1
Identification of irradiated foodstuffs using ESR microwave
saturation
Canan AYDAŞ,*, Semra Tepe Çam
Turkish Atomic Energy Authority, Sarayköy Nuclear Research and Training Center, Saray, 06983 Ankara, Turkey
Abstract
The aim of this study primarily is to investigate whether the alteration of the microwave saturation behaviour can be used for samples not having radiation spesific cellulose signals in order to identification of radiation treatment. Twenty different samples (dry plant, herbal, spice etc.) which are not having radiation specific satellite ESR signal were especially selected. It is not possible to detect radiation treatment on these samples by European standart (EN 1787, 2000). MW saturation studies were performed on all samples in the range of 0.01-160 mW. Our experimental results demonstrate that radiation identification can be possible for ten samples and can not be possible for the other ten samples by performing the microwave saturation studies.
2 1. Introduction
Exposing food to high-energy rays (gamma rays, x-rays, or electron beams) in order to kill harmful bacteria,viruses and insect, reducing contamination, extend shelf life. (Aydaş et al., 2008; Mangiacotti et al., 2013). Although food irradiation processing does not cause of the increasing of food temperature up to high temperature. The effect of this prosess is similar to heat pasteurization. Therefore, food irradiation is sometimes termed as "cold pasteurization" or "electronic pasteurization" (Crawford and Ruff, 1996). These terms are some times also used in order to avoid consumer fear of the "radiation" word. Presently, radiation processing is practiced in several countries for over 100 food items.
Application doses are classified as low (up to 1 kGy), medium (1 kGy to 10 kGy), and high-dose (above 10 kGy). Low high-dose are used to inhibition of sprouting of stored tubers, roots and bulbs. The medium dose range can reduce the microbial load, kill the insect eggs and larva. High doses are used to sterilaziation purposes. Although the radiation dose values applied to food vary depending on different purposes, the FAO/IAEA/WHO Expert Committee (1981) agreed the use of ionizing radiation up to an absorbed dose of maksimum 10 kGy inorder to the reduction of food losses and for improvement of the wholesomeness and nutritional quality. Recently, some countries have allowed irradiation at high dose values (>10 kGy) for decontamination of food additives and ingredients (Aydaş et al., 2008). Therefore, it is necessary to develop convenient methods to distinguish irradiated foods from unirradiated ones and also to establish the amount of irradiation dose applied to the foods.
Several detection methods based on physical, chemical and biological principles have been developed for identification of irradiated foods (Sanyal et al., 2011). One of the frequently used physical detection techniques is electron spin resonance (ESR). Three European standards for
3 detection of irradiated food by ESR spectroscopy have been released by the European Committee of Normalisation (CEN). These three standarts are for food containing bone (EN 1786, 1997) , crystalline sugar (EN 13708, 2001) and cellulose (EN 1787, 2000) respectively.
Identification of irradiated food containing cellulose is based on the detection of a characteristic pair of radiation-specific satellite ESR lines found at the symmetric positions on both sides of the single central ESR signal.The spacing of this γ- radiation-induced signal pair related with cellulose free radicals is about 6mT. ESR could be used for detection of radiation treatment, only if the radiation-induced radicals responsible from satellite peaks are stable for at least commercial storage time and corresponding resonance signals are clearly distinguishable from those of unirradiated food sample. Therfore, the ratio of radiation-induced signal intensity to noise intensity should be high enough for precisely detection of radiation treatment. In some cases, only singlet signal intensity increase. Radiation specific satelate peaks can not be observed even immediately after irradiation. (Raffi et al., 2000; Yordanov & Gancheva, 2000; Engin et al., 2011). This single ESR signal is overlapped the naturally present single signal.So, it is not possible to distinguish the signals from each other even by Q-band spectrometry (Yordanov & Aleksieva, 2004). Unfortunately, the increasing of singlet ESR peak intensity can not be sufficient to identification of radiation treatment according to European protocol (EN 1787, 2000). In these cases, recently, ESR MW saturation technique has been attempted for detection of previous radiation treatment (Yordanov et al., 2005; Engin et al., 2011).
