Research Article Received: 24 November 2014
Revised: 13 May 2015
Accepted article published: 1 June 2015
Published online in Wiley Online Library: 30 June 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7280
Impact of electron beam irradiation on quality of sea buckthorn (Hippophae rhamnoides L.) oil Monica R Nem¸tanu* and Mirela Bra¸soveanu* Abstract BACKGROUND: Sea buckthorn oil is a valuable product that can be incorporated into daily foodstuffs, cosmetics or pharmaceuticals. The effect of accelerated electron irradiation up to 8 kGy on quality characteristics of sea buckthorn oil was investigated in this study. RESULTS: Irradiation had no significant influence on phenolic content. Conversely, carotenoid content, antioxidant activity, and oxidative status suffered alterations as the irradiation dose increased. Although no colour changes were visible for oil irradiated up to 3 kGy, the total colour difference indicated clearly changes that involved a two-step pattern associated with slow degradation of oil colour up to 3 kGy, followed by a fast degradation up to 8 kGy. Some changes of the oil spectral features related to the frequency and intensity of some bands have been found after irradiation, indicating an alteration of the structural integrity induced by irradiation. CONCLUSION: The present investigation may be a useful starting point for irradiation processing of food or non-food matrices containing sea buckthorn oil. Thus, sea buckthorn oil safety can be ensured with minimal undesirable changes in its quality by applying irradiation doses up to 3 kGy, which allow control of the microbial contamination depending on microorganism type and initial microbial load. © 2015 Society of Chemical Industry Keywords: antioxidant activity; carotenoids; color; FTIR; irradiation; oil
INTRODUCTION
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Sea buckthorn (Hippophae rhamnoides L.) is considered a versatile nutraceutical product due to its unique cocktail of bioactive compounds like vitamins, carotenoids, flavonoids, organic acids, amino acids, micro and macronutrients.1 – 5 Xu et al.6 concluded that sea buckthorn (28 g day−1 berry or 5 g day−1 oil for humans) appears to be a promising plant for health benefits. Due to high amounts of natural antioxidants, different products based on this incredible multipurpose plant (e.g. sea buckthorn oil with coenzyme Q10, tea of sea buckthorn combined with bilberry and patience, pulp of sea buckthorn berries combined with Spirulina and different essential oils, juices, jams, purees, etc.) are considered food supplements and can be found in the world market (e.g. China, India, Pakistan, Russia, Ukraine, Romania, Poland, Germany, Finland, Sweden, Norway, etc.). However, sea buckthorn oil is the most valuable product in sea buckthorn derivatives. Although it is widely requested as a phototherapeutic product on the market, nowadays there is a real interest in it as a functional food due to its nutritive value and natural antioxidant character. Actually, the trend in the application of sea buckthorn oil is to incorporate the oil into daily foodstuffs, such as bread, juice and yogurts.7 Oil from the fruit (pulp/peel and seeds) is rich in carotenoids, tocopherols, tocotrienols, phytosterols and fatty acids.8 𝛽-Carotene, an important antioxidant in the food industry, is the most abundant in the pulp oil and constitutes 15–55% of the total amount of carotenoids, but 𝛼-carotene, 𝛾-carotene, dihydroxy carotene, lycopene, and zeaxanthin could be also found.9 The pulp/peel oil is more saturated containinig primarily palmitic acid (C16:0; most common saturated fatty acid), palmitoleic acid J Sci Food Agric 2016; 96: 1736–1744
(C16:1n-7, unsaturated fatty acid), and lower concentrations of polyunsaturated acids while the seed oil is rich in unsaturated fatty acids, mainly 𝛼-linolenic (C18:3n-3), linoleic (C18:2n-6) and oleic acids (C18:1n-9).10 – 15 Processing of food with ionising radiation (gamma radiation or electron beams) is a well-known and effective physical method used to control food spoilage and consequently extend the shelf life of foods. The European Food Safety Authority16 recently stated that food irradiation used in conjunction with an integrated food safety management program (Good Agricultural, Hygienic and Manufacturing Practices and HACCP), depending on the dose applied, can contribute to improved consumer safety by reducing food-borne pathogens. Also, the irradiation doses appropriate for a food are a compromise between several objectives and constraints. The effects of irradiation on edible vegetable oils depend on the chemical composition of oils and irradiation conditions such as irradiation dose, dose rate, etc.17 – 21 Lately, research has focused on quantifying the levels of phytochemicals found in vegetable oils after irradiation processing.21 – 24 Our preliminary tests22
∗
Correspondence to: Mirela Bra¸soveanu or Monica R Nem¸tanu, National Institute for Lasers, Plasma and Radiation Physics, Electron Accelerators Laboratory, 409 Atomi¸stilor St, P.O. Box MG-36, 077125, Bucharest–M˘agurele, Romania. E-mail: mirela.brasoveanu@inflpr.ro (Bra¸soveanu); monica.nemtanu@inflpr.ro (Nem¸tanu) National Institute for Lasers, Plasma and Radiation Physics, Electron Accelerators Laboratory, 409 Atomi¸stilor St, P.O. Box MG-36, 077125, Bucharest–M˘agurele, Romania
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Irradiation effect on quality of sea buckthorn oil
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on irradiation of sea buckthorn oil showed that the antioxidant activity can be affected by increasing the irradiation dose while the antimicrobial activity was unaffected. The purpose of this paper was to investigate the impact of electron beam (e-beam) irradiation on antioxidant chemical compounds, radical scavenging potential, oxidative status, as well as colorimetric and spectral features of sea buckthorn oil in order to find the suitable range of irradiation dose wherein the properties of sea buckthorn oil are minimally affected.
EXPERIMENTAL Material and chemicals Oil of sea buckthorn provided by the main Romanian producer of plant-based food supplements (SC Hofigal SA, Bucharest, Romania) was used in experiments. 𝛽-Carotene, gallic acid, Folin–Ciocalteu phenol reagent, sodium carbonate and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma–Aldrich (St. Louis, MO, USA), and n-hexane from Merck (Darmstadt, Germany). Other common solvents/chemicals were of analytical grade and purchased from SC Chimreactiv SRL (Bucharest, Romania). Electron beam irradiation Amber glass tubes with plastic cap (35 mm diameter) were filled with sea buckthorn oil (20 mL each) and then exposed to an electron beam generated by a linear electron accelerator facility (INFLPR, Bucharest-Magurele, Romania). The accelerator is a laboratory installation of travelling-wave type, driven by 2 MW peak power tunable S-band EEV M5125 type magnetrons. It generates an electron beam having an energy of 6.23 MeV, peak intensity of 75 mA for repetition frequency of 100 Hz and pulse duration of 3.5 μs. The one-sided exposures of samples were carried out in static mode and in vertical beam obtained by magnetic deflection, at room temperature (23 ∘ C) and at ambient pressure. The samples were irradiated at 1, 3, 6, and 8 kGy with mean rate of 1.6 ± 0.2 kGy min−1 , checked by chemical ceric–cerous sulfate dosimetry system. Non-irradiated samples were considered as controls. After irradiations the samples were stored in the dark conditions at room temperature. Irradiations were performed in duplicate. Determination of total carotenoid content Sea buckthorn oil was dissolved in hexane in a 25 mL volumetric flask. The absorbance of diluted oil was measured at 450 nm by using a Cary Bio 100 spectrophotometer (Varian Inc., Walnut Creek, CA, USA). Quantification of the amount of total carotenoids was performed by using calibration with 𝛽-carotene standard and amounts of carotenoids were expressed in mg kg−1 of oil.
