Received date: October 08, 2013; Accepted date: December 20, 2013; Published date: December 26, 2013
Citation: Hariharan B, Singaravadivel K, Alagusundaram K (2013) Identification of Volatile Compounds in Coconut Toddy by GC-MS - Assisted With Different Solvent System. J Microb Biochem Technol 6:017-023. doi:10.4172/1948-5948.1000115
Copyright: © 2013 Hariharan B, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
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Thirty one Volatile compounds from fermented coconut toddy were isolated by extracting with three different organic solvents viz., Diethyl ether, Dichloromethane and Chloroform simultaneously and analyzed by GC-MS. Eleven Distinct flavor compounds were extracted using diethyl ether in which greater quantity constitutes hydroquinone, ethyl hydrogen succinate, 2,4,6,8 – tetraazabicyclo [3.3.0] octon-3-one, 7-nitroimino, Phenylethyl alcohol is followed by oleic acid, hexanoic acid, squalene and n-Hexadecanoic acid in moderate level. 1, 2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester, Dibutyl phthalate and n-Decanoic acids were found to be very trace amount. Dichloromethane was able to find out another set of twelve compounds including ethyl hydrogen succinate, Di-n-octyl phthalate, Pentanoic acid (10-undecenyl ester) and nonanoic acid as a major compounds followed by 3-Pentanol, 2, 3-dimethyl, 1,2-Benzenedicarboxylic acid (butyl octyl ester and diheptyl ester) in moderate level and lower levels of dichloroacetic acid, 3,4-Hexanediol and 2-Buten-1-ol propanoate. Another set of extraction using chloroform repeatedly showed the presence of squalene as studied in diethyl ether extraction. It also shows the lupeol in higher level along with lower concentrations of 1,2-Benzenedicarboxylic acid, diisooctyl ester, 2,3-Epoxyhexanol, Propanedioic acid, and Aminocyanoacetic acid.
Cocos nucifera; Coconut toddy; Organic solvents; Volatile compounds; GC-MS
Toddy is a fermented sap collected from young inflorescence of tropical plants belonging to Palmae family, such as the oil palm (Elaeis guineensis), coconut palm (Cocos nucifera) date palm (Phoenix dactylifera) and raffia palm (Raphia hookeri). Nearly 30 different species of palm trees grown in various regions around the world are used for the production of toddy. Tapping toddy from the coconut palm tree is very ancient and skilled method. When the palm has reaches the normal bearing stage, the leaf axis produces a spadix or inflorescence (Figure 7). To prevent it from opening, the spathe is tied around with fiber. This unopened flower (spathe) is prepared by slightly brushing it with gentle taps of small mallet daily morning and evening. By the end of second or third week, the spathe would be almost ready to produce toddy. Now, about two to three inches is sliced from the tip of spathe. During the preparation, the spathe gradually bends over and the sap starts oozing out from the spathe, a receptacle (usually a pot) is placed underneath to collect it. The flow of sap increases gradually with time. The pot should be changed twice daily and at the same time, a thin slice is shaved from the end of the spathe, along with simultaneous slight tapping with a mallet and smearing a mixture of bruised leaves on the tip of the spathe to stimulate the flow of sap .
Fresh toddy is a sweet, oyster white and translucent fluid considered to be as pure as mother’s milk. It is a refreshing health drink traditionally believed to have many medicinal properties. Coconut toddy contains a small amount of protein, fat, minerals and vitamins as well as sugar components. These could possibly interact during processing and form some of the volatile components. If it is kept undisturbed, fermentation occurs and progresses, resulting in the continuous production of acetic acid. The microorganism responsible for this fermentation is dominated by yeast, especially Saccharomyces cerevesiae. Sources of fermenting organisms are generally the gourds, tapping vessels and air [2,3]. This process changes the characteristic smell of fresh toddy into fermented acid smell. Active contributors to the aroma of palm toddy were recently studied with identification of those compounds which induce the characteristic alcoholic, malty and floral-fruity notes of palm wine. Babasaheb Bhaskarrao Borse et al. have reported the chemical composition of fresh, clarified and fermented coconut sap  responsible for the unpleasant odor and converting toddy into unpalatable. The chemical composition is varying with place and exposure time of sugary contents to the microflora in the sap and tapping time. Besides its nutritional properties, the shelf life of the drink is very little which leads to the development of various technologies for the preservation of coconut toddy. But studies on the flavor compounds present in the coconut toddy were minimal. Hence this study was undertaken with an objective of creating a database on the volatile compounds which helps to understand the difference in the usage of organic solvents in extracting the various volatile compounds from a coconut toddy sample.
Toddy was collected by tapping the unopened spadix of the coconut trees (Cocos nucifera) available in the coconut plantation of Indian Institute of Crop Processing Technology, Thanjavur (Tamil Nadu, India.)
Collection of coconut toddy
Regularly irrigated coconut tree was used for collecting toddy samples. Toddy was collected in earthen pot covered with nets to avoid insect’s entry. About 750 ml of sample from the selected tree was collected every day. The toddy collected at 6 a.m. was used for the study and it was processed for the analysis within 2 hours after collection. For the whole study, single tree’s samples were used for the analysis in duplicates.
