Alcoholic Extraction Enables EPR Analysis To Characterize Radiation

Article pubs.acs.org/JAFC

Alcoholic Extraction Enables EPR Analysis To Characterize RadiationInduced Cellulosic Signals in Spices Jae-Jun Ahn,† Bhaskar Sanyal,† Kashif Akram,†,‡ and Joong-Ho Kwon*,† †

School of Food Science & Biotechnology, Kyungpook National University, Daegu 702-701, Republic of Korea Institute of Food Science and Nutrition, University of Sargodha, Sargodha 40100, Pakistan

‡

ABSTRACT: Different spices such as turmeric, oregano, and cinnamon were γ-irradiated at 1 and 10 kGy. The electron paramagnetic resonance (EPR) spectra of the nonirradiated samples were characterized by a single central signal (g = 2.006), the intensity of which was significantly enhanced upon irradiation. The EPR spectra of the irradiated spice samples were characterized by an additional triplet signal at g = 2.006 with a hyperfine coupling constant of 3 mT, associated with the cellulose radical. EPR analysis on various sample pretreatments in the irradiated spice samples demonstrated that the spectral features of the cellulose radical varied on the basis of the pretreatment protocol. Alcoholic extraction pretreatment produced considerable improvements of the EPR signals of the irradiated spice samples relative to the conventional oven and freeze-drying techniques. The alcoholic extraction process is therefore proposed as the most suitable sample pretreatment for unambiguous detection of irradiated spices by EPR spectroscopy. KEYWORDS: food irradiation, detection of irradiated food, EPR spectroscopy, spices

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INTRODUCTION Spices represent a potential source of microbial contamination for foodstuffs to which they are added. The exposure of spices to a high level of natural contamination by mesophilic, sporogenic, and asporogenic bacteria, hyphomycetes, and fecal coliforms during harvesting and storage is recognized as a realistic probability.1 Most spices are dried in the open air and can become seriously contaminated by air- and soil-borne bacteria, fungi, and insects. Spices and herbs are currently treated with ionizing radiation to eliminate microbial contamination. It has been unambiguously confirmed that treatment with ionizing energy is more effective against bacteria than thermal treatment, and does not leave chemical residue in the food product.2,3 Food irradiation technology is commercially employed in more than 55 countries around the world,4 and increasing amounts of irradiated food continue to circulate into the international trade market.5 However, various national and international regulations with mandatory labeling requirements restrict the general use of this technology. Reliable methods of identification to enforce regulations and traceability are mandatory for the acceptability of irradiated food commodities.6 Consequently, simple, reliable, and routine analytical identification techniques in compliance with regulations and consumer’s right of choice are of key importance. The interactions between biological materials and different forms of energy are very complex and depend on the irradiation and postirradiation conditions, which makes the detection of irradiated food a challenging task.7−9 Electron paramagnetic resonance (EPR) spectroscopy is a unique technique for the detection of paramagnetic species that are generated during the γ-irradiation process. The main advantage of the EPR technique lies in its nondestructive nature and the lack of requirement for sample preparation protocols. Three European standards for the detection of irradiated food via EPR spectroscopy have been released by the European Committee of Normalization (CEN) © XXXX American Chemical Society

and adopted by the Codex Alimentarius Commission as Codex Standards. These pertain to foods containing bone,10 crystalline sugar,11 and cellulose.12 Irradiation of foods of plant origin, such as spices, gives rise to a triplet EPR signal with a hyperfine coupling constant (hfcc) of 3 mT, attributed to the cellulose radical.13,14 However, the identification of radiation-induced cellulose radicals is limited by the lifetime of the paramagnetic species,15 especially in foods containing a high level of moisture. Raffi and Stocker15 observed that even though electron paramagnetic resonance is known to be a very sensitive method, in the case of spices, it did not lead to favorable results because the main radiation-induced signal decreased too fast with the storage time and disappeared before the maximal general commercial storage time. Thus, many researchers have attempted to address this problem by studying various irradiated foods using freeze-drying,16 oven-drying,17 or other techniques.18 However, very little is known about the EPR characteristics of irradiated spices subjected to alcoholic extraction processes for the detection of irradiation. In the present study, the effect of various sample pretreatments on the EPR spectra of spices (turmeric, oregano, and cinnamon) is evaluated by comparison of the spectra acquired before and after irradiation. The main objective of this investigation is to provide a comparative analysis of the EPR spectral features of the irradiated spices subjected to alcoholic extraction techniques with those generated by conventional pretreatments and to extend the applicability of EPR spectroscopy to the detection of irradiated spices. Received: May 13, 2014 Revised: October 26, 2014 Accepted: October 27, 2014