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AA (%) =
Acontrol − Asample Acontrol
× 100
(1)
where Acontrol and Asample are the absorbance values of the reaction in DPPH solution without or with the tested oil. Peroxide value The peroxide value of sea buckthorn oil was determined by the iodometric titration method.25 Colour measurement Colorimetric features of samples were measured by determining the absorbance in the visible region (360–830 nm), for standard illuminant D65 (daylight source), observer angel of 10∘ (perception angle of a human observer) (Cary Bio 100 spectrophotometer, Varian Inc.). The colorimetric attributes expressed as CIELAB and CIELCH parameters (L*, a*, b*, C*, h∘ ) were analysed by using the Colour Application of Cary Win UV v. 3.10 software (Varian Australia Pty Ltd., Victoria, Australia). Fourier transform infrared spectroscopy Fourier transform infrared (FTIR) spectra of oil samples were recorded on a Tensor 27 FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany) using the attenuated total reflectance (ATR) accessory. The samples were placed in contact with the ATR element. Spectra were recorded at room temperature (23 ∘ C) in the region 4000 to 650 cm−1 by an average of 64 scans at a resolution of 4 cm−1 . The background spectra was recorded in air (with no sample) and subtracted from the sample spectra. Between measurements, the crystal was carefully cleaned with hexane and ethanol. Two spectra were collected for each sample and then analysed with Opus v. 6.5. software ( Bruker Optik GmbH, Ettlingen, Germany). Regression analysis and statistical assessment The data reported are averages of triplicate observations except oil irradiation that was performed in duplicate. The regression analysis and statistics of experimental data were performed using OriginPro 8.1 (OriginLab Corporation, Northampton, MA, USA), Microsoft® Excel 2010 (Microsoft Corporation, Redmond, WA, USA), StatistiXL v.1.10 and InfoStat version 2013.26 The data were analysed by using analysis of variance (one-way ANOVA) with the Fisher LSD (least significant differences) post-hoc test to discern the statistical difference. A probability value P < 0.05 was considered as statistically significant.
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Estimation of total phenolic content Total phenolic compounds were quantified employing the Folin–Ciocalteu reagent. Sea buckthorn oil was mixed with 5 mL of 10-fold diluted Folin–Ciocalteu reagent and 4 mL of 1 mol L−1 sodium carbonate solution. The reaction mixture was shaken vigorously and then allowed to stand at room temperature for 30 min. The absorbance was measured at 765 nm (Cary Bio 100 spectrophotometer, Varian Inc.). Gallic acid was used as standard and thus the results were expressed as mg gallic acid equivalents (GAE) kg−1 of sample.
Evaluation of antioxidant activity The antioxidant effectiveness as a free radical-scavenging activity of sea buckthorn oil was determined by using the stable radical DPPH. A mixture containing 1 g of sea buckthorn oil and 8 mL of methanol:hexane (4:1) was shaken vigorously and allowed to stand overnight at room temperature. Then, 0.5 mL of this mixture was added to 2.5 mL of methanol:hexane (4:1) and 0.5 mL of 1 mmol L−1 DPPH solution in methanol; this reaction mixture was shaken and allowed to stand at room temperature for 30 min in the dark. The radical-scavenging activity of samples was quantified by decolorisation at 517 nm so that lower absorbance indicated stronger scavenging activity. The antioxidant activity (AA) of sea buckthorn oil was calculated using the equation:
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MR Nem¸tanu, M Bra¸soveanu
Table 1. Carotenoid and phenolic contents, antioxidant activity and peroxide value of sea buckthorn oil Total carotenoid content (mg · kg−1 oil)
Irradiation dose (kGy)
Total phenolic content (mg · kg−1 oil)
967 ± 9a 948 ± 18b 929 ± 6c 888 ± 3d 859 ± 8e
0 1 3 6 8
629 ± 10a 627 ± 15a 623 ± 24a 615 ± 10a 613 ± 9a
AA (%) 62 ± 5a 46 ± 7b 41 ± 4b 31 ± 2c 28 ± 3c
PV (meq O2 · kg−1 ) 1.5 ± 0.1a 2.1 ± 0.2b 3.6 ± 0.1c 5.0 ± 0.3d 5.5 ± 0.1e
Values within each column with different superscripts are significantly different (P < 0.05). AA, antioxidant activity; PV, peroxide value.
RESULTS AND DISCUSSION Influence of electron beam on carotenoid content Among the wealth of constituents of sea buckthorn oil, carotenoids are very important due to their antioxidant role. Besides, carotenoids are responsible for the orange colour typical for sea buckthorn oil, and their content is considered an important quality index for this type of oil. Because of their unsaturated carbon chain, these compounds are susceptible to some reactions, such as oxidation and isomerisation, during processing and storage of foods, and they consequently suffer changes in their nutritional value.27 Thus, the total carotenoid content was analysed to estimate the rate of degradation during the irradiation processing. The amount of carotenoids in raw sea buckthorn oil was 967 ± 9 mg kg−1 oil. The carotenoid content gradually decreased by e-beam irradiation (P < 0.05), the loss being about 11% for 8 kGy irradiation (Table 1). This reduction can be attributed to the oxidative processes caused by e-beam irradiation. A similar evolution was noticed for 𝛽-carotene in sunflower, soybean and palm oils exposed to irradiation,17,28,29 meaning that increasing the radiation dose, decreased the carotenoid level due to the radiation-induced breakdown of carotenoid molecules. Consequently, the reduction in the carotenoid level will result in lowering the oil stability to oxidative reactions.28,29 Oxidation leads to the formation of highly reactive species, such as alkyl and peroxyl radicals, that further react with carotenoid compounds, resulting in their degradation. The change of total content of carotenoids was adequately (R2 = 0.998) described by the zero-order kinetics equation [Eqn (2)] in the range of irradiation doses (0–8 kGy) tested (Fig. 1): C = C0 − kD
(2)
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where C and C 0 are the concentrations for irradiated and control sample, respectively, (mg kg−1 oil), k is the degradation rate constant (mg kg−1 kGy−1 ), and D is the irradiation dose (kGy). The model parameters (initial concentration of carotenoids and degradation rate constant) for degradation of carotenoid content of sea buckthorn oil exposed to electron beam irradiation in the range of 0–8 kGy were estimated as C 0 = 969 ± 2 mg kg−1 oil and k = 13.5 ± 0.4 mg kg−1 kGy−1 , respectively. Knowledge of a mathematical description of the dose–effect relationship and its model parameters might correctly enable the prediction of changes of the total carotenoid content during the irradiation and permit the proper selection of processing conditions for the acceptable level of carotenoid content loss.
