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Keywords:

  • Eleutherine americana;
  • food contamination;
  • natural preservative;
  • Staphylococcus aureus

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

ABSTRACT:  About 106 samples of ready-to-eat foods were purchased over a period of 3 mo out of which 76 (71.69%) were contaminated with Staphylococcus aureus. S. aureus isolated from the food samples were characterized phenotypically using traditional biochemical methods. Ninety-four percent of the isolates were mannitol fermenters, 86% positive for coagulase test, while 80% produced lipase enzyme. Antibiotic susceptibility test revealed that 21% and 63% of the food isolates were resistant to oxacillin and penicillin, respectively. The antibacterial activity of the bulb of Eleutherine americana used in Thai cuisine was investigated by agar disc diffusion using 2.5 mg of the crude extract and produced inhibition zone between 14.5 and 15.7 mm, while the minimum inhibitory concentration (MIC) value ranged from 0.06 to 1.00 mg/mL on both food isolates and reference strains. Growth curve in the presence of the crude ethanol extract at 4 MIC showed bacteriostatic effect by 5 log reduction relative to the control. Partially purified fractions tentatively identified by column chromatography were suspected to be responsible for the antibacterial property. This study suggests that E. americana bulb has potential for application as a natural preservative in foods.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

In developing countries, street vending of foods is common because they are cheap and the locations are convenient. However, foods consumed at public eating establishments and street foods have been implicated in food poisoning incidents (Bean and others 1990; CDC 1993). The lack of basic infrastructure, practice of good hygiene, and proper storage facilities contribute to a generally poor microbial quality of street foods (Akinyele 1987; Bryan and others 1992). Also, the year round high temperatures in tropical countries can enhance microbial multiplication in street foods.

Staphylococcus aureus is the most significant human pathogen among the staphylococci, as other species are considered opportunists (Schleifer 1986). Although S. aureus is an important pathogen, many healthy people carry it as part of the normal population of microorganism associated with the nose, throat, perineum, or skin. S. aureus has the ability to produce several exoenzymes that contribute to virulence such as coagulase, haemolysin, protease, and lipase and enterotoxin. Staphylococci can grow and produce heat-stable enterotoxins in foods at the temperature range from 7 to 48 °C (ICMSF 1996).

Staphylococcal food poisoning (SFP) is one of the most common types of foodborne disease worldwide. It has been identified as a causative agent in numerous outbreaks of food poisoning, but it is believed to be under reported due to the self-limiting nature of the illness (Dinges and others 2000). However, staphylococcal food poisoning represents a considerable social burden in terms of hospital expenses, loss of patients' working days and productivity, together with the cost of disposing the contaminated food (Normanno and others 2005). An outbreak of acute gastroenteritis in an Australian boarding school was reported where S. aureus was implicated. About 101 out of 113 cases were hospitalized, while the causative agent was isolated from stool specimens and swab of palmar skin lesion of one of the healthy kitchen workers (Schmid and others 2007). Foods that are frequently implicated in SFP include meat and meat products, salads, poultry and egg products, cream pies, éclairs, sandwich fillings as well as other foods that require considerable handling during preparation (Bremer and others 2004).

Food safety is of major concern both to consumer and food industries as there are increasing numbers of food-associated infections. Consumers prefer foods of high nutritional quality with little or no chemical preservatives due to their adverse effect (Brewer and others 1994). Herbs and spices have been safely used by people to impart desirable flavors and aroma to the local foods and some of these have been reported to possess antimicrobial activities thus preventing spoilage and extending shelf life of foods (Shelef 1984; Nakatani 1994). Many researchers have reported activities of a number of medicinal plants on S. aureus (Khan and others 2001; Somchit and others 2003; Oometta-aree and others 2006; Shan and others 2007).