Microwave saturation techniques are employed for many various purposes; the spin–lattice and spin-spin relaxation times of paramagnetic centres in inorganic solids are determined; the performance of potential dosimeter materialsis investigated; the radiation induced radicals in
4 materials are characterized;several new applications of quantitative ESR are performed.Such studies often is performed to obtain optimum spectral parameter in order to reach the maximum sensitivity (Lund & Shiotani, 2013) . Here, microwave saturation studies were used to identification of radiation treated foods.
The aim of the current study was to analyse ESR MW saturation behaviour of twenty samples and to test whether this methot could be used for detection of irradiated foods. For the first time with the present investigation, MW saturation behaviour was analysed on so many various food samples.
2. Materials and methods
2.1. Samples
All food samples (Anise, black cumin, black pepper, cardamon, clove, coconut, cumin, fennel, fig, garlic, grape seed, green tea, lentil, onion powder, poppy, red pepper, sumac, thyme, tumeric, wheat) used in this work were purchased from local markets in Ankara, Turkey which were not been irradiated earlier. All samples were stored at room temperature.
2.2. Irradiation
Food samples were irradiated, at room temperature, by a Co-60 gamma irradiator (PX-g-30 Isslodovateji) at the Saraykoy Establishment of the Turkish Atomic Energy Authority in Ankara (Turkey). All samples were sealed in polyethylene bags. The absorbed dose was measured by means of Harwell Amber Perspex Dosimeter (Batch R, Type 3042, range 1–30 kGy) and Bruker alanine-EPR dosimetry system with an average uncertainty of about 3%. Target dose applied to samples was of 1.8 kGy. We interested the special case of ESR spectra which not having
5 cellulose satellite peaks. Therefore, we especially prefered to applied low irradiation dose to the samples.
2.3. ESR measurements
ESR measurements were carried out on samples open to air at room temperature by the use of a computer interfaced Bruker EMX 131 X-Band (9.5 GHz) spectrometer provided with a TE102
rectangular resonance cavity and 100 kHz modulation field. Both irradiated and non-irradiated samples (300 mg) were then introduced into pyrex tubes (4 mm inner tube diameter). Each tube was centered in the MW cavity in exactly the same position. EPR spectra of empty tubes were recorded in order to eliminate any signal due to the sample holder. All EPR spectra were recorded under the same experimental conditions, i.e., microwave frequency 9.8 GHz, modulation field amplitude of 0.2 mT, modulation frequency of 100 kHz, scan range of 10 mT and time constant of 327.7 ms. These conditions have been set up by finding the best signal-to-noise ratio. The strong pitch (g = 2.0028)was used as a standard sample for measuring g-factor. Each data point was the average of at least four independent measurements. Thus, the experimental error was estimated to be ± 4%. During this work, the intensity of the ESR signal was measured as the peak-to-peak height of the signal, and is reported in arbitrary units (a.u.).
3. Results and discussion
3.1. ESR spectra of unirradiated (control) and irradiated samples
All unirradiated (control) samples examined here exhibit a ESR singlet centered at g2.0 is due to the organic free radical. This singlet ESR signal was attributed to semiquinone-like free radicals formed by the oxidation of polyphenolic compounds present in plants (Jezierski et al., 2002; Morsy & Khaled, 2001; Polovka et al., 2003; Polovka et al., 2006; Raffi & Agnel,1989). The similar singlet ESR spectra of unirradiated samples were also given in literature for black
6 pepper (Haire et al., 1997; Roberto et al., 2004), cloves and cardamom (Beshir, 2014: Duliu et al., 2007), cumin (Abdel-Fattah, 2002; Kim et al., 2009), fennel (Yamaoki et al., 2008), garlic (Haire et al., 1997), Tumeric (Chosduet al., 1995). Pair of radiation induced satellite ESR peaks were not observed even immediately after irradiation for all irradiated samples. Fig. 1 shows the represantative ESR spectra of black pepper both before and after 1.8 kGy irradiation. As can be seen from Fig. 1, intensity of singlet ESR peak was increased by irradiation. Signal intensity increase of the ESR singlet with irradiation can be explained by addition second radiation sensitive ESR singlet peak (Raffi et al., 2000; Engin et al, 2011). The origin of this second singlet is also unknown. This second ESR singlet may be attributed to radiation-induced free radicals of quinones, phenols, etc., present in the plant as previously mentioned by Engin et al. (2011). Pair of radiation induced cellulose peaks were not observed for even 10 kGy irradiated tumeric sample (Chosdu, 1995). Even at quite high doses (20 kGy) of radiation, radiation spesific cellulose satellite lines spacing 6.05 mT was not clearly visible in fennel spectrum given in literature (Yamaoki, et al., 2008). As a similar, radiation induced cellulose peaks were not observed for irradiated black pepper and garlic (Haire et al., 1997), for irradiated cloves (Beshir, 2014), for irradiated dry plants (Yordanov et al., 2005), irradiated cumin (Kim et al., 2009) and irradiated wheat (Murrieta et al., 1996) in literature.