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Figure 1. Evolution of total carotenoid content with irradiation dose.
Influence of electron beam on total phenolic content Phenolic compounds are known as very important constituents of vegetal materials with recognised capacity of radical scavenging due to their hydroxyl groups.30,31 According to Spiridon et al.,32 the antioxidant activity of phenolic compounds is mainly due to their redox properties, which enable them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. In raw sea buckthorn oil, 629 ± 10 mg GAE kg−1 oil of phenolic derivatives was detected. This value was slightly (P > 0.05) reduced by e-beam irradiation up to 8 kGy (Table 1). Influence of electron beam on antioxidant activity Sea buckthorn oil has been proved to be an outstanding free radical inhibitor acting as a primary antioxidant.14 In this study, the capacity of free radical scavenging of sea buckthorn oil calculated as percentage of DPPH radical inhibition was effective with value of 62 ± 5%. This ability reduced dramatically (P < 0.05) with the irradiation dose increase (Table 1). Even relative low doses of 1 kGy and 3 kGy caused the diminishing of about 30% in the antioxidant activity, while irradiation doses ≥ 6 kGy caused a more than 50% decrease in the initial value of antioxidant activity of unirradiated oil. The reduction of antioxidant activity might be correlated with the carotenoid level decreasing evinced in this study as well as with a possible deterioration (degradation and peroxidation) of the unsaturated fatty acids. The latter can also greatly contribute to the overall antioxidant activity as Ting et al.14 and Gruia et al.33 reported earlier.
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Figure 2. Visible spectra of unirradiated and irradiated samples sea buckthorn oil (inner window: variations of peak intensity at 670 nm with the red–green coordinate).
Influence of electron beam on peroxide value Peroxide value (PV) is an index of the oxidation degree of oil and fats subjected to stress factors able to initiate oxidative reactions. Lipid oxidation produces several intermediate chemical species like peroxides and hydroperoxides, which lead to rancidity of oil. Thus, PV indicates the amount of these compounds formed as a result of oxidation in the oil sample. A high PV confirms that the oxidation process occurred. The peroxide value for unirradiated sea buckthorn oil sample was 1.5 ± 0.1 meq kg−1 of oil, showing a low level of oxidation. A significant (P < 0.05) increasing trend was observed with increase in radiation dose (Table 1). Such behaviour might be due to oxidation and preferential radiolytic cleavage of bonds in the oils.20,21 The increase of the peroxide value with increasing radiation dose were also reported for sunflower, soybean, palm and canola oils17,28,29,34 or oils extracted from irradiated black cumin,35 peanuts20 and sunflower and maize seeds.21 A PV greater than 20 meq kg−1 of oil corresponds to very poor quality oils, even rancid, which normally would have significant off-flavours.36,37 Therefore, in our study, though the PV had an increasing trend with the irradiation dose, it was below the limit indicating oxidative rancidity.
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CIE L*, a*, b* coordinates According to the CIE L*, a*, b* colour space, the sea buckthorn oil has a rather dark (L* ∼35%) orange–reddish colour consisting of yellow (+b*) and red (+a*), 60.46 ± 0.02 and 54.62 ± 0.01, respectively. Table 2 shows the changes of the colour parameters according to the irradiation dose. After e-beam irradiation
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Influence of electron beam on colorimetric parameters Colour is one of the characteristics of a food product involved in the concept of food quality and can influence the appreciation and judgment of the product quality by the consumer. Thus, colour is one of the vital components of the visual quality of the foods and plays an important role in the consumer choice.38 As e-beam irradiation is able to induce changes in properties depending on the level of irradiation dose used and the structure and/or composition of the treated material, colour can also offer global information about the changes of some constituents or properties. In this study, colour change of oil samples was evaluated by chromatic parameters calculated in two colour spaces CIE L*, a*, b* and
CIE L*, C*, h∘ . These systems are recommended by the CIE (Commission Internationale de l’Eclairage), and are widely adopted by the food industry for colour measurement.39 Thus, the colorimetric attributes were expressed as CIELAB L* (lightness: 0 (black) → 100 (white)), a* (red–green coordinate: +a* → red, −a* → green) and b* (yellow–blue coordinate: +b* → yellow, −b* → blue) as well as CIELCH L* (lightness), C* (chroma) and h∘ (hue). The colour of untreated oil sample was used as reference for determination of the total colour difference, ΔE ab, of each irradiated sample. Although the colour of vegetable oils is associated with the total pigment content,40 the dark orange–red colour typical for sea buckthorn oil is mainly due to the carotenoids as a result of their conjugated double-bond system (chromophore). For all investigated samples the spectra showed absorption peaks between 360 and 560 nm, which can be attributed to yellow–red pigments, i.e. carotenoids and polyphenols.41,42 Also, the oil had two absorption maxima: a weak peak at 610 nm and a stronger one at 670 nm (Fig. 2). Nikolova et al.43 identified only one absorption peak at 670 nm in the visible region of sea buckthorn oil. The well-defined absorption bands centered for most of the oils at wavelengths 670 and 610 nm show the presence undoubtedly of chlorophyll compounds known as a green pigment group.44 Electron beam irradiation at doses higher than 1 kGy clearly had influence (P < 0.05) on these maxima expressed as a hypochromic effect. A small hypsochromic shift of 1 nm for both absorption peaks was also detected in samples irradiated with the highest applied dose. These observations suggest that the chlorophyll pigment level diminished by irradiation.
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Table 2. CIE L*, a*, b* parameters of sea buckthorn oil Irradiation dose (kGy) 0 1 3 6 8
L* 35.07 ± 0.01a 35.17 ± 0.11b 35.87 ± 0.01c 37.71 ± 0.06d 40.39 ± 0.02e
a* 54.62 ± 0.01a 54.73 ± 0.05b 54.89 ± 0.05c 55.22 ± 0.03d 56.78 ± 0.04e
b* 60.46 ± 0.02a 60.63 ± 0.18b 61.84 ± 0.02c 65.01 ± 0.11d 69.62 ± 0.03e
Values within each column with different superscripts are significantly different (P < 0.05).