Eleutherine americana Merr. is a herbal plant whose red bulb has been used as folk medicine (Ding and Huang 1983) and commonly employed in Thai cuisine. Bioactive compounds from bulbs of this plant such as anthraquinones, bi-eleutherol, and elecanacin have been isolated and identified (Hara and others 1997; Xu and others 2006). Preliminary data from our research group indicate that the ethanol extract of this plant species possesses good antibacterial activity against most Gram-positive pathogenic bacteria (Voravuthikunchai and others 2007). However, no scientific study has been reported on the activity of the bulb of this plant against foodborne strains. The aims of this study therefore were to investigate ready-to-eat food for S. aureus contamination and to assay antibacterial properties of crude extract from the bulbs of E. americana. The ultimate goal of this study was to develop an effective food preservative, which is able to inhibit the growth of S. aureus and prevent further enterotoxin production.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Preparation of crude extract from Eleutherine americana

Bulbs of Eleutherine americana were collected and washed with distilled water. Classified reference voucher specimens were deposited at the Herbarium of Faculty of Pharmaceutical Sciences, Prince of Songkla Univ., Hat Yai, Songkhla, Thailand. Samples were washed with distilled water, dried in the oven (BINDER GMBH ED, Bangkok, Thailand) at 60 °C, for 2 d, and then ground to obtain fine particles using a blender. They were soaked in different solvents including, 95% ethanol, hexane (Lab-Scan Analytical Sciences, Bangkok, Thailand), and acetone (Fisher Scientific UK Ltd., Loughborough, Leicestershire, U.K.) in the ratio of 1 : 2 (w/v) (500 g of extract : 1000 mL), mixed together, and left at room temperature for 7 d to allow proper dissolution of the active compounds in the plant extract. The extract was filtered by a Whatman filter paper 125 mm (Whatman Int. Ltd., Maidstone, U.K.). under vacuum at room temperature. The filterates were evaporated under reduced pressure in a rotary evaporator (BUCHI Rotavapor R-114, Switzerland) at 45 °C until the extracts became completely dry. After evaporation, a yellow-brown solid of hexane extract and brown solids of ethanol and acetone extracts were obtained and the extracts were stored at −20 °C until use. The percentage yield of the extract was obtained by dividing final weight of the dried extract with the weight of plant material × 100. All extracts were dissolved in 10% dimethylsulfoxide (DMSO; Sigma-Aldrich, St. Louis, Mo., U.S.A.) before use.

To separate protein fragments of crude hexane extracts, column chromatography was performed by using silica gel 100 (70-120 Mesh ASTM, Merck, Darmstadt, Germany) or silica gel 60 (230-400 Mesh ASTM, Merck). Hexane extract was chromatographed on column chromatography over silica gel 100 H using hexane–dichloromethane (1 : 9), dichloromethane, dichloromethane–methanol (49 : 1) and methanol as eluents. For thin layer chromatography (TLC), aluminum sheets of silica gel 60 254 GF (20 × 20 cm, layer thickness 0.2 mm, Merck) were used for analytical purposes and the compounds were visualized under ultraviolet light (200 to 400 nm). Fractions with similar TLC patterns were pooled.

Food samples

A total of 106 food samples were bought over a period of 3 mo from 2 food centers, (Hospital and student cafeteria) at Prince of Songkla Univ. and a local market at Hat Yai, Songkhla, Thailand. All samples were taken immediately to the laboratory for analyses.

Isolation and identification of S. aureus

Twenty-five grams of each of the food samples were suspended in 225 mL of buffered peptone water (Difco Labs, Division of Becton Dickinson and Co., Sparks, Md., U.S.A.) and aseptically homogenized in a stomacher. Serial 10-fold dilutions were prepared and 0.1 mL each of the different dilutions was transferred onto the surface of Baird-Parker agar (Difco), and incubated at 35 °C for 48 h. Typical colonies from the plates were subcultured on tryptic soy agar (TSA, Merck KGaA) and incubated at 35 °C for 24 h (Lee 2003). For long-term storage, isolates were kept at – 80 °C in brain heart infusion broth (BHIB, Difco) containing 20% glycerol (Vidhyasom Co. Ltd., Bangkok, Thailand) as a cryoprotectant (Hammer and others 2006).

