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Effect of Electron Beam Irradiation on the Volatile Flavor Profile of Elettaria Cardamomum (L.) Maton., from Pakistan.

Byline: Naeem Khan, Nargis Jamila, Ji Yeon Choi, Eun Yeong Nho, Muhammad Imran, Iqbal Hussain, In Min Hwang and Kyong Su Kim

Summary: This study was aimed to investigate the effects of electron beam irradiation on volatile flavor profile of Elettaria cardamomum (L.) Maton., from Pakistan. The volatile flavor compounds of non-irradiated and 1, 5, 10 and 20 kGy E-beam irradiated samples were extracted by simultaneous distillation-solvent extraction (SDE) and analyzed by gas chromatography mass spectrometry (GC-MS). Volatile flavor compounds were slightly changed by E-beam irradiation as some of the components showed a minor increase or decrease in concentration after ionization treatment. A total of 136 (10684.15 mg/kg) volatile flavor compounds were detected in non-irradiated control samples which decreased to 133 for 1 kGy (11068.61 mg/kg), remained the same 136 for 5 kGy (10884.10 mg/kg) and 10 kGy (11762.52 mg/kg), and increased to 137 for 20 kGy (10973.78 mg/kg) E-beam irradiated samples. There was no major change observed in the application of E-beam irradiation treatment of the samples compared to the non-irradiated ones.

E-beam irradiation was therefore concluded to have no adverse effect on the volatile flavor of Elettaria cardamomum (L.) Maton and doses up to 20 kGy were declared not to interfere with the flavor perception of consumers.

Keywords: Volatile flavor, E. cardamomum (L.) Maton., E-beam irradiation, simultaneous distillation-solvent extraction (SDE), gas chromatography-mass spectrometry (GC-MS)

Introduction

The "Queen of Spices", Elettaria cardamomum (L.), Maton., is one of the world's most ancient spices and the third most expensive spice, after saffron and vanilla [1, 2]. It is commonly known as small cardamom, green cardamom or true cardamom [3]. Originally E. cardamomum belong to the tropical regions of Asia [4], but presently cultivated around the world especially in India, Sri Lanka, Nepal, Pakistan, Bhutan, Iran, Vietnam and Costa Rica [1, 5, 6]. This spice is a non-perishable perennial herbaceous plant belonging to the ginger family Zingiberaceae, growing from thick rootstalk up to 4 m in height. The commercial part of the plant is the fruit, which is used as spice and a flavoring agent [7]. E. cardamom is used for flavor in tea and coffee in Pakistan, the Kakakule in Turkey and Masala tea in India. As a flavoring agent it is also used in the preparation of curries, cakes, breads, pickles, rice, and drinks [3, 6].

In the Middle-East countries, traditionally cardamom is used along with coffee in making of a beverage Gahwa [8]. The essential oil of fruit is known for its traditional health care, fine perfumery application, and the use in cosmetic preparation [6]. As a traditional herbal medicine, it has been prescribed in the treatment of gastrointestinal, stomachic, digestive, antiemetic, and carminative disorders [3, 9, 10].

Spices are often originated in developing countries where harvest and storage conditions are inadequately controlled with respect to food hygiene. Mostly they are dried in the open air and become seriously contaminated by air-and soil-borne bacteria, fungi, and insects. Bacterial plate counts of one to 100 million per gram of spice have been reported in literature [11]. According to WHO, good manufacturing practices during harvest and processing could improve their hygienic quality, but frequently not to an extent sufficient to obtain an acceptable microbiological purity level [12]. Therefore irradiation of spices is recommended around the world to extend their shelf life, improve hygienic quality and promote international trade.

Literature survey revealed that in recent years, there have been several research studies on the chemical composition of cardamom fruits from around the world. These included studies from Costa Rica [13], Italy [14], India and Sri-Lanka [10, 15, 16], Pakistan [17], Turkey [2], and Iran [18]. All these studies on the chemical compositions of cardamom are still limited and require further analyses.