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dx.doi.org/10.1021/jf502258r | J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Journal of Agricultural and Food Chemistry

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and Planck’s constant (h) in appropriate units into the EPR resonance equation.

MATERIALS AND METHODS

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Samples and Irradiation. Dried spices (turmeric, oregano, and cinnamon) packed in polyvinyl chloride (PVC) film were purchased from a local market in Daegu, South Korea, and stored in a refrigerator at 5 °C. These spices were handpicked from carefully selected crops and air-dried before being ground to the perfect texture by accurately calibrated machines during the process of manufacturing. The samples were γ-irradiated (1 and 10 kGy at a dose rate of 2.1 kGy/h) at room temperature using a Co-60 γ-ray source (AECL, IR-79, MDS Nordion International Co. Ltd., Ottawa, ON, Canada) at the Korean Atomic Energy Research Institute (KAERI), in Jeongeup, Korea. The absorbed doses were confirmed using alanine dosimeters with a diameter of 5 mm (Bruker Instruments, Rheinstetten, Germany). The free-radical signals were measured using a Bruker EMS 104 EPR analyzer (Bruker Instruments, Rheinstetten, Germany). After irradiation until EPR measurements the spice samples were stored at normal laboratory conditions, temperature (23 ± 3 °C), and relative humidity (58−64%) in the dark. However, the sample pretreatments were carried out in normal white fluorescent illumination of laboratory. Moisture Contents Determination. The moisture contents of the spices samples were measured after various sample pretreatments. One gram of the powder sample was placed in an aluminum disc and kept in an electrically heated dry oven at 105 °C for 2−3 h under normal atmospheric pressure. The samples were then transferred to a desiccator for 30 min followed by weight measurements within 2 min time to cool to ambient temperature. Finally, the moisture content was calculated until their absolute differences did not exceed 0.002 g. For each sample, three determinations were carried out. EPR Spectroscopy. To investigate the radiation-induced free radicals in the dried spice samples, three different sample pretreatments were conducted to decrease the moisture content of the spices prior to the EPR measurements: (i) FD: Freeze-drying (Bondiro, Ilsin Bio Base, Yangju, Kyunggido, Korea) of the samples.16 (ii) OD: Oven-drying (HB-502M, Hanbaek Scientific Co., Bucheon, Kyunggido, Korea).17 (iii) AE: Alcoholic extraction of the samples was carried out as described by de Jesus et al.19 Approximately 6 g of the spice samples was put into a beaker containing 40 mL of an 80% ethyl alcohol solution in deionized water. The samples were mixed adequately using a glass rod and kept for 30 min at ambient temperature (23 ± 3 °C). The solid fraction was then separated by filtration, pressed further to reduce the moisture, and then dried in air for 1 h at ambient temperature before being put into the EPR tube. (iv) WAE: Washing of the finely chopped sample for 20 min with distilled water using a nylon sieve (150 mm). The residues were used after alcoholic extraction as described above.20,21 The sample (approximately 0.1 g after the pretreatments) was placed in a quartz EPR tube (5 mm diameter). The open end of the sample tube was then sealed with plastic film. EPR was performed after 15 days of irradiation in accordance with the European standard, EN 1787,10 using a X-band EPR spectrometer (JES-TE200, Jeol Co., Tokyo, Japan) at room temperature under the following conditions: power, 0.4 mW; frequency, 9.10−9.21 GHz; center field, 324 ± 2 mT; sweep width, 10− 25 mT; modulation frequency, 100 kHz; modulation width, 1−2 mT; amplitude, 50−400; sweep time, 30 s; and time constant, 0.03 s. All analyses were conducted in triplicate (n = 3), and the mean values (±standard deviation) are reported. Microsoft Excel (Microsoft Office 2010 version) and Origin (version 8) software were used for data analysis and presentation. The EPR signal height was computed using ESPRIT-425 software (Jeol Co.) as the peak-to-peak amplitude of the first derivative spectrum, whereas the signal intensity was presented in arbitrary units per unit sample weight (AU/mg). The g-values were determined using an internal Mn(II) standard attached to the EPR cavity. In addition, g values were also confirmed by calculation using the relation g = 71.448 × microwave (GHz)/magnetic field (mT). This relation was obtained by substituting the values of Bohr magneton (βe)