a* values slightly increased (P < 0.05) as a function of the irradiation dose, and oil was less ‘green’, reflecting the possible loss of chlorophyll-derived pigments. An example to support this allegation is related to absorption peak at 670 nm assigned to chlorophyll compounds. Thus, the increase in the a* value with concomitant decrease in this peak intensity (inset graph of Fig. 2) were highly positive correlated (R2 = 0.998) confirming the reduction of chlorophyll pigment content. These findings are in agreement with those of other investigators45 who proved that chlorophyll content of the linoleic acid solution containing chlorophyll b affected greenness, resulting in an increase in the a*-value. Actually, according to Byun and co-workers,45 by its ability to breakdown chlorophyll, irradiation technology could be applied to reduce or eliminate the residual chlorophyll in oil processing without developing lipid oxidation during the irradiation process. The proportions of yellow had instead a greater increase (P < 0.05) with the irradiation dose, up to 69.62 ± 0.03 for 8 kGy, as a probable consequence of rearrangements of chemical structure, mainly of conjugated double bonds typical for carotenoid pigments. The lightness, L*, values had increasing evolution (P < 0.05) with the irradiation dose increase up to 40.39 ± 0.02 for 8 kGy in a similar manner as yellow coordinate. The positive variation in L* values indicates that the oil became lighter with increasing irradiation dose. Briefly, the greatest differences in L*, a* and b* between each irradiated sample and control one were in b* followed by L*, the colour of oil being more yellow and lighter as irradiation dose increased. These results suggest that oil suffered alteration of colour through bleaching caused by oxidation process because of irradiation. CIE L*, C*, h∘ coordinates For the CIE L*, C*, h∘ colour system, lightness, L*, is similar to that
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of CIE L*, a*, b* space, chroma, C*, is the quantitative attribute of colorfulness while hue h∘ is the qualitative attribute of colour being expressed in degrees: 0∘ (red), 90∘ (yellow), 180∘ (green), 270∘ (blue). In these terms, for raw sea buckthorn oil, the values of chroma and hue were 81.48 ± 0.01 and 47.91 ± 0.01∘ , respectively. This location is equivalent to reddish–orange colour on CIE L*, a*, b* space. Figure 3 illustrates a significant (P < 0.05) shift of chroma as the irradiation dose increases, showing a value of 89.84 ± 0.05 for irradiation dose of 8 kGy. The positive values of chroma differences for all irradiated samples show that e-beam irradiation led to greater intensity of oil colour. In a similar fashion, hue moved further away from the origin and in the upward direction with ∼3∘ (50.80 ± 0.01∘ ) at 8 kGy, indicating the movement of colour toward yellow as the irradiation dose increased. In this colour space, the greatest differences in chromatic parameters
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Figure 3. CIE L*, C* and h∘ parameters of sea buckthorn oil.
between each irradiated sample and control one were in chroma, C*, followed by L*, the colour of oil being more intense and lighter as irradiation dose increased. Therefore, the increase of the irradiation dose caused the colour bleach of investigated samples suggesting the modification of the colored compounds (i.e. carotenes, xanthophylls) as a probable combined result of isomerisation and oxidative reactions. As mentioned, carotenoids are susceptible to degradation during processing and storage. Their common degradation pathways are isomerisation, oxidation, and fragmentation of the carotenoid molecules promoting changes in colour as a result of the rearrangement or formation of cis-isomers, epoxides, short chain products and, in some cases, volatile compounds.46 – 48 Total colour difference 𝚫Eab Total colour difference is important when evaluating the relationship between the visual and the numerical analyses.42 Colour difference is an indicator of global changes and interaction among components of a material. According to several research groups,42,49,50 the perception of the colour difference, ΔEab , varies depending on the observed colour and the sensitivity of the human eye so that the following limits for human eye perception of ΔEab were nominated: • 3 – perceptible colour difference. Experimental data achieved in this study showed clearly that radiation-induced changes of oil chromatic parameters are irrefutably reflected in the total colour difference ΔEab , which increased (P < 0.05) with irradiation dose. The colour change involved two steps described by zero-order kinetics, having different rates in the range of tested irradiation doses (Fig. 4). Slow degradation of oil colour occurred with rate of 0.51 ± 0.06 kGy−1 (R2 = 0.971) up to 3 kGy, followed by a fast degradation with significant higher rate (1.79 ± 0.42 kGy−1 , R2 = 0.948) up to 8 kGy. In addition, colour differences distinguishable by the human eyes were noticed for samples irradiated with 6 kGy (ΔEab = 5.29 ± 0.12) and 8 kGy (ΔEab = 10.81 ± 0.05). These findings are in agreement with the imposed limits previously mentioned. Sea buckthorn oil is a complex compositional matrix in which the components
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Figure 4. Evolution of ΔEab of sea buckthorn oil after irradiation.
interact among them exerting a certain protection to stress factors. Taking into account this aspect, it can be inferred that such protection could act against irradiation up to 3 kGy, explaining thus the slow rate of degradation of irradiated oil. One can consider the colour of sea buckthorn oil as a two-component system containing two principal groups of pigment compounds: yellow–red and green. As we have already seen, both chlorophyll pigments and carotenoid ones reduced by e-beam irradiation; the green pigments diminishing more than carotenoids. However, when the yellow–red pigments are present in considerable proportion, the removal of the green pigments does not have a large effect on the colour.44 Moreover, the intensity of the colour of sea buckthorn oil depends mainly upon the presence of carotenoids, which can explain the important variation of chroma simultaneously with degradation of carotenoid pigment as increasing of irradiation dose. Although from visual perception, which is a subjective way to assess color, no colour changes were perceptible for oil irradiated up to 3 kGy, the total colour difference indicated clearly considerable (P < 0.05) changes as increasing the irradiation dose. Changes in colour were previously reported for sunflower, soybean, and canola oils exposed to low or high levels of gamma radiation.28,34
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Pearson’s correlation analysis Pearson’s correlation test has been applied to analyse the relationship between investigated properties and irradiation dose. The correlation coefficients are shown in Table 3. The peroxide value, total colour difference and chromatic parameters such as L*, b*, C* and h∘ were strongly positive correlated (r = 0.95) with irradiation dose whereas the red–green coordinate a* had only good positive correlation (r ∼ 0.881) with irradiation dose. Conversely, the total carotenoid and phenolic contents as well as antioxidant activity were strongly negatively correlated (r = −0.93) with irradiation dose. Carotenoids and phenolic compounds have high contribution to the antioxidant activity indicated by the strong positive correlation coefficients (r = 0.987 and r = 0.925, respectively). According to Spiridon et al.,32 the synergism between the antioxidants in the mixture makes the antioxidant activity not only
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Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy is an analytical tool often applied to qualitatively classify edible oil and fat products or to verify the authenticity of edible oils and fats, due to its simplicity, rapidity, and ease of sample preparation.51,52 The analytical evaluation of the FTIR spectrum of sea buckthorn oil showed bands typical to oils and fats according to existing literature.51 – 54 In the region of hydrogen stretching, the characteristic bands were identified around 3007 cm−1 (C—H stretching vibration of the cis-double bond —CH CH), 2953 and 2874 cm−1 (C—H asymmetric and symmetric stretching vibrations of the aliphatic —CH3 group), and 2924 and 2854 cm−1 (C—H asymmetric and symmetric stretching vibrations of the aliphatic —CH2 group). In the region of double bond stretching, the spectrum displayed a strong absorption band around 1744 cm−1 (ester carbonyl C O stretching) and a very weak peak around 1655 cm−1
(C C stretching vibration of cis-olefins). The spectral region of other bonds deformations and bendings showed bands at 1466 cm−1 (C—H bending (scissoring) vibrations of the —CH2 and —CH3 aliphatic groups), 1417 cm−1 ( C—H bending (rocking) vibration of the cis-disubstituted olefins) and 1378 cm−1 (C—H symmetric deformation of the aliphatic —CH3 group). In the fingerprint region, the peaks appeared around 1240 and 1166 cm−1 (stretching vibrations of the C—O ester groups and C—H bending vibrations of the aliphatic —CH2 groups), 1140 cm−1 , 1097 cm−1 and 1031 cm−1 (stretching vibrations of the C—O ester groups), 964 cm−1 and 912 cm−1 (out-of-plane bendings of trans- and cis-disubstituted olefinic groups), 722 cm−1 (overlapping of the C—H bending (rocking) vibration of the aliphatic —CH2 group and the out-of-plane vibration of the cis-disubstituted olefins). The spectral features of the e-beam irradiated oil samples were apparently similar to unirradiated oil (Fig. 5). No new functional groups were found in the FTIR spectra of irradiated samples. However, some differences related to the frequency and intensity of some bands have been found after irradiation. In the characteristic contour from 1300 to 900 cm−1 , a small peak shift of 1 cm−1 toward lower wavenumbers were detected for peaks centred at 1240, 1097 and 964 cm−1 even after the smallest irradiation dose applied. The weak peak at 1031 cm−1 assigned to the vibration of the O—C—C band of esters derived from primary alcohols suffered a larger shift to 1028 cm−1 after irradiation with 3 kGy. At the same time, the widening of the band (P < 0.05) took place with the increase of irradiation dose resulting in its disappearance at higher irradiation doses. The frequency of this band changes without specific pattern as the oxidation process advances.55 Conversely, the characteristic peak at 912 cm−1 related to the bending vibration of cis-disubstituted olefinic groups shifted to higher wavenumbers (915 cm−1 ) with broadening of band as increasing the irradiation dose. But according to Guillen and Cabo,55 the changes observed in the frequency of this band as the oxidation progresses are not relevant. On the other hand, decrease (P < 0.05) in the intensity (inset graph of Fig. 5) and width of the maximum absorption at 2924 cm−1 ascribed to the asymmetric stretching vibration of the aliphatic —CH2 functional group was also observed by increasing the irradiation dose. The changes identified in the spectral features of irradiated oil samples indicated that the structural integrity of sea buckthorn oil has been affected as consequence of oxidative degradation induced by irradiation. Although the alterations of spectral features occurred as irradiation dose increased, any specific pattern of changes could not be identified.