Most importantly, to the best of our knowledge, no research work has been reported so far on the effects of irradiation on the volatile flavor compounds of spices such as Elettaria cardamomum (L.), Maton. Research studies on the effects of irradiation techniques on volatile flavor constituents of spices are urgently required to ascertain their safety, acceptability and promote international trade. In view of these objectives this study was designed to investigate the volatile flavor profile compounds of E. cardamomum and to further evaluate any possible effects of sterilization by irradiations such E-beam on these flavor compounds. From literature the well-known method of choice for volatile flavor studies involves extraction by simultaneous distillation-extraction (SDE) and analyses by gas chromatography-mass spectrometry (GC-MS) [19-21].

This advanced SDE-GC-MS technique was applied for the analysis of volatile flavor compounds of E. cardamomum spice fruits collected from Pakistan. The identified volatile flavor compounds of the E-beam irradiated samples at 1, 5, 10 and 20 kGy doses were compared with those from non-irradiated (0 kGy) control ones to find out any possible effects of these irradiation doses on the overall flavor of the subject spice.

Experimental

Samples Collection

E. cardamomum fruit samples were collected in triplicate from a local super market in Peshawar, Khyber Pakhtunkhwa, Pakistan. These were identified by a plant taxonomist at Kohat University of Science and Technology, Kohat, Pakistan. A voucher specimen of the samples was deposited at the university herbarium center for future reference. The collected samples were labeled properly in plastic bags and stored at -22 oC in refrigerator (MICOM CFD-0622, Samsung, Korea) until required for analysis.

Reagents

All the reagents used in this research work were purchased from sigma Co. (St Louis, MO, USA) otherwise stated. The extraction solvents; n-pentane and diethyl ether were purchased from Fisher Scientific (Waltham, MA., USA) and were redistilled using a wire spiral packed double distilling apparatus (Normschliff Geratebau, Wertheim, Germany) before use. The purified water (Milli-Q) was obtained through a water purification system (Millipore Corp., Bedford, USA). Anhydrous Na2SO4 was used for dehydration of organic solvents after burning for overnight at 650 oC in a furnace (F 6000, Barnstead Thermolyne Co., IA, USA) and allowing cooling down in desiccator.

Samples Irradiation

Samples were irradiated at 1, 5, 10 and 20 kGy doses by E-beam accelerator (model ELV 4, 2.5 MeV, EB-Tech., Ltd., Daejeon, Korea). The beam currents used were 0.47 mA (0.25 kGy), 1.3 mA (0.5 kGy), 2.96 mA (1 kGy) and 3.7 mA (3 kGy), and velocites of 20 m/min (0.25, 0.5, 1 kGy) and 10 m/min (3 kGy). The absorbed doses were investigated with cellulose triacetate (CTA) dosimeter.

The irradiated samples were stored at -22 degC in refrigerator until required for analysis. The non-irradiated samples were used as control [22, 23].

Extraction of Volatile Flavor Compounds

Each 30.0 g of E. cardamomum sample was homogenized using a blender (MR 350CA, Braun, Spain) and mixed well with 500 mL distilled water. The pH was adjusted at 7.0, using 0.1 N NaOH and 0.1 N HCl through pH meter (HM-30P, DKK-TOA Corp., Tokyo, Japan). Then as internal standard, 10 mL of n-butyl benzene (110 ppm in n-pentane) was added and the resultant slurry so obtained was used for the extraction of flavor compounds. The volatile compounds were extracted for 3 hours with 100 mL redistilled n-pentane/diethyl ether mixture (1:1, v/v), using a simultaneous steam distillation and extraction (SDE, Likens and Nickerson type) apparatus [24], as modified under atmospheric pressure by Schultz et. al. [25]. The extract obtained was dehydrated with 10 g anhydrous Na2SO4 for overnight and concentrated to final volume of approximately 2.5 mL using a vigreux column.

Finally it was concentrated to 0.5 mL under mild stream of N2 gas. This sample obtained was used for the gas chromatography-mass spectrometry (GC-MS) analysis [26, 27].