Biochemical tests

S. aureus isolates were subjected to further tests for characterization. These include Gram staining, catalase reaction, production of acid from mannitol salt agar (MSA, Merck), anaerobic fermentation of mannitol, production of acetoin with methyl red Voges-Proskauer medium (Difco), and tube coagulase test using rabbit plasma with EDTA (Difco) (Schleifer 1986).

Preliminary characterization of virulence factors

Haemolysins, lecithinase, and lipase activity were investigated following modified methods (Udo and Jacob 2000). Inocula were prepared by subculturing isolates on TSA and incubated overnight at 35 °C. Well-isolated colonies were then suspended in 0.85% NaCl (Merck) and adjusted to a concentration of approximately 108 CFU/mL. A multipoint inoculator was used to deliver drops of bacterial suspension onto the surface of each pre-dried plate as described subsequently. Haemolysins were detected by spot-inoculating isolates onto TSA agar containing 5% horse blood and incubated plates at 35 °C for 24 h. Haemolytic isolates demonstrated a zone of clearing on the blood agar adjacent to the bacterial growth. Egg yolk agar was used to investigate the lecithinase activity. The plates were prepared by adding egg yolk emulsion (Difco) at a final concentration of 10% (v/v) to nutrient agar (NA, Difco) supplemented with 1% glucose (Merck). The inoculated plates were incubated at 35 °C for 72 h and isolates demonstrating opaque growth were considered lecithinase positive (Baron and others 1994). The nutrient agar plates containing 1% tween 80 (BDH Laboratory Supplies, Poole, U.K.) (v/v) were used to study the lipase activity. They were incubated at 35 °C for 72 h and colonies showing a clear zone adjacent to growth were recorded as lipase positive. Casein agar plates were prepared by adding 2% casein (Sigma-Aldrich) to NA. The plates were incubated at 35 °C for 24 h and isolates producing opalescent zones around the growth were protease positive.

Reference strains

Three reference strains including ATCC 25923, ATCC 23235, and ATCC 27664 were used. ATCC 23235 and ATCC 27664 are enterotoxin-producing strains. All strains were routinely grown in tryptic soy agar (TSA, Merck).

Antibiotic susceptibility

The isolates were tested for antibiotic susceptibility with selected antibiotics using the disc agar diffusion method on Mueller–Hinton agar following the guidelines of CLSI (2006a). The antibiotic discs (Difco) included, erythromycin (15 μg), gentamicin (10 μg), oxacillin (1μg), penicillin (10 IU), sulfamethoxazole trimethoprim (25 μg), tetracycline (30 μg), vancomycin (30 μg). The experiments were performed in duplicate and the strains were classified as susceptible or resistant according to the chart supplied with the discs.

Antibacterial activity of crude extract on S. aureus

Paper disc agar diffusion method was used (CLSI 2006a). Ten microliters of the crude extracts (250 mg/mL) dissolved in DMSO were added to sterile filter paper disc (Macherey-Nagel GmbH) containing 2.5 mg of the extract. The discs were dried at 35 °C overnight and applied on the surface of Muller Hinton agar (MHA, Difco) seeded with 5 h broth culture of the test bacteria adjusted to 108 CFU/mL. The plates were then incubated at 35 °C for 24 h. The experiments were performed in duplicate and the means of the diameters of the inhibition zones were calculated.

Determination of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

A modified microdilution method (CLSI 2006b) was used to determine the MIC of E. americana extracts which produced inhibition zones. One hundred microliters (1: 200) of a 5 h culture of each bacterial strain, containing approximately 106 CFU/mL were applied onto Mueller Hinton broth (MHB, Difco) supplemented with the medicinal plant extracts (2-fold dilution) at concentrations ranging from 0.12 to 1.00 mg/mL. Controls with 1% DMSO and without the extract were set up under the same condition. The microtiter plates were then incubated at 35 °C for 24 h. Minimal inhibitory concentrations were observed at least in duplicate as the lowest concentration of plant extracts that produce a complete suppression of colony growth. Minimal bactericidal concentrations were performed by streaking contents from microtiter wells that gave significant MIC values on fresh MHA and incubated at 35 °C for 24 h. Concentration at which there was no visible bacteria growth after 24 h incubation was regarded as MBC.