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Figure 5. FTIR spectra of unirradiated and irradiated samples of sea buckthorn oil (inner window: variation of peak intensity at 2924 cm−1 with irradiation dose). Table 3. Pearson’s correlation coefficients Parameter Irradiation dose (D) Total carotenoid content (TCC) Total phenolic content (TPC) Antioxidant activity (AA) Peroxide value (PV) Lightness (L*) Red–green coordinate (a*) Yellow–blue coordinate (b*) Chroma (C*) Hue (h∘ ) Total colour difference (ΔEab )
D
TCC
TPC
– −0.998 −0.993 −0.930 0.987 0.959 0.881 0.959 0.949 0.978 0.957
– – 0.987 0.937 −0.977 −0.964 −0.894 −0.964 −0.956 −0.980 −0.963
– – – 0.925 −0.990 −0.933 −0.829 −0.933 −0.919 −0.964 −0.930
dependent on the concentration, but also on the structure and the interaction between the antioxidants. Antioxidant activity had also negative correlations with peroxide value, chromatic parameters, and total colour difference. On the other hand, peroxide value was positive correlated with chromatic parameters and total colour difference. Total colour difference showed negatively correlation with carotenoid and phenolic contents and consequently with antioxidant activity while it was positively correlated with peroxide value.
CONCLUSIONS
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The influence of e-beam irradiation up to 8 kGy on antioxidant chemical compounds, radical scavenging potential, oxidative status, as well as colorimetric and spectral features of sea buckthorn oil was evaluated and discussed in correlation with irradiation dose. Irradiation exerted no considerable effect on phenolic
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AA – – – – −0.949 −0.820 −0.732 −0.820 −0.807 −0.848 −0.818
PV – – – – – 0.903 0.801 0.903 0.888 0.934 0.900
L* – – – – – – 0.971 1.000 0.999 0.994 1.000
a*
b*
C*
– – – – – – – 0.971 0.980 0.939 0.973
– – – – – – – – 0.999 0.994 1.000
– – – – – – – – – 0.988 0.999
h∘ – – – – – – – – – – 0.993
content. Degradation of total content of carotenoids followed zero-order kinetics with rate constant of 13.5 ± 0.4 mg kg−1 kGy−1 . Antioxidant activity was also negatively affected especially at higher irradiation doses (≥6 kGy). The peroxide value was below the limit indicating the oxidative rancidity though it had an increasing trend with the irradiation dose. Chromatic parameters changed by irradiation, the colour of oil being more yellow, intense and lighter as irradiation dose increased. No colour changes were distinguishable by the human eye for oil irradiated up to 3 kGy, but the total colour difference indicated significant changes involving a two-step pattern associated with slow degradation of oil colour (rate of 0.51 ± 0.06 kGy−1 ) up to 3 kGy, followed by a fast degradation (rate of 1.79 ± 0.42 kGy−1 ) up to 8 kGy. Therefore, colour measurement that is non-destructive and requires only a small sample can be suitable tool to evaluate the aesthetic aspects and to monitor the quality in industrial processing of sea buckthorn oil.
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Irradiation effect on quality of sea buckthorn oil
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Although the spectral features of the irradiated oil samples were apparently similar to control, some differences related to the frequency and intensity of some bands have been found after irradiation indicating radiation-induced alteration of the oil structural integrity. In conclusion, electron beam irradiation up to 3 kGy can be an effective eco-friendly mean of ensuring oil safety with minimal undesirable changes in its quality. Therefore, an appropriate balance between favourable and side effects of oil irradiation might be achieved by responsible selection of processing parameters. In addition, it is important to accept that some potential loss of chemical compounds with bioactive role can be compensated by the improved hygiene of the irradiated food.
ACKNOWLEDGEMENTS This work was supported by project Nucleu (2014) and IIN (Electron Accelerators Laboratory of the National Institute for Lasers, Plasma and Radiation Physics).
REFERENCES
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41 Socaciu C, Ranga F, Fetea F, Leopold L, Dulf F and Parlog R, Complementary advanced techniques applied for plant and food authentication. Czech J Food Sci 27:S70–S75 (2009). 42 Sant’Anna V, Gurak PD, Marczak LDF and Tessaro IC, Tracking bioactive compounds with colour changes in foods – A review. Dyes Pigments 98:601–608 (2013). 43 Nikolova K, Panchev I and Sainov S, Optical characteristics of oil, obtained from sea-buckthorn (Hippophae rhamnoides L. – Elaeagnaceae). Eur Food Res Technol 223:843–847 (2006). 44 McNicholas HJ, The color and spectral transmittance of vegetable oils. Oil Soap 12:167–178 (1935). 45 Byun M-W, Jo C, Lee K-H and Kim K-S, Chlorophyll breakdown by gamma irradiation in a model system containing linoleic acid, J Am Oil Chem Soc 79:145–150 (2002). 46 Bonnie TYP and Choo YM, Oxidation and thermal degradation of carotenoids. J Oil Palm Res II:62–78 (1999). 47 Rodriguez-Amaya DB, A Guide to Carotenoid Analysis in Foods. ILSI Press, Washington (2001). 48 Mercadante AZ, Food colorants – Chemical and functional properties, in Carotenoids in Foods: Sources and Stability During Processing and Storage, ed by Socaciu C. CRC Press, New York, pp. 213–235 (2008).