Isolation of volatiles from coconut toddy
The collected samples were filtered using filter paper to remove the foreign matters if any, present in the sample and stored. 50 ml of coconut toddy was taken into the separating funnel and 50 ml of organic solvent was added to it. The funnel was stoppered tightly and shaken well. Gas was produced inside the separating funnel which was released at equal intervals by opening the knob. The shaking was done until there was less pressure inside the funnel. The funnel was kept undisturbed for 10 minutes in a stand. Separation of two layers viz, aqueous layer and organic layer inside the funnel was observed. The aqueous layer was drained and 25 ml of the organic solvent was added to the organic layer and the first step was repeated. Then the extract was collected in a conical flask through a Whatmann filter No. 4 paper using sodium sulphate to get clear solution. The organic layer was concentrated to about 2 ml by giving nitrogen flushing to the sample further to avoid the presence of moisture in the sample if any and then it was injected into the GC-MS for analysis .
GC-MS analysis was carried out on a GC CLARUS 500 PerkinElmer system comprising a gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions: column Elite-1 fused silica capillary column (30×0.25 mm ID×1EM df, composed of 100% Dimethyl poly siloxane), helium (99.999%) was used as carrier gas at a constant flow of 1ml/min and an injection volume of 0.5μl was employed (split ratio of 10:1) injector temperature 250°C; ion-source temperature 280°C. The oven temperature was programmed from 50°C (isothermal for 2 min), with an increase of 10°C/min, to 200°C [5,6]. Then 5°C/min to 280°C, ending with a 9 min isothermal at 280°C. Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 40 to 550 Da. The total MS running time was 36 minutes. The comparative percentage of each chemical constituent was calculated by comparing the average peak area to the total areas. This comparison may give an idea about the range of compounds dominated in the sample. Turbomass 5.2 software was used. The identification was done by matching the mass spectra obtained with those present in the “NIST 2005” Library (Figure 1).
In this study, 31 volatile components were identified in fermented coconut toddy among which 2 were recurrent. They are Squalene and Ethyl hydrogen succinate. Borse et al. have reported that around 25 volatile components were identified in unfermented coconut sap . They also noted that the major volatile components in coconut sugar were dodecanoic acid, acetic acid, 2-undecanone-decanoic acid, 2- nonanone and 2-furfural . Eleven distinct flavor compounds were extracted using diethyl ether (Table 1) in which greater contribution was by hydroquinone (31.71%) followed by ethyl hydrogen succinate (31.03%), 2,4,6,8 – tetraazabicyclo [3.3.0] octon-3-one, 7-nitroimino (15.06%), Phenylethyl alcohol (11.69%) as observed from Figure 1. Compounds namely oleic acid (4.18%), hexanoic acid (2.72%), squalene (1.20%) and n-Hexadecanoic acid (1.10%) were observed in moderate level. Apart from these, trace amounts of 1,2-Benzenedicarboxylic acid (0.54%), Dibutyl phthalate (0.39%) and n-Decanoic acid (0.32%) were noticed. Analysis of the coconut toddy by GC-MS exposed some positive facts also. It was reported that the major compound responsible for the fermented odor of coconut toddy could be succinic acid as a byproduct after sugar fermentation and hexanoic acids along with other compounds such as decanoic acid, Phenyl ethyl alcohol. Flavor such as 3-hydroxy-2-butanone was reported to be responsible for sweet-odour as reported by Naknean et al. . This volatile flavour compound in palm sugar sap as reported by Taiapaiboon . In addition some esters including isoamyl acetate, ethyl acetate, ethyl hexanoate, ethyl octanoate, ethyl decanoate and ethyl-9-decanoate have been detected in palm sap. This result is similar to the result reported by Samarajeewa et al.  and Uzochukwu et al. . Normally, many esters are formed during yeast fermentation. Isoamyl acetate in palm sap contributed to fruity and sweet aroma. This compound is derived from isoamyl alcohol and acetyl coenzyme A by alcohol acetyl transferase in yeast .
|S.No||RT||Compounds identified in Diethyl ether extract||Molecular Formula||MW||Peak Area %|
|4||6.12||Ethyl hydrogen succinate||C6H10O4||146||31.03|
|11||25.22||1,2-Benzenedicarboxylic acid, mono (2-ethylhexyl) ester||C16H22O4||278||0.54|
|Compounds identified in Dichloromethane extract|
|12||6.05||Ethyl hydrogen succinate||C6H10O4||146||32.44|
|14||11.63||Dichloroacetic acid, 2,2-dimethylpropyl ester||C7H12Cl2O2||198||0.46|
|16||15.08||1,2-Benzenedicarboxylic acid, diheptyl ester||C22H34O4||362||0.93|
|17||16.43||1,2-Benzenedicarboxylic acid, butyl octyl ester||C20H30O4||334||1.85|
|19||17.16||3-Pentanol, 2-chloro-4-methyl-, (R*,S*)-(ñ)-||C6H13ClO||136||1.39|
|21||19.26||Pentanoic acid, 10-undecenyl ester||C16H30O2||254||18.54|
|23||29.58||1-Monolinoleoylglycerol trimethylsilyl ether||C27H54O4Si2||498||13.90|
|Compounds identified in Chloroform extract|
|2||10.60||Butanoic acid, 2-oxo-, methyl ester||C5H8O3||116||0.00|
|3||14.11||D-Mannopyranoside, methyl 3,6-anhydro-||C7H12O5||176||0.00|
|4||16.56||Propanedioic acid, propyl-||C6H10O4||146||0.51|
|6||25.20||1,2-Benzenedicarboxylic acid, diisooctyl ester||C24H38O4||390||3.05|
Table 1: Compounds identified by GC-MS in Diethyl ether, Dichloromethane extract, Chloroform extract of coconut toddy.