Killing curves in the presence of the extract from E. americana

This experiment was performed using the modified method of Lorian (1996) and Carson and others (2002). One milliliter of 5 h MHB culture (1.5 × 106 CFU/mL) was added to MHB containing 1 mL of plant extract at 1/2 MIC, MIC, 2 MIC, and 4 MIC concentrations respectively in sterile test tubes. The tubes were incubated with shaking at 35 °C and the growth of bacteria was measured by enumeration after 0, 2, 4, 6, 8, 12, 16, 20, and 24 h. Surviving bacteria were enumerated and tubes without the extract but with 1% DMSO were incubated under the same conditions which served as control. All assays were carried out in triplicate.

Results and Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

Food isolates

The types of ready-to-eat foods that were analyzed are shown in Table 1. About 70% of the foods were contaminated with S. aureus. Seventeen of the cake samples (73.9%), and 28 out of the 30 (93.3%) meat samples were contaminated with S. aureus, whereas only 10 (45.4%) of chicken sausage were contaminated while no S. aureus was isolated from milk desserts. The contamination rate of ready-to-eat foods that are left at slightly elevated temperatures after preparation was higher than that of sausages and milk samples. The sausages were fried in hot oil at the time of purchase while the milk had been pasteurized and packed in a sealed container. An average count of at least 4 log10 (CFU/g) was obtained from each of the contaminated food samples (data not shown). S. aureus is present on the skin and mucosa of humans, animals, and in the environment (Jay 2000). As a consequence, food products may originally be contaminated during or after processing. Growth of S. aureus is encouraged in foods kept at slightly elevated temperatures. All the food samples bought except the sausage and milk samples were kept at room temperature (30 °C), which is likely to support the growth of any S. aureus that survived the cooking processes or recontamination from the body of the handler after processing (Bremer and others 2004). It may be concluded that the sanitation or temperature control or both were inadequate and under this suitable condition S. aureus present in foods can multiply and produce enterotoxin which when consumed would result to SFP. Lack of personal hygiene among food handlers, poor hand, and surface hygiene has also been reported as factors that contribute to foodborne illness (Cogan and others 2002).

Table 1—.  Sources of Staphylococcus aureus test strains.
Food typeNumber of samplesNumber of sample with S. aureus
Cakes 2317 (73.9%)
Meat 3028 (93.3%)
Pork soup  99 (100%)
Papaya salad  77 (100%)
Steamed fish cake 10 5 (50.0%)
Chicken sausage 2210 (45.4%) 
Milk dessert  50
Total106 76 (71.69%)

Phenotypic characteristics of food isolates

Traditional biochemical techniques were used for the isolation and identification of S. aureus in this study (Schleifer 1986). S. aureus which failed to produce halo on Baird–Parker were subcultured on mannitol salt agar, tested for mannitol fermentation and finally tube coagulase test (Adesiyun and others 1999; Sandel and McKillip 2004). The characteristics of S. aureus isolated from the food samples are shown in Table 2. Ninety-four percent of the isolates were mannitol fermenters and positive for acetoin production while 86% were positive for tube coagulase test. About 25% of the isolates secreted all the 3 enzymes protease, lipase, and hemolysin. At least each of the isolates secreted 1 or 2 enzymes. Most isolates were capable of producing lipase enzyme (81.1%), contrastly, only few isolates possessed lecithinase enzyme (10.3%). The result showed that lipase enzyme was produced more than any other enzyme. This corresponds with Hammer and others (2006) where 32 out of 44 strains tested were positive for lipase enzyme. It has been suggested that one of the important factors enabling S. aureus to survive, colonize, proliferate, and cause human infection is the expression of these virulence factors.