49 Bodart M, De Penaranda R, Deneyer A and Flamant G, Photometry and colorimetry characterisation of materials in daylighting evaluation tool. Build Environ 43:2046–2058 (2008). 50 Castagna A, Chiavaro E, Dall’Asta C, Rinaldi M, Galaverna, G and Ranieri A, Effect of postharvest UV-B irradiation on nutraceutical quality and physical properties of tomato fruits. Food Chem 137:151–158 (2013). 51 Yang H, Irudayaraj J and Paradkar MM, Discriminant analysis of edible oils and fats by FTIR, FT-NIR and FT-Raman spectroscopy. Food Chem 93:25–32 (2005). 52 Vlachos N, Skopelitis Y, Psaroudaki M, Konstantinidou V, Chatzilazarou A and Tegou E, Applications of Fourier transform-infrared spectroscopy to edible oils. Anal Chim Acta 573–574:459–465 (2006). 53 Safar M, Bertrand D, Roberst P, Devaux MF and Genot C, Characterization of edible oils, butters and margarines by Fourier transform infrared spectroscopy with attenuated total reflectance. J Am Oil Chem Soc 71:371–377 (1994). 54 Guillen MD and Cabo N, Infrared spectroscopy in the study of edible oils and fats. J Sci Food Agric 75:1–11 (1997). 55 Guillen MD and Cabo N, Usefulness of the frequency data of the Fourier transform infrared spectra to evaluate the degree of oxidation of edible oils. J Agric Food Chem 47:709–719 (1999).
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J Sci Food Agric 2016; 96: 1736–1744
Revised: 13 May 2015
Accepted article published: 1 June 2015
Published online in Wiley Online Library: 30 June 2015
(wileyonlinelibrary.com) DOI 10.1002/jsfa.7280
Impact of electron beam irradiation on quality of sea buckthorn (Hippophae rhamnoides L.) oil Monica R Nem¸tanu* and Mirela Bra¸soveanu* Abstract BACKGROUND: Sea buckthorn oil is a valuable product that can be incorporated into daily foodstuffs, cosmetics or pharmaceuticals. The effect of accelerated electron irradiation up to 8 kGy on quality characteristics of sea buckthorn oil was investigated in this study. RESULTS: Irradiation had no significant influence on phenolic content. Conversely, carotenoid content, antioxidant activity, and oxidative status suffered alterations as the irradiation dose increased. Although no colour changes were visible for oil irradiated up to 3 kGy, the total colour difference indicated clearly changes that involved a two-step pattern associated with slow degradation of oil colour up to 3 kGy, followed by a fast degradation up to 8 kGy. Some changes of the oil spectral features related to the frequency and intensity of some bands have been found after irradiation, indicating an alteration of the structural integrity induced by irradiation. CONCLUSION: The present investigation may be a useful starting point for irradiation processing of food or non-food matrices containing sea buckthorn oil. Thus, sea buckthorn oil safety can be ensured with minimal undesirable changes in its quality by applying irradiation doses up to 3 kGy, which allow control of the microbial contamination depending on microorganism type and initial microbial load. © 2015 Society of Chemical Industry Keywords: antioxidant activity; carotenoids; color; FTIR; irradiation; oil
INTRODUCTION
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Sea buckthorn (Hippophae rhamnoides L.) is considered a versatile nutraceutical product due to its unique cocktail of bioactive compounds like vitamins, carotenoids, flavonoids, organic acids, amino acids, micro and macronutrients.1 – 5 Xu et al.6 concluded that sea buckthorn (28 g day−1 berry or 5 g day−1 oil for humans) appears to be a promising plant for health benefits. Due to high amounts of natural antioxidants, different products based on this incredible multipurpose plant (e.g. sea buckthorn oil with coenzyme Q10, tea of sea buckthorn combined with bilberry and patience, pulp of sea buckthorn berries combined with Spirulina and different essential oils, juices, jams, purees, etc.) are considered food supplements and can be found in the world market (e.g. China, India, Pakistan, Russia, Ukraine, Romania, Poland, Germany, Finland, Sweden, Norway, etc.). However, sea buckthorn oil is the most valuable product in sea buckthorn derivatives. Although it is widely requested as a phototherapeutic product on the market, nowadays there is a real interest in it as a functional food due to its nutritive value and natural antioxidant character. Actually, the trend in the application of sea buckthorn oil is to incorporate the oil into daily foodstuffs, such as bread, juice and yogurts.7 Oil from the fruit (pulp/peel and seeds) is rich in carotenoids, tocopherols, tocotrienols, phytosterols and fatty acids.8 𝛽-Carotene, an important antioxidant in the food industry, is the most abundant in the pulp oil and constitutes 15–55% of the total amount of carotenoids, but 𝛼-carotene, 𝛾-carotene, dihydroxy carotene, lycopene, and zeaxanthin could be also found.9 The pulp/peel oil is more saturated containinig primarily palmitic acid (C16:0; most common saturated fatty acid), palmitoleic acid J Sci Food Agric 2016; 96: 1736–1744
(C16:1n-7, unsaturated fatty acid), and lower concentrations of polyunsaturated acids while the seed oil is rich in unsaturated fatty acids, mainly 𝛼-linolenic (C18:3n-3), linoleic (C18:2n-6) and oleic acids (C18:1n-9).10 – 15 Processing of food with ionising radiation (gamma radiation or electron beams) is a well-known and effective physical method used to control food spoilage and consequently extend the shelf life of foods. The European Food Safety Authority16 recently stated that food irradiation used in conjunction with an integrated food safety management program (Good Agricultural, Hygienic and Manufacturing Practices and HACCP), depending on the dose applied, can contribute to improved consumer safety by reducing food-borne pathogens. Also, the irradiation doses appropriate for a food are a compromise between several objectives and constraints. The effects of irradiation on edible vegetable oils depend on the chemical composition of oils and irradiation conditions such as irradiation dose, dose rate, etc.17 – 21 Lately, research has focused on quantifying the levels of phytochemicals found in vegetable oils after irradiation processing.21 – 24 Our preliminary tests22
∗
Correspondence to: Mirela Bra¸soveanu or Monica R Nem¸tanu, National Institute for Lasers, Plasma and Radiation Physics, Electron Accelerators Laboratory, 409 Atomi¸stilor St, P.O. Box MG-36, 077125, Bucharest–M˘agurele, Romania. E-mail: mirela.brasoveanu@inflpr.ro (Bra¸soveanu); monica.nemtanu@inflpr.ro (Nem¸tanu) National Institute for Lasers, Plasma and Radiation Physics, Electron Accelerators Laboratory, 409 Atomi¸stilor St, P.O. Box MG-36, 077125, Bucharest–M˘agurele, Romania
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Irradiation effect on quality of sea buckthorn oil
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on irradiation of sea buckthorn oil showed that the antioxidant activity can be affected by increasing the irradiation dose while the antimicrobial activity was unaffected. The purpose of this paper was to investigate the impact of electron beam (e-beam) irradiation on antioxidant chemical compounds, radical scavenging potential, oxidative status, as well as colorimetric and spectral features of sea buckthorn oil in order to find the suitable range of irradiation dose wherein the properties of sea buckthorn oil are minimally affected.