RESULTS AND DISCUSSION Spectral Features of the Nonirradiated Samples. Figure 1 shows the EPR spectra of all of the nonirradiated spice samples subjected to different extraction techniques. The spectra were invariably characterized by a central line at g0 = 2.006 with a line width (ΔBpp) of 0.63 mT as a native signal, plausibly attributable to the photooxodation of the existing polyphenols. Experimental parameters of the spectrometer were kept identical during

Figure 1. EPR spectra of the nonirradiated spices after different drying treatments. FD, freeze-drying; OD, oven-drying; AE, alcoholic extraction; and WAE, water washing alcoholic extraction. B



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Figure 2. Radiation-induced EPR signals in 10 kGy-irradiated turmeric after different sample drying pretreatments (FD, freeze-drying; OD, ovendrying; AE, alcoholic extraction; WAE, water washing and alcoholic extraction): (a) superposed EPR spectra and (b) magnified spectra.

measurements of all of the samples. Several reports have suggested that these free radicals are semiquinone radicals produced by the oxidation of plant polyphenolics22 or lignin.23

The intensity of the singlet (g0 = 2.006) was almost identical for the spice samples subjected to FD, OD, AE, and WAE. Recently, Akram et al. reported changes in the signal intensity of sauce C



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Figure 3. Radiation-induced EPR signals in 10 kGy-irradiated oregano after different sample drying pretreatments (FD, freeze-drying; OD, oven-drying; AE, alcoholic extraction; WAE, water washing and alcoholic extraction): (a) superposed EPR spectra and (b) magnified spectra.

samples subjected to different sample pretreatments.21 However, no such effect was observed for the present spice samples,

suggesting that the stability of the existing paramagnetic centers depends on the type of food. Thus, complex matrixes of all of the D



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Figure 4. Radiation-induced EPR signals in 10 kGy-irradiated cinnamon after different sample drying pretreatments (FD, freeze-drying; OD, ovendrying; AE, alcoholic extraction; WAE, water washing and alcoholic extraction): (a) superposed EPR spectra and (b) magnified spectra.

Spectral Features of Irradiated Samples. Figures 2, 3, and 4 show the spectral features of the three spice samples subjected to post irradiation sample pretreatments. Complex EPR spectra

spice samples with different moisture contents even after sample pretreatments could be responsible for the inconsistent manifestations of the signal intensities without any defined trend. E