Analysis of Volatile Organic Compounds by GC/MS

The quantitative analysis was done by using gas chromatography-mass spectrometry instrument (GC-MS-QP2010, Shimadzu, Japan) in the electron impact ionization (EI) mode. The voltage of ionization and temperature of injector and ion source were 70 eV, 230 oC and 250 oC, respectively. The mass spectrometer was scanned from 45 to 450 m/z. The capillary column used for separation was DB-WAX, 60 m length x 0.25 mm diameter, 0.25 um film thickness (Agilent, JandW, USA). The oven temperature was programmed as: 40 oC (isothermal for 3 min) which was ramped to 180 oC (isothermal for 5 min) at 2 oC /min, to 200 oC (isothermal for 10 min) at 4 oC /min, to 220 oC (isothermal for 5 min) at 5 oC/min and then to 250 oC (isothermal for 10 min) at 5 oC/min. Helium was used as the carrier gas at a flow rate of 1.0 mL/min and the sample injector volume was 1.0 uL using a 1:30 split ratio (Table-1) [26, 27].

Table-1: GC-MS conditions and operating parameters as applied for volatile flavor compounds of Elettaria cardamomum (L.) Maton., fruits from Pakistan

###GC-MS###GCMS-QP2010, Shimadzu, Japan

###Column

###DB-Wax (60 m Length, 0.25 mm Diameter,

###0.25 um thickness)

###Carrier gas###He (1.0 mL/min)

###40 oC (3 min), to 2 oC/min-180oC(5 min),

###to 4 oC/min-200 oC (10 min),

Temperature program

###to 5oC/min-220 oC (5 min) and to

###5 oC/min-250 oC (10 min)

Injector temperature###240 oC

Ion source temperature###250 oC

###Ionization###Electron Impact (EI)

###Ionization voltage###70 eV

###Mass range###50 ~ 400 (m/z)

###Injection volume###1uL

###Split ratio###1:100

Identification and Quantification of Volatile Flavor Compounds

Retention index was used as parameter for checking of a solute from chromatogram by comparing the retention time of standard n-alkanes (C7-C40), which appeared above and below the solute [28]. Mass spectra of volatile organic compounds were identified with the help of our own mass spectral data and those contained within the FFNSC 2.0, NIST 05 and Willey 7spectral libraries provided with the GCMS instrument and mass spectral data books [29, 30], as well as by the comparison of retention indices to reference data [31] and online available data from The Pherobase [32]. The similarity ratio more than 90% was considered effective for the identification of volatile compounds in the samples. The quantitative analysis was carried out with the help of peak area percent of internal standard (n-butylbenzene) by using following formula [26, 27]:

Component content (mg/kg) = C x 1000 g/A x B g

Where: A = Peak area of each sample of internal standard; B g = Amount of sample and C = Peak area of each component in sample

Statistical Analysis

Data were reported as average of three replicates (n=3) as mg/kg of the sample. Significant differences (p < 0.05) within means of same compounds among different irradiated dose samples were analyzed by one way analysis of variance (ANOVA), following Tukey's honestly significant difference (HSD) test and Duncan test, in the Statistical Package for Social Sciences (SPSS), Software Version 20 (IBM, New York, USA). The concentrations of chemical compounds significantly different (p 10 kGy), the decrement of some of volatile flavor compounds was also reported [34]. These research findings, verify the slight variation in volatile flavor contents of E. cardamomum fruits by E-beam irradiation in present study from Pakistan.

Conclusion

The E-beam irradiation of E. cardamomum (L.) Maton., fruit samples at 1, 5, 10, and 20 kGy doses caused only slight variations in the number and relative contents of volatile flavor profile compounds. To a greater extent similarity in occurrence of volatile flavor constituents in non-irradiated and irradiated samples was reported. The slight variation reported in the number of volatile compounds was belonging to aldehydes, ketones, oxygenated monoterpenes, sesquiterpene hydrocarbons and oxygenated sesquiterpenes. There was no major change and the volatile flavor of the subject spice remained unaffected by E-beam irradiation technique. All the major flavor compounds responsible for characteristic flavor remained unchanged to a greater extent. Therefore irradiation was declared quite safe up to the recommended dose of 10 kGy.

The application E-beam for sterilization was therefore recommended for spices which will extend their shelf life and additionally would also slightly increase their volatile flavor profile compounds.

Acknowledgments

This research study was financially supported by research grants from Chosun University, Gwangju 61452, Republic of Korea.

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Publication:Journal of the Chemical Society of Pakistan
Article Type:Report
Geographic Code:9PAKI
Date:Jun 30, 2017
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