EXPERIMENTAL Material and chemicals Oil of sea buckthorn provided by the main Romanian producer of plant-based food supplements (SC Hofigal SA, Bucharest, Romania) was used in experiments. 𝛽-Carotene, gallic acid, Folin–Ciocalteu phenol reagent, sodium carbonate and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma–Aldrich (St. Louis, MO, USA), and n-hexane from Merck (Darmstadt, Germany). Other common solvents/chemicals were of analytical grade and purchased from SC Chimreactiv SRL (Bucharest, Romania). Electron beam irradiation Amber glass tubes with plastic cap (35 mm diameter) were filled with sea buckthorn oil (20 mL each) and then exposed to an electron beam generated by a linear electron accelerator facility (INFLPR, Bucharest-Magurele, Romania). The accelerator is a laboratory installation of travelling-wave type, driven by 2 MW peak power tunable S-band EEV M5125 type magnetrons. It generates an electron beam having an energy of 6.23 MeV, peak intensity of 75 mA for repetition frequency of 100 Hz and pulse duration of 3.5 μs. The one-sided exposures of samples were carried out in static mode and in vertical beam obtained by magnetic deflection, at room temperature (23 ∘ C) and at ambient pressure. The samples were irradiated at 1, 3, 6, and 8 kGy with mean rate of 1.6 ± 0.2 kGy min−1 , checked by chemical ceric–cerous sulfate dosimetry system. Non-irradiated samples were considered as controls. After irradiations the samples were stored in the dark conditions at room temperature. Irradiations were performed in duplicate. Determination of total carotenoid content Sea buckthorn oil was dissolved in hexane in a 25 mL volumetric flask. The absorbance of diluted oil was measured at 450 nm by using a Cary Bio 100 spectrophotometer (Varian Inc., Walnut Creek, CA, USA). Quantification of the amount of total carotenoids was performed by using calibration with 𝛽-carotene standard and amounts of carotenoids were expressed in mg kg−1 of oil.
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AA (%) =
Acontrol − Asample Acontrol
× 100
(1)
where Acontrol and Asample are the absorbance values of the reaction in DPPH solution without or with the tested oil. Peroxide value The peroxide value of sea buckthorn oil was determined by the iodometric titration method.25 Colour measurement Colorimetric features of samples were measured by determining the absorbance in the visible region (360–830 nm), for standard illuminant D65 (daylight source), observer angel of 10∘ (perception angle of a human observer) (Cary Bio 100 spectrophotometer, Varian Inc.). The colorimetric attributes expressed as CIELAB and CIELCH parameters (L*, a*, b*, C*, h∘ ) were analysed by using the Colour Application of Cary Win UV v. 3.10 software (Varian Australia Pty Ltd., Victoria, Australia). Fourier transform infrared spectroscopy Fourier transform infrared (FTIR) spectra of oil samples were recorded on a Tensor 27 FTIR spectrometer (Bruker Optik GmbH, Ettlingen, Germany) using the attenuated total reflectance (ATR) accessory. The samples were placed in contact with the ATR element. Spectra were recorded at room temperature (23 ∘ C) in the region 4000 to 650 cm−1 by an average of 64 scans at a resolution of 4 cm−1 . The background spectra was recorded in air (with no sample) and subtracted from the sample spectra. Between measurements, the crystal was carefully cleaned with hexane and ethanol. Two spectra were collected for each sample and then analysed with Opus v. 6.5. software ( Bruker Optik GmbH, Ettlingen, Germany). Regression analysis and statistical assessment The data reported are averages of triplicate observations except oil irradiation that was performed in duplicate. The regression analysis and statistics of experimental data were performed using OriginPro 8.1 (OriginLab Corporation, Northampton, MA, USA), Microsoft® Excel 2010 (Microsoft Corporation, Redmond, WA, USA), StatistiXL v.1.10 and InfoStat version 2013.26 The data were analysed by using analysis of variance (one-way ANOVA) with the Fisher LSD (least significant differences) post-hoc test to discern the statistical difference. A probability value P < 0.05 was considered as statistically significant.
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Estimation of total phenolic content Total phenolic compounds were quantified employing the Folin–Ciocalteu reagent. Sea buckthorn oil was mixed with 5 mL of 10-fold diluted Folin–Ciocalteu reagent and 4 mL of 1 mol L−1 sodium carbonate solution. The reaction mixture was shaken vigorously and then allowed to stand at room temperature for 30 min. The absorbance was measured at 765 nm (Cary Bio 100 spectrophotometer, Varian Inc.). Gallic acid was used as standard and thus the results were expressed as mg gallic acid equivalents (GAE) kg−1 of sample.
Evaluation of antioxidant activity The antioxidant effectiveness as a free radical-scavenging activity of sea buckthorn oil was determined by using the stable radical DPPH. A mixture containing 1 g of sea buckthorn oil and 8 mL of methanol:hexane (4:1) was shaken vigorously and allowed to stand overnight at room temperature. Then, 0.5 mL of this mixture was added to 2.5 mL of methanol:hexane (4:1) and 0.5 mL of 1 mmol L−1 DPPH solution in methanol; this reaction mixture was shaken and allowed to stand at room temperature for 30 min in the dark. The radical-scavenging activity of samples was quantified by decolorisation at 517 nm so that lower absorbance indicated stronger scavenging activity. The antioxidant activity (AA) of sea buckthorn oil was calculated using the equation:
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MR Nem¸tanu, M Bra¸soveanu
Table 1. Carotenoid and phenolic contents, antioxidant activity and peroxide value of sea buckthorn oil Total carotenoid content (mg · kg−1 oil)
Irradiation dose (kGy)
Total phenolic content (mg · kg−1 oil)
967 ± 9a 948 ± 18b 929 ± 6c 888 ± 3d 859 ± 8e
0 1 3 6 8
629 ± 10a 627 ± 15a 623 ± 24a 615 ± 10a 613 ± 9a
AA (%) 62 ± 5a 46 ± 7b 41 ± 4b 31 ± 2c 28 ± 3c
PV (meq O2 · kg−1 ) 1.5 ± 0.1a 2.1 ± 0.2b 3.6 ± 0.1c 5.0 ± 0.3d 5.5 ± 0.1e
Values within each column with different superscripts are significantly different (P < 0.05). AA, antioxidant activity; PV, peroxide value.
