Antimicrobial spectrum of different solvent based extraction from rhizomes of species of the two genera Curcuma and Zingiber
4.3 Materials and Methods
Bacterial pathogens transmitted commonly through foods are responsible for a significant portion of food-related illnesses (Mead et al. 1999) and pose a high risk to public health. Pathogenic strains from Campylobacter, Salmonella, Listeria and Escherichia coli are well recognized as important food borne pathogens. Bacterial food borne illnesses are still a global health concern and present a continuous challenge for food safety. Assurance of food safety, and prevention and control of bacterial food borne diseases necessarily rely on the ability to detect the pathogens especially in a low number in foods. Increasing investigations on rapid, sensitive and specific detection of bacterial food borne pathogens have advanced the development of detection methods from conventional culture plating techniques to newer techniques. Raman spectroscopy is such a technique based on molecular vibrations and is emerging as an important nondestructive, noninvasive, analytical tool for biologic materials including whole bacteria (Naumann 2000) because of the high specificity and high resolution of vibrational spectra and weak background signal from the aqueous environment (Maquelin et al. 2002). A number of studies employing the Raman spectroscopic technique for the microbiologic analysis (detection, identification and characterization) of bacteria in forms of single cells, colonies or aqueous culture (after drying) have been reported (Kirschner et al. 2001; Grow et al. 2003; Zeiri et al. 2004).
Researchers have examined the bactericidal activity of some metal nanoparticle using Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) (Lindsay et al.
2007; Peter et al. 2002; Raffi et al. 2010). However, reports are scanty in evaluating antibacterial potential of biological samples like essential oils till date. Hence, another aim of the study was to characterize the rhizome essential oils of the selected gingers against Gram-positive and Gram-negative bacteria with the help of micro-Raman spectroscopy.
collected plants (around 1 kg each) were dried at 65 oC in an oven for approximately 4-5 days till the dry weight was constant, manually crushed and stored at 4 oC until all further use. All experiments were performed with the same dried material.
Table 4.1: Four selected Zingiberaceae species collected from NE India and their characteristics used in the study
Species Tag no.
Collection Site
Latitude &
altitude
Habitat Rhizome character
C. amada ZSD 01 Kamrup (Assam)
26° 11'N, 56 m
Hilly slopes and
moist grasslands
Light yellow, creeping, soft, strong smell like raw mango C. longa ZSD 07 Kahikuchi
(Assam)
26° 9'N, 55.5 m
Shady , moist places
Dark Yellow, small tuber, with characteristic smell Z. moran ZSH 02 Tizu
(Nagaland)
27° 4'N, 900 m
Shady , moist, hilly
places
Blackish white in color, much smaller
in size, tuber has strong pungent odor Z.
zerumbet
ZSH 04 Kokrajhar (Assam)
26° 24'N, 65 m
Shady , hot and
humid places
Yellowish in color, larger tubers with strong characteristic
smell
4.3.2 Extraction of crude oil
The rhizome crude oil was extracted from 50 g of each of the oven-dried and coarsely powdered plant material following the Soxhlet extraction method as per the standards AOAC (American Oil Chemical Society) procedure (Arlington 1995) using different solvents viz, n-Hexane, petroleum ether, acetone, isopropanol and methanol at their boiling points. Extracted oils were subjected to simple distillation and evaporation under reduced pressure to remove the solvent. The crude oil was expressed as % vol wt-1 and was stored at 4oC until further use (max. 2 months) for all chemical analysis and bioactivity studies.
Moreover, essential oil of the rhizomes was also extracted by the process of hydro distillation using Clevenger apparatus. This part of oil was washed with anhydrous sodium- sulfate and stored at 4oC.
4.3.3 Antibacterial assay 4.3.3.1 Disc diffusion method
Bacterial cultures viz. Listeria monocytogenes, Salmonella paratyphi, Yersinia enterocolitica (MTCC 859), and E. coli ETEC (enterotoxic) were grown in nutrient agar media (stored slants maintained at 4-5 oC) were transferred to 10 ml nutrient broth and incubated overnight at 37 oC. A preculture was prepared by transferring 1 ml of this culture to 9 ml nutrient broth (Hi media) and incubated for 48 h. The cells were harvested by centrifugation (2795 g for 5 min), washed and suspended in saline. Inoculum (100 µl) was then spread onto the solidified agar plate. The assay was carried out by disc diffusion technique (Bauer et al. 1966). Observations for growth inhibition zone were recorded after 24 h.