Table 2—.  Characteristics of Staphylococcus aureus isolated from food.
Biochemical tests and enzyme productionPositive isolates (%)
Anaerobic fermentation of mannitol94.0
Acetoin production from Voges-Proskauer medium94.0
Tube coagulase test86.0
Protease enzyme35.8
Haemolysin55.6
Lipase81.1
Lecithinase10.3

Antibiotic susceptibility patterns of food isolates

Figure 1 shows the antibiotic susceptibility patterns of the tested strains. In the antimicrobial resistance trials, 80.2% of the isolates demonstrated antimicrobial resistance properties to at least one of the antibiotics but none was resistant to vancomycin. About 67 (63.2%) exhibited resistance to penicillin while 22 (20.75%) were resistant to oxacillin (Figure 1). A relatively high number of isolates tested in this study were resistant to 1 or 2 antibiotics commonly used in the therapeutic protocols of many human infections. Normanno and others (2007) also reported that about 68.8% strains isolated from meat and dairy products showed antimicrobial resistance properties to all the antibiotics tested except vancomycin. This study revealed that methicillin-resistant strains could be isolated from food. This could be an indication that the source of S. aureus in food samples were the food handlers (Soriano and others 2002), or the food-producing animals (Lee 2003).

image

Figure 1—. Antibiotic susceptibility patterns of Staphylococcus aureus isolated from food. OX = oxacillin, P = penicillin, VA = vancomycin, GM = gentamicin, E = erythromycin, TE = tetracycline, SXT = trimethoprim-sulfamethoxazole.

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Inhibitory activities of the crude extract of E. americana on food isolates

The agar disc susceptibility test of the crude extract from E. americana is shown in Table 3. The average percentage yield of the extract for all the solvents was approximately 4%. The diameter of inhibition zone showed that the crude extract from 3 solvents produced similar activities. Ethanol and hexane extracts were mixed together at same proportion to check if they could produce synergistic effect. The results of the susceptibility of the food isolates when subjected to statistical analysis (ANOVA) revealed that the activities of the ethanol and acetone extract were significantly different from that of hexane and hexane and ethanol. Ethanol extract was used for further studies because of the future plan to add the extract into food. Food isolates produced average inhibition zone between 14.5 and 15.7 mm, while the reference strains produced diameter range of 12.3 to 17.0 mm. The minimum inhibitory concentration for all the reference strains was 0.25 mg/mL while the minimum bactericidal concentration was more than 1.00 mg/mL. The MIC values of the food isolates ranged from 0.06 to 1.00 mg/mL (Table 4). Only two isolates (2.63%) had MIC value of 1.00 mg/mL. About 97% of the isolates had their MIC values within the range of 0.06 to 0.5 mg/mL. The results of the agar susceptibility test and MIC of the crude extract are within the range that has been reported by other researchers where medicinal plants have been shown to possess inhibitory activities against S. aureus (Oometta-aree and others 2006; Shan and others 2007; Voravuthikunchai and others 2007).

Table 3—.  Agar disc susceptibility test of extracts (2.5 mg/disc) of Eleutherine americana on Staphylococcus aureus isolates from food samples and reference strains.
S. aureusInhibition zone (mm)1
EthanolHexaneAcetoneEthanol + Hexane
  1. Values in the same row with different superscript (a-b) are significantly different (P < 0.05).

  2. 1Mean values ± standard error from 76 strains of food isolates.

  3. 2Mean values ± standard error of triplicate results.

Food isolates15.35 ± 0.15a114.51 ± 0.15b115.75 ± 0.14a114.59 ± 0.15b1
ATCC 2592316.37 ± 0.08a216.37 ± 0.08a215.75 ± 0.02a215.87 ± 0.03a2
ATCC 2323516.75 ± 0.14a212.25 ± 0.25b216.75 ± 0.25a217.00 ± 0.25a2
ATCC 2766414.37 ± 0.37a212.50 ± 0.50b215.25 ± 0.25a214.50 ± 0.16a2
Table 4—.  The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the ethanol crude extract from Eleutherine americana against Staphylococcus aureus.
Eleutherine americana (mg/mL)Number of isolates (%)
MICMBC
0.063 (3.94)NA
0.1216 (21.05)NA
0.2530 (39.47)NA
0.5035 (32.89)NA
1.002 (2.63)14 (18.42)
> 1.00NA62 (81.57)