A limitation of the EN 1787 ESR cellulose method is that observation of the cellulose peaks is evidence of irradiation, but absence of the cellulose signals does not constitute evidence that the sample is unirradiated. Therefore, application of EN 1787 standard is not suitable in the case of absence of cellulose peaks after irradiation. In such case, the differency in the MW saturation curves of irradiated and unirradiated samples may be used as a idendificator of radiation treatment (Yordonov et al., 2006).
7
3.2. Saturation behavior of ESR Signal
In order to find differency in saturation feature between irradiated and unirradiated samples, room temperature MW saturation behaviour of the ESR signal of unirradiated (control) and irradiated (1.8 kGy) samples were investigated in the MW power (P) range of 0.01–159 mW. To achieve this goal, power saturation curves were obtained by measuring the ESR signal intensity (I) of the first derivative ESR signal as a function of the microwave and plotted against the square root of the applied MW power (√𝑃 ).
Fig. 2a and Fig. 2b shows the power saturation behaviours of the ESR signal intensities before and after radiation treatment for twenty different samples. Plots which have quite different MW saturation features of the resonance signals for un-irradiated and irradiated samples are given at Fig. 2a. As it is seen from Fig. 2a, MW saturation features of the resonance signals of un-irradiated and un-irradiated samples are not similar. The ESR detection of un-irradiated dry plants using MW saturation has been proposed by Yordanov et al. (2005). In their work, typical curves of saturation of unirradiated and irradiated plants versus 𝑃1/2 were studied and un-irradiated
samples were saturated at MW power higher than 15 mW while irradiated samples were saturated at MW power of around 8 mW.
In our present study, some of irradiated samples (coconut, garlic, lentil, red pepper, sumac, wheat) showed early saturation compared to un-irradiated samples and revealed a significant difference in saturation behaviour from un-irradiated samples as in Yordanov's study. It can be explained by references (Bolton & Wertz, 1994; Nakamura et al., 2006) in which saturation modes are defined by the line broadening scheme. The measured signals exhibited the characteristic behavior of a homogeneously broadened resonance line, which the ESR signal intensity increased with increasing of MW power and reached maximum value at threshold
8 MW value (𝑃𝑚𝑎𝑥), then decreased above this MW power value for these irradiated samples. Experimental 𝑃𝑚𝑎𝑥 values for these irradiated samples were given in Table 3. Whereas ESR signal intensity of irradiated samples showed power saturation behavior as mentioned above, ESR signal intensity of un-irradiated samples signifies the inhomogenous broadening, which the signal intensity increased with MW power and approached to almost at a constant value. We get an extraordinary result for un-irradiated redpepper sample that ESR signal intensity increased linearly with MW power without any saturation in the studied MW power range. However, the signal intensity of irradiated redpepper sample leveled off quickly. Namely, the signal of un-irradiated red pepper sample reflects the shorter relaxation time than the signal of irradiated one.