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Table 1. EPR Signal Information from Irradiated Spices with Different Drying Pretreatments g-valueb sample tumeric

dose (kGy)

treatment

0

FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE

1

10

oregano

0

1

10

cinnamon

0

1

10

a

g1

g0 2.006 ± 0.00015 2.007 ± 0.00012 2.007 ± 0.00007 2.007 ± 0.00009 2.007 ± 0.00011 2.007 ± 0.00023 2.007 ± 0.00018 2.007 ± 0.00012 2.007 ± 0.00010 2.007 ± 0.00037 2.007 ± 0.00007 2.007 ± 0.00012 2.007 ± 0.00010 2.007 ± 0.00012 2.007 ± 0.00007 2.007 ± 0.00006 2.007 ± 0.00017 2.007 ± 0.00009 2.007 ± 0.00015 2.007 ± 0.00011 2.006 ± 0.00027 2.007 ± 0.00025 2.007 ± 0.00028 2.006 ± 0.00031 2.007 ± 0.00004 2.007 ± 0.00019 2.007 ± 0.00016 2.007 ± 0.00009 2.007 ± 0.00008 2.007 ± 0.00012 2.007 ± 0.00007 2.007 ± 0.00015 2.007 ± 0.00016 2.007 ± 0.00021 2.007 ± 0.00017 2.007 ± 0.00011

2.024 ± 0.00042 2.024 ± 0.00031 2.024 ± 0.00017 2.024 ± 0.00042 2.024 ± 0.00002 2.024 ± 0.00033 2.024 ± 0.00017 2.024 ± 0.00042

2.024 ± 0.00017 2.024 ± 0.00023 2.024 ± 0.00050 2.025 ± 0.00027 2.024 ± 0.00025 2.024 ± 0.00028 2.024 ± 0.00031

2.024 ± 0.00030 2.024 ± 0.00011 2.024 ± 0.00016 2.024 ± 0.00013 2.024 ± 0.00017 2.025 ± 0.00026 2.024 ± 0.00012 2.024 ± 0.00023

g2

g1−g2 distance (mT)

1.987 ± 0.00013 1.987 ± 0.00055 1.987 ± 0.00020 1.987 ± 0.00027 1.986 ± 0.00010 1.987 ± 0.00013 1.986 ± 0.00055 1.986 ± 0.00053

6.0 ± 0.1 6.1 ± 0.0 6.2 ± 0.2 6.1 ± 0.1 6.1 ± 0.1 6.1 ± 0.1 6.2 ± 0.1 6.2 ± 0.1

1.987 ± 0.00051

6.0 ± 0.1

1.987 ± 0.00026 1.987 ± 0.00022 1.987 ± 0.00062 1.987 ± 0.00051 1.987 ± 0.00019 1.987 ± 0.00029

6.1 ± 0.0 6.0 ± 0.1 6.1 ± 0.0 6.0 ± 0.1 6.1 ± 0.1 6.1 ± 0.1

1.987 ± 0.00012 1.987 ± 0.00005 1.988 ± 0.00020 1.987 ± 0.00017 1.987 ± 0.00015 1.987 ± 0.00012 1.987 ± 0.00003 1.987 ± 0.00012

5.9 ± 0.0 5.8 ± 0.1 5.8 ± 0.1 5.9 ± 0.1 6.0 ± 0.0 6.0 ± 0.1 6.0 ± 0.1 6.0 ± 0.0

a

FD = freeze-drying, OD = oven-drying, AE = alcoholic extraction, WAE = water-washing and alcoholic extraction. bg value (g1 = left, g0 = central, g2 = right) = 71.448 × microwave (GHz)/magnetic field (mT).

pretreatments as depicted in Figure 2. This signal was possibly because of the axially symmetric radiation-induced doublet of carbohydrate radicals and was masked in case of OD samples. A similar observation has recently been reported in case of the irradiated medicinal plant products where irradiated EPR spectra were the superposition of three paramagnetic centers of polyphenols, cellulose, and carbohydrate radicals.7 Therefore, in case of turmeric, all three sample pretreatments, FD, AE, and WAE, were successful in identifying radiation-specific radicals beyond doubt. Characteristics of EPR Spectra of the Irradiated Spices after Drying Treatments. To enhance the distinct visibility of the radiation-specific EPR lines for the spices, the samples were subjected to various pretreatments (OD, FD, AE, and WAE) prior to EPR analysis. The intensity of the central signal (g0) of all of the irradiated spice samples was enhanced. The most prominent increase in intensities was observed for FD and OD samples (Figures 2, 3, and 4). However, in case of OD samples, the spectral profile changed minimally following irradiation, although the two radiation-induced side lines (g1 = 2.024 and g2 =

