Antimicrobial and Antioxidant Activities of Betel Oil
Total Page：16
File Type：pdf, Size：1020Kb
Kasetsart J. (Nat. Sci.) 40 (Suppl.) : 91 - 100 (2006) Antimicrobial and Antioxidant Activities of Betel Oil Panuwat Suppakul*, Nutcha Sanla-Ead and Panchuti Phoopuritham ABSTRACT Betel oil has been studied for its antimicrobial and antioxidant activities against ten pathogenic and spoilage bacteria and three strains of yeast using an agar well diffusion assay and against oxidative bleaching using a b-carotene agar well diffusion assay, respectively. The minimum inhibitory concentration (MIC) and minimum oxidative bleaching inhibitory concentration (MOBIC) of betel oil were determined using an agar dilution method. At the concentration of 50 mL mL-1, betel oil showed a zone of inhibition, ranging from 9.15 to 17.30 mm in diameter. The MICs of betel oil in a range of 12.5- 100 mL mL-1 could inhibit the growth of all test microorganisms except Pseudomonas aeruginosa, which was not sensitive to this oil even at the highest concentration (200 mL mL-1). The most sensitive bacteria to betel oil were Listeria monocytogenes and Salmonella Enteritidis. Betel oil (100 mL mL-1) also revealed ability to inhibit the oxidation of b-carotene, yielding a yellow zone surrounding the well with a 8.40 mm in diameter. The MOBIC of betel oil was 100 mL mL-1. Betel oil might have a potential application in controlled and released food packaging technology as antimicrobial and antioxidant agents. Key words: betel; antimicrobial; antioxidant; MIC; MOBIC INTRODUCTION Prevention of pathogenic and spoilage microorganisms in foods is usually achieved by The appearance of foods is one of the using chemical preservatives. These chemical major determinants of its sensory appeal to preservatives act as antimicrobial compounds consumers and consequently, sales of the product. which inhibit the growth of undesirable Microbial contamination and lipid oxidation are microorganisms. However, the onset of increasing the main factors that determine food quality loss demand for minimally-processed, extended shelf- and shelf-life reduction. Therefore, preventing life foods and reports of chemical preservatives microbial contamination and delaying lipid as having potential toxicity demand food oxidation are highly relevant to food processors. manufacturers to find alternative means (Conner, The growth of microorganisms in food products 1993; Nychas, 1995). There is a currently strong may cause spoilage or foodborne diseases. debate about the safety aspects of chemical Oxidative processes in food products lead to the preservative since they are considered responsible degradation of lipids and proteins which, in turn, for many carcinogen and teratogenic attributes as contribute to the deterioration in flavour, texture well as residual toxicity. For these reasons, and colour of the products (Decker et al., 1995). consumers tend to be suspicious of chemical Department of Packaging Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok 10900, Thailand. * Corresponding author: e-mail: [email protected] Received date : 24/07/06 Accepted date : 26/09/06 92 Kasetsart J. (Nat. Sci.) 40 (Suppl.) additives and thus the exploration of naturally Bhattacharya et al., 2006). occurring antimicrobials for food preservations The objectives of this study are aimed at receives increasing attention due to consumer investigating for antimicrobial activity of betel awareness of natural food products and a growing leaves oil against several pathogenic and spoilage concern of microbial resistance towards bacteria and yeasts and at assessing for its conventional preservatives (Skandamis et al., antioxidant activity against oxidative bleaching of 2001; Schuenzel and Harrison, 2002). b-carotene. Oxidative deterioration of fat components in food products is responsible for off- MATERIALS AND METHODS flavours and rancidity which decrease nutritional and sensory qualities. An addition of antioxidants Materials is required to preserve product quality. Synthetic Betel oil from fresh leaves was purchased antioxidants (e.g. butylate hydroxytoluene (BHT), from Thai-China Flavours and Fragrances Industry butylate hydroxyanisole (BHA), tert- Co.,Ltd. (Bangkok, Thailand). This oil is obtained butylhydroxyhydroquinone (TBHQ) and propyl by distillation of the fresh leaf of Piper betel Linn. gallate (PG)) are widely used as antioxidants in The company claimed that major constituents of the food industry. Their safety, however, has been betel oil were chavibetol, chavibetol acetate and questioned. BHA was revealed to be carcinogenic caryophyllene. in animal experiments. At high doses, BHT may Dimethyl sulfoxide (DMSO), sodium cause internal and external hemorrhaging, which dihydrogen orthophosphate (NaH2PO4.2H2O), di- leads to death in some