Time–kill assay

Figure 2A shows the time–kill curve of one of the reference strains (ATCC 27664) used in this study. Treatment with crude extract at 4 MIC (1 mg/mL) reduced the viability of S. aureus by at least 5 log relative to control and kept the inoculum at lag phase throughout the period of study. The isolates from food were named Natural Product Research Centre (NPRC 401-476) and were selected for the time–kill curve based on the MIC results. NPRC 412 (Figure 2B), NPRC 438 (Figure 2C), and NPRC 475 (Figure 2D) had MIC values of 1.00, 0.06, and 0.12 mg/mL, respectively. The response of isolate with 0.06 mg/mL to the assay was different, at 4 MIC (0.25 mg/mL) the extract could only produce 3 to 4 log difference compared with the control (Figure 2C). At 2 MIC and 4 MIC, the extract had bacteriostatic effect on inoculum. The inoculum remained at lag phase of growth and there were 5 to 6 log reduction when compared with the control. (Figure 2B and 2D). The results indicated that each strain behaved differently at above MIC concentrations. Time–kill curve is an example of bacteriostatic or bactericidal activity expressed as the rate of killing by a fixed concentration of an antimicrobial agent. Large numbers of isolates tested demonstrated that the extract used at 4 MIC brought about at least 5 to 6 log cycle reduction with respect to the control and kept the inoculum at lag phase at 4 MIC after 24-h exposure.

image

Figure 2—. Time–kill curve of Staphylococcus aureus ATCC 27664 (A), NPRC 412 (B), NPRC 438 (C), and NPRC 475 (D) after treatment with ethanol extract of Eleutherine americana, respectively. The control suspensions (+), the 4 MIC (□), the 2 MIC (▴), the MIC (▪), and 0.5 MIC (◆). The lower detection threshold was 102 CFU/mL.

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Inhibitory activity of partially purified fractions from E. americana

The partially purified fractions, Ea 6 and Ea 9 produced MIC value of 0.125 to 0.25 mg/mL with minimum bactericidal concentrations (MBC) values of 0.25 and 0.5 mg/mL, respectively (Table 5). It is expected that the semi-purified fractions of E. americana should contain higher levels of active compounds whose components possessed better inhibitory activity compared to the crude extract. Bioactive compounds from bulbs of E. americana plants such as naphthoquinones, bi-eleutherol, and elecanacin have been isolated (Hara and others 1997; Xu and others 2006). Medicinal plants rich in naphthoquinones have been reported to possess antibacterial activity (Machado and others 2003). Naphthoquinone derivatives can act as antibacterial agents by participating competitively in electron transport with the cell components (Holmes and others 1964).

Table 5—.  The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the partially purified fractions from Eleutherine americana on Staphylococcus aureus (ATCC 25923).
FractionsMIC/MBC (mg/mL)
Ea 10.5/>1.0
Ea 20.25/>1.0
Ea 30.125/>1.0
Ea 4S> 1/>1.0            
Ea 50.125/1.0
Ea 60.125/0.25
Ea 7S0.5/>1.0
Ea 80.0625/>1.0
Ea 90.25/0.5
Ea 9S0.5/>1.0
Ea 100.0625/>1.0
Ea 110.5/>1.0
Ea 121.0/>1.0

Conclusions

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

This study that was carried out with relatively high numbers of isolates and from different sources demonstrated that the ethanol extract of E. americana produced inhibitory effect on S. aureus isolated from food. At the beginning of this study, we were not expecting such a high rate of contamination of the foods. This could be an indication that the food handlers needed to be given better education in sanitary practices focusing on their potential roles as reservoir of foodborne pathogen. It is established from our study that the crude extract from E. americana could inhibit S. aureus isolated from foods. However, further studies are being carried out to examine the antibacterial effect of the crude extract in food systems along with the sensory evaluation studies.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References

The authors are grateful to the Graduate School, Prince of Songkla Univ. and Thailand Research Fund (DBG 5180021, fiscal year 2008–2011) for funding this study.

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  2. Abstract
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
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