were observed, with an increase in the signal intensity of the existing weak singlet (g = 2.006). Raffi et al. and Ahn et al. previously reported similar observations, where an intense signal was noticed in the spectrum of irradiated spice and vegetable; the relatively high intensity was attributed to irradiation treatment.24,25 The line width (ΔBpp) of the EPR signal was observed to increase from 0.74 to 0.91 mT, and was possibly due to the induction of multiple paramagnetic centers in the matrix of the spice samples. In addition, a radiation specific triplet signal with a hyperfine coupling constant (hfcc) of 3 mT was observed for all samples and identified as originating from the cellulose radical. The triplet was characterized by g0 (central line) at 2.006, g1 (left line) at 2.024, and g2 (right line) at 1.986. Deighton et al.26 reported that the left line is strictly due to radiation-induced cellulose radicals, whereas the right line, induced by lignin radicals, is sensitive to irradiation as well as the drying processes.27 The triplet signal of the cellulosic radical is a wellestablished signature of radiation processing of foods.12 In case of the irradiated turmeric, an additional weak line at the extreme right of the central signal was observed after FD, AE, and WAE F



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Table 2. Moisture Contents (%) of Dried Spices at Different Drying Treatments treatmenta sample tumeric

oregano

cinnamon

a

dose (kGy)

FD

OD

AE

WAE

0 1 10 0 1 10 0 1 10

1.204 ± 0.063 1.072 ± 0.042 1.261 ± 0.191 1.969 ± 0.249 1.546 ± 0.074 1.455 ± 0.266 2.896 ± 0.164 2.516 ± 0.099 2.409 ± 0.268

1.692 ± 0.250 1.405 ± 0.085 1.757 ± 0.155 2.011 ± 0.728 2.013 ± 0.050 2.369 ± 1.241 2.614 ± 0.589 2.454 ± 0.395 3.197 ± 1.925

5.441 ± 0.234 5.922 ± 0.173 5.999 ± 0.320 5.602 ± 0.384 5.798 ± 0.067 5.844 ± 0.403 6.190 ± 0.231 6.418 ± 0.390 6.277 ± 0.337

4.738 ± 0.574 4.822 ± 0.486 5.363 ± 0.093 5.831 ± 0.393 5.885 ± 0.372 6.164 ± 0.788 6.844 ± 0.452 6.729 ± 0.511 7.296 ± 0.937

FD = freeze-drying, OD = oven-drying, AE = alcoholic extraction, WAE = water-washing and alcoholic extraction.

Table 3. EPR Signal Ratios (Intensity) of Irradiated Spices at Different Sample Drying Pretreatments signal ratio (gn intensity/g1 intensity)b sample tumeric

dose (kGy)

treatmenta

left (g1)

center (g0)

right (g2)

1

FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE FD OD AE WAE

1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 c

41.2 ± 5.0 b 102.7 ± 20.9 a 16.1 ± 0.7 c 21.0 ± 0.8 c 41.3 ± 2.7 b 55.4 ± 4.6 a 13.9 ± 0.8 c 14.9 ± 0.7 c 119.7 ± 45.8 a

1.1 ± 0.0 d 1.3 ± 0.4 d 1.0 ± 0.0 d 1.0 ± 0.1 d 1.0 ± 0.0 d 1.2 ± 0.1 d 1.0 ± 0.1 d 1.0 ± 0.0 d 1.0 ± 0.0 c

1.0 ± 0.0 c 1.0 ± 0.0 c 1.0 ± 0.0 e 1.0 ± 0.0 e 1.0 ± 0.0 e 1.0 ± 0.0 e 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d 1.0 ± 0.0 d