Unlike the aforementioned samples (coconut, garlic, lentil, red pepper, sumac, wheat), un-irradiated samples incluiding cumin, fig and grape seed showed early saturation compared to their irradiated ones. The second extreme case was observed for irradiated fig sample. The intensity of singlet ESR signal did not show almost any saturation with increasing MW power. Differency between saturation curves of un-irradiated and irradiated fig samples could be used for identification of irradiated food. For the irradiated and un-irradiated onion powder samples, it can be seen from Fig. 2a, MW power saturation curves at the low MW power are completely overlapped with each other. Differences in the saturation curves at high MW power could be used as a tool to distinguish between irradiated and un-irradiated onion powder.
There are also many food samples which exhibiting same MW power behavior for both irradiated and unirradiated cases in the literature. For example MW power saturation curve obtained for the irradiated and non-irradiated soybean samples were also found to be identical (Sanyal & Sharma, 2009). In our study, we get parallel results for samples includig anise, black
9 cumin, black pepper, cardamom, clove, fennel, green tea, poppy, thyme, tumeric. Their un-irradiated and un-irradiated samples exhibited almost same relaxation characteristics with no significant difference in saturation behaviors. Plots which have similar MW saturation features of the resonance signals for these samples are given at Fig. 2b. As it is seen from the Fig. 2b, the homogeneously broadened behavior is observed for both of irradiated and un-irradiated anise, black cumin, clove, fennel, poppy, thyme and tumeric samples. In the case of black pepper, cardamom and green tea, unhomogenous broadened behavior is observed for irradiated and un-irradiated samples.
Saturation mechanism includes two limiting cases called homogeneous and inhomogeneous broadening. However saturation curve was obtained as a different shape for these two extreme cases, ESR signal intensity is proportional to √𝑃 for low microwave range. This assumes that the MW power is low enough so that no saturation occurs (Wertz & Bolton, 1972). If the MW power to be increased to higher power values, ESR signal intensity reaches a maximum value at a particular MW power and decreased with increasing MW power in the case of homogenous broadening. On the other hand, for inhomogenous broadening, the ESR signal intensity increases and remains almost a constant value with increasing MW power. It should be noted that power dependence of homogeneously broadened line is different for the absorbtion and first derivative. The ESR signal intensity achieves the maximum value at 𝑃𝑜 for absorption
spectrum, whereas the ESR signal intensity reaches a maximum value at 𝑃𝑜
2 for first derivative. Homogeneous (Ihom) and inhomogeneous (Iinhom) first derivative lines intensities are given by
Eq. (1) as function of MW power P (Lund & Shiotani, 2013).
𝐼
ℎ𝑜𝑚.∝
√𝑃 (1+𝑃 𝑃0) 3 2⁄, 𝐼
𝑖𝑛ℎ𝑜𝑚.∝
√𝑃 (1+𝑃 𝑃0) 1 2⁄(1)
10 Data of ESR signal intensity vs. √P graph can not be usually fitted practically using only one of the these limiting case. In this case, First derivative line intensity is given below equation (Lembicz et al., 2004; Lund & Shiotani, 2013)
𝐼 = 𝐴√𝑃 (1 +𝑃𝑃 𝑜)
−𝛼
(2)
In this equation, A is scaling factor, 𝑃0 and are adjustable parameters. 𝑃0
2 is the power at which
the first derivative amplitude reaches maximum value. is the measure of the homogeneity of saturation of resonance signal. parameter can take any value in the range of 1 2⁄ ≤ 𝛼 ≤ 3 2⁄ for the recorded first derivative spectra (Lund & Shiotani, 2013; Lembicz et al., 2004). Eq. 2 gives the homogenous and inhomogenous saturation limits for 𝛼 = 3 2⁄ and 𝛼 = 1 2⁄ , respectively. In our present study, obtained experimental power saturation roll-over curves were fitted using Eq. (2).
The calculated saturation curves data and experimental counterparts were given in Fig. 2a and Fig. 2b. As is seen from this figures, the agreement between the theoretical and experimental saturation curves is fairly good. For un-irradiated and irradiated samples, , 𝑃0, A parameter
values yielded by the fitting are presented in Table 1 and Table 2. Theoric 𝑃𝑚𝑎𝑥 values were
calculated using ∝, 𝑃0 parameters according to Eq (3) (Hedin et al., 2004).