strain of mice and guinea sodium hydrogen orthophosphate (Na2HPO4), pigs (Ito et al., 1986). There is much interest butylate hydroxyanisole (BHA), butylate among food manufacturers in natural antioxidants, hydroxytoluene (BHT) and sodium chloride to act as replacements for synthetic antioxidants (NaCl) were purchased from Fluka Chemie currently used (Plumb et al., 1996). (Buchs, Switzerland). Glycerol, linoleic acid and Piper betel Linn. is a tropical plant b-carotene were purchased from Sigma-Aldrich closely related to the common pepper. It is (Singapore). Ethanol was supplied by Liquor extensively grown in Sri Lanka, India, Malaysia, Distillery Organization, Excise Department, Thailand, Taiwan and other Southeast Asian Ministry of Finance (Chachoengsao, Thailand). countries. Its common names are betel (in English), The media used in the present studies pan (in Indian), phlu (in Thai) and sirih (in Bahasa were nutrient broth, yeast malt broth and Indonesian). It has been historically known as bacteriological agar were obtained from Hi-media traditional herb used as mouth wash, dental (India). The count plates used in the experiments medicine, cough medicine, astringent, tonic and were 3M Petrifilm‘ aerobic count plates and 3M others (Farnsworth and Bunyapraphatsara, 1992). Petrifilm‘ yeast and mould count plates. All these Recently, Bhattacharya et al. (2006) have found count plates were supplied by 3M Microbiology that betel ethanolic extract appears to be a Products, USA. promising formulation for further investigation as a new natural photo-protector. Several researchers Microbial strains and inoculum preparation have reported that betel extract and betel oil The microorganisms used in this study showed antimicrobial and antioxidant activities in were Aeromonas hydrophila DMST 2798, Bacillus model systems (Salleh et al., 2002; Lei et al., 2003; cereus DMST 5040, Escherichia coli DMST 4212, Dilokkunanant et al., 2004; Suliantari et al., 2005; Escherichia coli 0157:H7 DMST 12743, Listeria Kasetsart J. (Nat. Sci.) 40 (Suppl.) 93 monocytogenes DMST 17303, Micrococcus luteus plate using a sterile cork borer. Fourty microlitres DMST 15503, Pseudomonas aeruginosa DMST of betel oil in DMSO to give final concentration 4739, Salmonella enterica serotype Enteritidis of 50 mL mL-1 was added in each well and DMSO DMST 15676 and Staphylococcus aureus DMST blank was used as control. The plates of bacteria 8840 were obtained from the Depatment of and yeasts were incubated at 37 êC for 24 h and at Medical Sciences (DMSC), Thailand whereas 30 êC for 48 h, respectively. The zone of inhibition Enterococcus faecalis TISTR 379, Candida surrounding the tested sample well was measured albicans TISTR 5779, Saccharomyces cerevisiae as diameter (mm) using Vernier calipers. The TISTR 5240 and Zygosaccharomyces rouxii antimicrobial index of betel oil was expressed as: TISTR 5044 were obtained from the culture (diameter of clear zone – diameter of well)/ collection at Thailand Institute of Scientific and diameter of well (Villaseñor et al., 2004). Technological Research (TISTR) , Thailand. Stock cultures were stored at frozen temperature in 40 Determination of minimum inhibitory % (v/v) glycerol-either nutrient or yeast malt broth. concentrations Working bacterial culture and yeast culture were The agar dilution method of the grown at 37 êC for 24 h on nutrient agar and at 30 European Society of Clinical Microbiology and êC for 48 h in yeast malt agar, respectively. To Infectious Diseases (2000) was adopted for obtain cells in the stationary growth phase, determination of minimum inhibitory bacterial culture and yeast culture were concentrations (MICs). Petri dishes contained 15 subcultured twice at 37 êC for 24 h on nutrient ml of nutrient agar for bacteria or yeast malt agar broth and at 30 êC for 48 h in yeast malt broth, for yeasts, supplemented by test strains at a density respectively. Cells were harvested by of 1 ¥ 106 CFU mL-1. Four wells (diameter 5 mm) centrifugation at 6,000 ¥ g for 2 min and washed were made in each agar plate using a sterile cork once with a 5 mM NaCl solution. The supernatant borer. Betel oil was dissolved in DMSO in two- was discarded and the cells were washed again. fold serial dilutions from 0.5 to 200 mL mL-1. Bacterial cells and yeast cells were re-harvested Fourty microlitres of betel oil dilutions were and suspended in fresh nutrient broth and yeast individually added in wells and DMSO blank was malt broth, respectively. Cell densities of used as control. The plates of bacteria and yeasts approximately 1 ¥ 106 CFU mL-1 were calculated were incubated at 37 êC for 24 h and at 30 êC for and prepared from cultures by dilution with 0.1 48 h, respectively. Minimum inhibitory M sodium phosphate buffer (pH 7.0). Cell densities concentration was defined