further studies.
Selection of Microorganisms
Stock cultures of six different species of food-borne bacteria such as Escherichia
coli, Salmonella enteriditis, Clostridium perfringens,
Staphylococcus aureus, Campylobacter jejuni and Bacillus cereus
and four different species of fungi such as Saccharomyces cerevisiae,
Hansenula anomala, Mucor mucedo and Candida albicans were
obtained from the microbiology laboratory, Masterskill University College of
Health Sciences, Selangor, Malaysia. The food-borne pathogens were maintained
separately in different stock culture for bacteria (nutrient broth) and fungi
(sabouraud dextrose broth). A loop full of each culture was inoculated individually
into the respective agar broth and incubated at 37±1°C for 24 h for
bacteria and 28±1°C for 48 h for fungi.
Antibacterial Assay by Disc Diffusion Method
All the extracts were concentrated and dissolved in dimethyl sulfoxide (DMSO)
to a final concentration of 10 mg mL-1 and sterilized by filtration
through 0.45 μm millipore filters. Screening of antimicrobial activity
was carried out by disc diffusion method described by Murray
et al. (2007) using 100 μL of suspension containing 108
colony forming unit (cfu) mL-1 of bacteria, 104 spore
mL-1 of fungi spread on nutrient agar and SDA medium, respectively.
Discs of 6 mm in diameter were impregnated with 10 μL of the extracts (300
μg disc-1) at a concentration of 100 μg mL-1
and placed on the inoculated agar. Ofloxacin (10 μg disc-1)
and fluconazole (10 μg disc-1) were used as reference standards
for the antibacterial and anti fungal activities, respectively (Karaman
et al., 2003; Amerjothy et al., 2007).
All the inoculated plates were incubated at 37±1°C for 24 h for the
bacteria and at 28±1°C for 72 h for fungus. The zones of inhibition
were measured for determining the antimicrobial activity and the findings were
tabulated.
Statistical Analysis
Data are expressed as Mean±SEM of triplicates. Students’s t-test
was used to compare the antimicrobial activity of the extracts against the standard
antimicrobial agents. All statistical analysis were conducted with SPSS software
(V. 12, SPSS, USA) at significant levels of 0.05, 0.01 and 0.001 (Sunilson
et al., 2008).
RESULTS
The antimicrobial activity of various extracts of Z. officinale, C. longa and A. galanga against the food-borne microorganisms examined in the present study were assessed by the diameter of zone inhibition (Table 1-3). On comparing various extracts, methanol extract of Z. officinale, C. longa and A. galanga exhibited moderate (p<0.01) to maximum (p<0.001) zone of inhibition against all food-borne pathogens whereas, petroleum extract exhibited lower (p<0.05) zone of inhibition against E. coli, S. aureus, M. mucedo and C. albicans. The chloroform and water extracts showed moderate zone of inhibition (p<0.01) against S. aureus and H. anomala, respectively.
| Table 1: | Antimicrobial activity of various extracts of Zingiber
officinale |
PE: Petroleum either extract, CE: Chloroform extract, ME:
Methanol extract, WE: Water extract, O: Ofloxacin (10 μg disc-1),
F: Fluconazole (10 μg disc-1). Values are means of three
times ±SEM. *p<0.05, **p<0.01, ***p<0.001, zone of inhibition
of extracts against bacteria and fungus vs. the normal diameter of disc |
|
| Table 2: | Antimicrobial activity of various extracts of Curcuma longa |
PE: Petroleum either extract, CE: Chloroform extract, ME:
Methanol extract, WE: Water extract, *O: Ofloxacin (10 μg disc-1),
F: Fluconazole (10 μg disc-1). Values are means of three
times ±SEM. *p<0.05, **p<0.01, ***p<0.001, zone of inhibition
of extracts against bacteria and fungus vs. the normal diameter of disc |
|
| Table 3: | Antimicrobial activity of various extracts of Alpina galanga |
PE: Petroleum either extract, CE: Chloroform extract, ME:
Methanol extract, WE: Water extract, *O: Ofloxacin (10 μg disc-1),
F: Fluconazole (10 μg disc-1). Values are means of three
times ±SEM. *p<0.05, **p<0.01, ***p<0.001, zone of inhibition
of extracts against bacteria and fungus vs. the normal diameter of disc |
|
The results revealed that all the extracts had significant antibacterial activity, among which methanol extracts (100 μg mL-1) exhibited maximum effect. When comparing the antimicrobial activity among different rhizomes, Z. officinale and C. longa possessed significant activity than A. galanga which was well comparable with reference standards ofloxacin and fluconazole.
DISCUSSION
The use of naturally occurring preservatives has gained momentum in the pharmaceutical and food industries due to their ability in prolonging the storage life of foods by preventing rancidity through their antioxidant or bacteriostatic activity (Beuchat and Golden, 1989).