𝑃𝑚𝑎𝑥 = 𝑃0/(2. ∝ −1) ( 3 )
It should be point out that the 𝑃𝑚𝑎𝑥 value calculated from Eq. (3) is equal to infinity in the case of inhomogeneously broadened (∝= 0.5). It is not possible to determine of 𝑃𝑚𝑎𝑥 value from
11 experimental data for inhomogenous broadened line. So, it is not comparable of theoretical and experimental 𝑃𝑚𝑎𝑥 values for inhomogeneous broadened line. Experimental 𝑃𝑚𝑎𝑥 values were determined from plot of the ESR intensity versus √𝑃 for homogeneously broadened line. Experimental and calculated 𝑃𝑚𝑎𝑥 values were given in Table 3 and Table 4. The differency between theoritical and experimental 𝑃𝑚𝑎𝑥 values may be originated from the MW power
values for two consecutive measurements can not be setted enough closer. For exaple un-irradiated cumin, experimental 𝑃𝑚𝑎𝑥 value was determinated as 15.9 mW, whereas theoretical 𝑃𝑚𝑎𝑥 value was calculated as 12.8 mW from fitting procedure. Experimental MW power was able to setted 10.0 mW and 20.0 mW as a most closer to 𝑃𝑚𝑎𝑥 value. Theoretical 𝑃𝑚𝑎𝑥 value
calculated as 12.8 mW is in the interval of consequtive experimental MW power values (10.0 mW-15.9 mW). If it was possible to adjust MW power with a smaller interval, experimental and theoretical 𝑃𝑚𝑎𝑥 values would be more closer each other.
In literature, a new protocol for ESR detection of irradiated food containing cellulose was proposed in the studies of Shimoyoma et al., 2007; Ukai et al., 2006 and Ukai et al., 2008. They claimed that 𝑃𝑚𝑎𝑥 (threshold) is the only the reliable value for the saturation and one may treat the threshold as a universal constant and added that threshould can be used as a measure of radical quantity.They investigated experimental 𝑃𝑚𝑎𝑥 values for dry vegetables, japanese
pepper and black pepper, then obtained the same 𝑃𝑚𝑎𝑥 (threshould) value (4 mW) for irradiated and un-irradiated long green and perilla. Otherwise, they observed different 𝑃𝑚𝑎𝑥 values for irradiated and un-irradiated parsley. These results are supported ours which the same or different 𝑃𝑚𝑎𝑥value can be obtained for the same sample before and after irradiation. Also,
𝑃𝑚𝑎𝑥 value can be changed depends on the sample kind and its origin. For instance, for black
pepper, we were observed inhomogeneous MW saturation behaviour , but Shimoyoma et al. were obtained homogenous behaviour (Shimoyoma et al., 2007). As a similar, for irradiated
12 fennel, saturation microwave value was found as 19.98 mw in our present study, while it was found as 32 mw in yordanow’s study ( Yordanov et al., 2005).
Conclusion
Our observations clearly revealed that application of European standard (EN 1787, 200) is not enough to identify our irradiated samples. That’s why feasibility of the ESR MW saturation technique to detection of irradiated food samples was investigated. For this purpose, the method of MW power saturation was carried out for twenty different food samples. MW saturation behaviour was found to be succesful to identify our samples including coconut, cumin, fig, garlic, grape seed, lentil, onion powder, red pepper, sumac and wheat. However, it was failed in the case of anise, black cumin, black pepper, cardamom, clove, fennel, green tea, poppy, thyme and turmeric samples. Based on our experimental results, it was concluded that this method could be used for detection of some irradiated samples in case cellulose satellite peaks in irradiated foodstuffs could not be observed clearly in distance of 6 mT. By the way,this study contributes to improve EN 1787 standard. Also, fitting studies were performed by using experimental data of twenty samples, so theoretical curves and 𝑃𝑚𝑎𝑥 values were obtained. The agreement between experimental and theoretical value of 𝑃𝑚𝑎𝑥 was seen fairly well.
We recommend that firstly; further study is also required to improve MW power saturation method by investigating thoroughly so many food samples, secondly; in future studies, MW saturation behaviour of those food samples should also be compared with the behaviour of the same kind of many samples ,but get from different origin.
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