4.3.3.2 Growth kinetics
The effect of the rhizome oil fractions were also examined by determining the growth kinetics of bacteria. Bacterial cultures under study were grown in liquid NB media for 12 h.
After two subcultures, 100 µl of 6 h grown fresh culture was inoculated in 2 ml fresh liquid NB and treated with rhizome oil fractions at concentrations 10, 20, 30, 40 and 50 µg /ml.
Cultures were incubated at 28 oC and 180 rpm in orbital shaking incubator for 24 hrs.
Optical density (OD) measurements were taken at 600 nm using a UV-Vis
spectrophotometer (Carry 100) to monitor the bacterial concentration after 8h of growth and every two hours thereafter.
4.3.3.3 Viability assay
To investigate the effect of rhizome oils on viability of bacteria the three bacterial strains viz. Listeria monocytogenes, Salmonella paratyphi, and Escherchia coli ETEC (enterotoxic) were grown in liquid nutrient broth (NB) for 12 h. These were subcultured twice and 6 h grown fresh cultures were used for viability test. Aliquots of 100 µl of the cultures were subcultured in fresh NB media (2 ml) and treated with rhizome oils at various concentrations (10, 20, 30, 40, 50 µg /ml). Cultures were kept for incubation at 28 oC and 180 rpm in orbital shaking incubator for 4 hrs. After incubation, 100 µl of the growth mixtures (bacteria + rhizome oil + media) were inoculated on solid NB agar (2 %) plates and kept at 37 oC for 24 hrs. Control plate contained only inoculum without the test oil fractions. Bacterial growth was observed and numbers of colonies were counted after 12 h and every 2 h thereafter.
4.3.3.4 Raman spectroscopy
Antibacterial efficacy was also characterized by examining the characteristic Raman spectra of test bacteria both gram positive (L. monocytogenes) and gram negative (E. coli) after treating with the rhizome oil. Bacterial cultures raised from single colony were grown in nutrient broth for 12 h. After one subculture, the 6 h grown fresh culture were taken for experiment. The cultures were treated with the rhizome oil fractions (10 µg each) and kept for incubation at 28 oC and 180 rpm in an orbital shaker. The excitation wavelength for recording Raman spectra was 488 nm Argon ion laser. Readings were taken after 12 h of incubation from inoculation. Changes in the Raman shift of the bacteria were observed.
4.3.4 Antifungal assay
Fungal slant cultures viz. Aspergillus niger (NRRL 3), A. niger (NRRL 326) and A. terreus were maintained in potato dextrose agar (PDA) medium. Inoculum (300 µl, 106 spores ml-1) from the spore suspension cultures of the different fungal strains was then spread onto the Czapek Dox Agar (CDA) plates. The assay was done out in the same way as described for bacterial cultures and observations were recorded after 48 h. Ampicillin (USB Amersham
-1 -1
The results were represented as the diameter of zone of inhibition (ZOI) (mm) ± SD, excluding the disc diameter (4 mm). Minimal inhibitory concentration (MIC) of all the fractions was determined according to (Lennette et al. 1974). The different concentrations (25-100%) of rhizome oil fractions were obtained by diluting it in pure dimethyl sulphoxide (DMSO). The minimum inhibitory concentration (MIC) was recorded as the lowest dilution of the tested sample inhibiting the visible growth of the organism after 24-48 h for the bacterial and fungal cultures respectively on the agar plate. The response of the positive control (antibiotic) was treated as 100% inhibition and with respect to that the percentage of inhibition were calculated for each fraction.
4.3.5 Statistical Analysis
Each assay was performed three times and the results were expressed as their mean ± standard error. The data were analyzed by one way analysis of variance (ANOVA) using Instat 3.036 version (http://www.ssc.rdg.ac.uk/software/download.html). Differences between means were tested by LSD tests and were considered significance at p <0.05.