Many medicinal plant extracts have been known to possess antimicrobial activity and are used for the purpose of food preservation (Shelef, 1983; Cowan, 1999; Zaika, 1988; Aktug and Karapinar, 1986). In this study, the methanol extracts of Z. officinale, C. longa and A. galanga displayed effective antimicrobial activity against food spoilage and food-borne pathogens. The zone of inhibition against sensitive microorganisms was in the range of 10-21 mm. These results are in parallel with the findings of previously reported studies that methanol is a better solvent for consistent extraction of antimicrobial components from medicinal plants compared to other solvents (Eloff, 1998).
The methanol extract of Z. officinale showed significant (p<0.001) zone of inhibition against all the microorganisms such as E. coli, S. aureus, (Archana et al., 2009) S. enteriditis, C. perfringens, B. cereus, S. cerevisiae, H. anomala, M. mucedo and C. albicans and moderate inhibition (p<0.01) against C. jejuni. Water extracts showed moderate inhibition (p<0.01) against E. coli, S. enteriditis, S. aureus, C. jejuni, B.cereus, S. cerevisiae, H. anomala and C. albicans and less inhibition (p<0.05) against C. perfringens and M. mucedo. Chloroform extract exhibited moderate (p<0.01) zone of inhibition against S. aureus, B. cereusia, S. cerevisiae, H. anomala and M. mucedo whereas petroleum ether extract exhibited lower (p<0.05) zone of inhibition against all food-pathogens except C. perfrigens and S. cerevisiae. Z. officinale is known to contain resins and volatile oils such as borneol, camphene, citral, eucalyptol, linalool, phenllandrene, zingiberine and zingiberol phenols (Ahmad et al., 2008; Hirasa and Takemasa, 1998) which may be responsible for its potent antimicrobial activities.
Methanol extract of C. longa showed significant (p<0.001) zone of inhibition against all strain of tested microorganisms except C. perfringens which was moderately (p<0.01) inhibited, followed by water extract that revealed significant inhibition (p<0.001) against S. aureus, B. cereus and S. cerevisiae and moderate (p<0.01) zone of inhibition against E. coli, C. prefringens, C. albicans, H. anomala and M. mucedo. The chloroform extract of C. longa revealed moderate (p<0.01) inhibition against all micro organisms except C. jejuni, B. cereus, S. cerevisiae and M. mucedo which displayed lower susceptibility. Petroleum ether extract exhibited moderate (p<0.001) zone of inhibition against S. enteriditis, C. jejuni, B. cereus and H. anomala. Also, previous scientific studies on C. longa reported that ethanol and chloroform extract possess potent antifungal activity (Misra and Sahu, 1971). The antibacterial activity of various extracts of C. longa may be attributed to the presence of active ingredients p-tolymethyl-carbinol, curcumin (Lutomoski et al., 1974; Ramprasad and Sirsi, 1956; Huhtanen, 1980) and essential oils (Banerjee and Nigam, 1978).
All the extracts of A. galanga displayed moderate (p<0.01) to lower zone (p<0.05) of inhibition except methanol extract which showed significant (p<0.001) inhibition against E. coli, C. prefringens, S. aureus and H. anomala. Alpinia galanga contains volatile oils, resins, galangol, kaempferid, galangin, alpinin and starch. The major constituents of its essential oils are β-farnesene, myrcene and 1, 8 cineole, β-bisabolene, β-caryophyllene and β-selinene (Kubota et al., 1999) which may be responsible for its antibacterial and antifungal activities.
Generally, the high concentrations of phenolic compounds in these natural products accounts for their antioxidant property (Burt, 2004; Lin et al., 2005; Delaquis et al., 2002; Nevas et al., 2004). Addition to the enormous scientific studies reported by several researchers on the antimicrobial activities of these herbs, there is also enough documented data suggesting the high positive correlation between antimicrobial activity, total phenolic content and antioxidant property (Shan et al., 2005; Wu et al., 2006; Kudo et al., 2004). Several studies have reported on the antioxidant property of Z. officinale (Dhuley, 1999; Shobana and Naidu, 2000; Halvorsen et al., 2002), C. longa (Jay, 2006; Cai et al., 2004) and A. galanga (Poh and Hasim, 2000; Juntachote and Berghofer, 2005).
CONCLUSION
The results of the present study reveals that all the selected species of Zingiberaceae possess potent antimicrobial activity against selected food borne pathogens which might be due to the presence of phenolic compounds and is well substantiated with the evidence of previously documented study. Hence, due to its strong antimicrobial activity imparting extended shelf-life with less harmful effects, they can be used as natural preservatives with considerable market prospects in the food industry.
ACKNOWLEDGMENTS
The authors are thankful to Dato’ Prof. Dr. Ishak Bin Tambi Kechik, Vice-Chancellor and Dato’ Edmund Santhara, CEO, Masterskill University College of Health Sciences, Malaysia, for their funding, continuous encouragement and support.