16.6 ± 19.9 b 18.6 ± 16.2 b 45.8 ± 2.2 b 77.8 ± 1.9 a 19.9 ± 0.7 c 16.2 ± 0.4 d 153.5 ± 15.4 b 368.9 ± 167.8 a 21.1 ± 3.3 c 22.3 ± 1.2 c 68.8 ± 2.6 b 95.7 ± 7.2 a 19.1 ± 1.2 c 20.9 ± 1.6 c

1.0 ± 0.1 c 1.1 ± 0.2 c 1.0 ± 0.0 e 1.0 ± 0.0 e 1.0 ± 0.1 e 1.0 ± 0.0 e 1.3 ± 0.0 d 2.8 ± 1.9 d 1.0 ± 0.1 d 1.1 ± 0.1 d 1.0 ± 0.0 d 1.2 ± 0.1 d 1.0 ± 0.1 d 1.0 ± 0.1 d

10

oregano

1

10

cinnamon

1

10

a

Treatments: FD = freeze-drying, OD = oven-drying, AE = alcoholic extraction, WAE = water-washing and alcoholic extraction. bValues within a row followed by different letters are significantly different based on Duncan’s multiple range test.

irradiated sauce samples, an enhancement in the central line signal intensity after sample pretreatment has also been reported. However, in the present case of the spices, no such increase in the central line intensity was observed, probably because of the considerable differences in the moisture contents between the samples subjected to conventional drying methods (FD, OD) and alcohol assisted extraction treatments (AE and WAE) as shown in Table 2. After alcoholic extraction, both the nonirradiated and the irradiated samples showed an increase in moisture content enhancing the chance of recombination of the paramagnetic centers in the spice moiety. The low starch content (ca. 7−12%) may also influence the EPR spectral profile.28 It has recently been reported that the presence of starch and the initial water content of a food sample are responsible for the observed differences between the initial and final spectra of the irradiated samples.7 In the case of cereals, the water content was shown to affect the initial spectra, particularly during irradiation, but the

1.986) were still apparent for all of the samples at a radiation dose of 1 kGy. Table 1 shows that the g-values (g1 = 2.024, g0 = 2.006, and g2 = 1.986) and the distance (g1−g2 = 6.0 ± 0.1 mT) between the two satellite lines did not vary significantly with variation of the samples and the pretreatments, which indicates that the radiation-induced free radicals remained unchanged following different pretreatments. In other words, the detection marker identified as cellulose radicals was apparent for all samples and indicated the irradiation history as per EN 1787.12 However, the intensity of the signal varied detectably with different pretreatment procedures. The side lines were not prominent, especially for the 1 kGy-irradiated OD samples. The signal intensity of the side EPR lines of the cellulose radical increased significantly in the case of all of the AE samples (Figures 2, 3, and 4). However, no further enhancement in the signal intensity was observed after the incorporation of the water-washing step, as reported by Akram et al. in the case of sauce samples.21 In the case of the G