Twelve compounds were extracted using Dichloromethane, the major compounds are ethyl hydrogen succinate (32.44%), Di-n-octyl phthalate (23.17%), Pentanoic acid [10-undecenyl ester] (18.54%) and nonanoic acid (4.63%) (Table 2) Compounds like 3-Pentanol 2, 3-dimethyl (1.85%), 1,2-Benzenedicarboxylic acid (butyl octyl ester (1.85%) and diheptyl ester (0.93%) were present in moderate level and compounds like dichloroacetic acid (0.46%), and 3,4-Hexanediol and 2-Buten-1-ol propanoate (0.37%) were observed in trace amounts (Figure 2).
|S.no||Solvent used||Name of the compound||Activity|
|1||Chloroform, diethyl ether||Squalene (41.16%)||Squalene is ahydrocarbonand atriterpene, and is a natural and vital part of the synthesis of cholesterol, steroid hormones, and vitamin D in the human body. It helps to stimulate the immune response through production ofCD4memory cells as a vaccine adjuvant.|
|2||Chloroform||Lupeol (53.86%)||Antibacterial, Antioxidant, Antitumor, Cancer preventive, Immuno stimulant, Chemo preventive, Lipoxygenase inhibitor, Pesticide|
|3||Diethyl ether||1-Monolinoleoylglycerol trimethylsilyl ether (23.17%)||Antimicrobial and antifouling activities.|
|4||Chloroform||1,2-Benzenedicarboxylic acid, diisooctyl ester (3.05%)|
|5||Dichloromethane||1,2-Benzenedicarboxylic acid, butyl octyl ester (1.85%)|
Table 2: Activity of important phyto-components identified in Coconut toddy analyzed by GC-MS .
addition, extraction using chloroform revealed the important compounds such as Lupeol (53.86%) and Squalene (41.16%) in higher concentrations (Table 1) distinctly observed from the peak area obtained in GC-MS (Figure 3). These compounds are reported to have medicinal properties as well as promoting physiological functions. Also, other compounds like 1,2-Benzenedicarboxylic acid diisooctyl ester (3.05%), and 2,3- Epoxyhexanol (1.02%) were observed in low level and compounds like Propanedioic acid, propyl- and Aminocyanoacetic acid were present in trace amount extracted during the process. Extraction using different solvents followed by analysis using GC-MS facilitated the identification of important phytochemicals (Table 2) such as Lupeol, Squalene, 1-Monolinoleoylglycerol trimethylsilyl ether, 1,2-Benzenedicarboxylic acid, diisooctyl ester and 1,2-Benzenedicarboxylic acid, butyl octyl ester. It was reported that squalene (Figure 4) extracted using Chloroform in the present study plays a vital role in the synthesis of cholesterol, and vitamin D in the human body . It is also used as a vaccine adjuvant in most of the vaccine preparation to develop the immune response. Lupeol (Figure 5) possess antibacterial, antioxidant, chemo preventive activities . The remaining compounds like 1-Monolinoleoylglycerol trimethylsilyl ether (Figure 6), 1,2-Benzenedicarboxylic acid, diisooctyl ester and 1,2-Benzenedicarboxylic acid, butyl octyl ester shows antimicrobial and antifouling activities .
During the studies, solvents like Acetone and Acetonitrile were used for standardization; both the solvents were miscible with the toddy samples taken for the analysis. However, the compounds extracted using dichloromethane and chloroform were observed different. This may be due to the difference in their boiling point, polarity difference and their density nature of the solvents. The volatile compounds identified in coconut toddy from this constituency with three solvent systems such as diethyl ether, dichloromethane and chloroform. The presence of the identified compounds were responsible for the flavor formation which is different from the compounds isolated from fresh, clarified, fermented neera . This variance in the compounds may be due to the method of collection of neera and toddy. In our experiment, the fermented toddy was collected directly without exposing to the sun light and extraneous matter like insects and other organisms. GC-MS of the coconut toddy assisted with different solvent systems exposed some medicinally important compounds (Table 2) too along with the compounds responsible for the off odor was the important findings in this present study.
The authors are grateful to Dr. K. Alagusundaram, Director, Indian Institute of Crop Processing Technology, Thanjavur for providing all the facilities, encouragement and support used to carry out the work. Also thanks are due to National Agricultural Innovation Project and Indian Council of Agricultural Research for the funds provided.