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final spectra were independent of hydration.29 Table 3 shows the ratio of the signal intensities of the central line (g0) and right line (g2) to the intensity of the left line (g1). The signal ratios were also observed to vary with the irradiation doses and the sample types. The ratio of the intensity of the side signals (corresponding to radiation-induced cellulose radicals) to the total intensity of the central EPR signal was reported to be ca. 5% for irradiated Foeniculi fructus and 50% for irradiated citrus fruits.30,31 In general, the signal intensity ratio of the OD and FD samples was 1:6:1 and 1:7:1, respectively. However, the ideal signal intensity ratio in case of radiation-induced cellulose radicals has been reported as 1:2:1.32 The ratio of the signal intensity of the right line to that of the left line was observed to be higher for the OD samples in comparison with that of other pretreated samples, and the maximum value was obtained for cinnamon. However, the spectra of the OD and FD samples were not as clear as those of the AE and WAE samples, and thus identification was difficult for the 1 kGy-irradiated sample. The samples subjected to AE and WAE exhibited improved and distinct radiation-induced triplets, as shown in the cases of turmeric (Figure 2b), oregano (Figure 3b), and cinnamon (Figure 4b), possibly because of the removal of the short-lived polyphenolic radicals (g = 2.006) during the sample pretreatments. Consequently, the intensity of the central lines for AE and WAE treated spice samples was found to be reduced, resulting in relative enhancement of the cellulosic triplet as depicted in Figures 2a, 3a, and 4a. The intensity ratio of the central signal to the left signal of all of the AE samples was observed to be less than that of the OD samples, confirming the aforementioned behavior of the EPR signals as demonstrated in Table 3. The behavior of the EPR lines (g1 and g0) with increase in dose from 1 to 10 kGy after different sample pretreatments was also studied. A dosedependent increase in the radiation-induced cellulose radical signals in the flesh of irradiated vegetables after different sample pretreatments has already been reported by de Jesus et al.33 In case of the spice samples, the signal intensities were measured by the peak to peak amplitude of the EPR lines. To take into account the variable sample quantities in EPR tubes, the signal intensities in arbitrary unit (AU) of each aliquot were divided by the respective sample weight. The percentage EPR signal intensity was then calculated by normalizing the intensities with respect to 100 as shown in Figure 5. In the case of the OD and FD spice samples, the percentage intensity of the g0 line showed a sharp increase at 1 kGy, followed by a slower enhancement up to 10 kGy. On the contrary, in most of the irradiated food samples sharp enhancements in signal intensities have been observed with increasing dose because food samples have not been subjected to any postirradiation treatments prior to EPR measurements.7,34 Therefore, in those food samples, the radiation-induced radicals at g0 responsible for intensity enhancement have not been removed. In this study, the spice samples were subjected to postirradiation pretreatments (FD, AE, and WAE) to eliminate the radiation-induced paramagnetic centers at g0 to enhance the signatures of the radiation-specific radicals in EPR spectra. In case of AE and WAE samples, the intensities of the g0 line did not show any sharp enhancement with the increasing dose because the majority of the radiation-induced polyphenolic radicals were eliminated during chemical extraction processes as depicted in Figure 5. A similar observation of a slow increase in EPR signal intensity in case of irradiated sea vegetables after pretreatments has recently been reported in our earlier communication.35 In addition, at very high dose the intensity normally showed saturation probably because of the increased recombination rate

Figure 5. Response of the EPR signal intensity with increasing radiation dose for the spices (turmeric, oregano, and cinnamon) after different sample drying pretreatments (FD, freeze-drying; OD, oven-drying; AE, alcoholic extraction; WAE, water washing and alcoholic extraction). The percentage EPR signal intensity was calculated by dividing the weight normalized EPR intensities by 100.

of the radiation-induced radicals due to the enhanced spin density. A similar observation has been reported in the case of irradiated guar gum samples, where the EPR signal intensity did not exhibit any distinct trend with increasing dose, possibly because of the fast recombination of the induced free radicals.36 However, the qualitative detection of irradiation features was possible for all of the irradiated spice samples on the basis of changes in EPR spectra, and the change was most prominent in the case of the samples subjected to AE pretreatment. H



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In conclusion, an advanced approach to the EPR analysis of irradiated food samples incorporating the alcoholic-extraction process resulted in improvement in the identification of irradiation status for irradiated spice samples relative to samples pretreated with conventional oven and freeze-drying protocols. The alcoholic-extraction method gave rise to enhanced EPR spectral features, and particularly improved signal intensity, and thus enabled clear detection of irradiation. An addendum waterwashing step (in addition to the alcoholic extraction) produced no further enhancement of the EPR spectra. These results suggest that the signature of the paramagnetic radicals in cellulose, produced by irradiation, can be enhanced by the alcoholic extraction process, and this protocol is useful for the identification of γ-irradiated spices.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +82 53 950 5775. Fax: +82 53 950 6772. E-mail: jhkwon@ knu.ac.kr. Funding

This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (no. 2013R1A1A4A03006993). Notes

The authors declare no competing financial interest.

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Alcoholic Extraction Enables EPR Analysis To Characterize Radiation

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