RESULTS AND DISCUSSION Influence of electron beam on carotenoid content Among the wealth of constituents of sea buckthorn oil, carotenoids are very important due to their antioxidant role. Besides, carotenoids are responsible for the orange colour typical for sea buckthorn oil, and their content is considered an important quality index for this type of oil. Because of their unsaturated carbon chain, these compounds are susceptible to some reactions, such as oxidation and isomerisation, during processing and storage of foods, and they consequently suffer changes in their nutritional value.27 Thus, the total carotenoid content was analysed to estimate the rate of degradation during the irradiation processing. The amount of carotenoids in raw sea buckthorn oil was 967 ± 9 mg kg−1 oil. The carotenoid content gradually decreased by e-beam irradiation (P < 0.05), the loss being about 11% for 8 kGy irradiation (Table 1). This reduction can be attributed to the oxidative processes caused by e-beam irradiation. A similar evolution was noticed for 𝛽-carotene in sunflower, soybean and palm oils exposed to irradiation,17,28,29 meaning that increasing the radiation dose, decreased the carotenoid level due to the radiation-induced breakdown of carotenoid molecules. Consequently, the reduction in the carotenoid level will result in lowering the oil stability to oxidative reactions.28,29 Oxidation leads to the formation of highly reactive species, such as alkyl and peroxyl radicals, that further react with carotenoid compounds, resulting in their degradation. The change of total content of carotenoids was adequately (R2 = 0.998) described by the zero-order kinetics equation [Eqn (2)] in the range of irradiation doses (0–8 kGy) tested (Fig. 1): C = C0 − kD
(2)
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where C and C 0 are the concentrations for irradiated and control sample, respectively, (mg kg−1 oil), k is the degradation rate constant (mg kg−1 kGy−1 ), and D is the irradiation dose (kGy). The model parameters (initial concentration of carotenoids and degradation rate constant) for degradation of carotenoid content of sea buckthorn oil exposed to electron beam irradiation in the range of 0–8 kGy were estimated as C 0 = 969 ± 2 mg kg−1 oil and k = 13.5 ± 0.4 mg kg−1 kGy−1 , respectively. Knowledge of a mathematical description of the dose–effect relationship and its model parameters might correctly enable the prediction of changes of the total carotenoid content during the irradiation and permit the proper selection of processing conditions for the acceptable level of carotenoid content loss.
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Figure 1. Evolution of total carotenoid content with irradiation dose.
Influence of electron beam on total phenolic content Phenolic compounds are known as very important constituents of vegetal materials with recognised capacity of radical scavenging due to their hydroxyl groups.30,31 According to Spiridon et al.,32 the antioxidant activity of phenolic compounds is mainly due to their redox properties, which enable them to act as reducing agents, hydrogen donors, and singlet oxygen quenchers. In raw sea buckthorn oil, 629 ± 10 mg GAE kg−1 oil of phenolic derivatives was detected. This value was slightly (P > 0.05) reduced by e-beam irradiation up to 8 kGy (Table 1). Influence of electron beam on antioxidant activity Sea buckthorn oil has been proved to be an outstanding free radical inhibitor acting as a primary antioxidant.14 In this study, the capacity of free radical scavenging of sea buckthorn oil calculated as percentage of DPPH radical inhibition was effective with value of 62 ± 5%. This ability reduced dramatically (P < 0.05) with the irradiation dose increase (Table 1). Even relative low doses of 1 kGy and 3 kGy caused the diminishing of about 30% in the antioxidant activity, while irradiation doses ≥ 6 kGy caused a more than 50% decrease in the initial value of antioxidant activity of unirradiated oil. The reduction of antioxidant activity might be correlated with the carotenoid level decreasing evinced in this study as well as with a possible deterioration (degradation and peroxidation) of the unsaturated fatty acids. The latter can also greatly contribute to the overall antioxidant activity as Ting et al.14 and Gruia et al.33 reported earlier.
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Figure 2. Visible spectra of unirradiated and irradiated samples sea buckthorn oil (inner window: variations of peak intensity at 670 nm with the red–green coordinate).
Influence of electron beam on peroxide value Peroxide value (PV) is an index of the oxidation degree of oil and fats subjected to stress factors able to initiate oxidative reactions. Lipid oxidation produces several intermediate chemical species like peroxides and hydroperoxides, which lead to rancidity of oil. Thus, PV indicates the amount of these compounds formed as a result of oxidation in the oil sample. A high PV confirms that the oxidation process occurred. The peroxide value for unirradiated sea buckthorn oil sample was 1.5 ± 0.1 meq kg−1 of oil, showing a low level of oxidation. A significant (P < 0.05) increasing trend was observed with increase in radiation dose (Table 1). Such behaviour might be due to oxidation and preferential radiolytic cleavage of bonds in the oils.20,21 The increase of the peroxide value with increasing radiation dose were also reported for sunflower, soybean, palm and canola oils17,28,29,34 or oils extracted from irradiated black cumin,35 peanuts20 and sunflower and maize seeds.21 A PV greater than 20 meq kg−1 of oil corresponds to very poor quality oils, even rancid, which normally would have significant off-flavours.36,37 Therefore, in our study, though the PV had an increasing trend with the irradiation dose, it was below the limit indicating oxidative rancidity.
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CIE L*, a*, b* coordinates According to the CIE L*, a*, b* colour space, the sea buckthorn oil has a rather dark (L* ∼35%) orange–reddish colour consisting of yellow (+b*) and red (+a*), 60.46 ± 0.02 and 54.62 ± 0.01, respectively. Table 2 shows the changes of the colour parameters according to the irradiation dose. After e-beam irradiation
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Influence of electron beam on colorimetric parameters Colour is one of the characteristics of a food product involved in the concept of food quality and can influence the appreciation and judgment of the product quality by the consumer. Thus, colour is one of the vital components of the visual quality of the foods and plays an important role in the consumer choice.38 As e-beam irradiation is able to induce changes in properties depending on the level of irradiation dose used and the structure and/or composition of the treated material, colour can also offer global information about the changes of some constituents or properties. In this study, colour change of oil samples was evaluated by chromatic parameters calculated in two colour spaces CIE L*, a*, b* and
CIE L*, C*, h∘ . These systems are recommended by the CIE (Commission Internationale de l’Eclairage), and are widely adopted by the food industry for colour measurement.39 Thus, the colorimetric attributes were expressed as CIELAB L* (lightness: 0 (black) → 100 (white)), a* (red–green coordinate: +a* → red, −a* → green) and b* (yellow–blue coordinate: +b* → yellow, −b* → blue) as well as CIELCH L* (lightness), C* (chroma) and h∘ (hue). The colour of untreated oil sample was used as reference for determination of the total colour difference, ΔE ab, of each irradiated sample. Although the colour of vegetable oils is associated with the total pigment content,40 the dark orange–red colour typical for sea buckthorn oil is mainly due to the carotenoids as a result of their conjugated double-bond system (chromophore). For all investigated samples the spectra showed absorption peaks between 360 and 560 nm, which can be attributed to yellow–red pigments, i.e. carotenoids and polyphenols.41,42 Also, the oil had two absorption maxima: a weak peak at 610 nm and a stronger one at 670 nm (Fig. 2). Nikolova et al.43 identified only one absorption peak at 670 nm in the visible region of sea buckthorn oil. The well-defined absorption bands centered for most of the oils at wavelengths 670 and 610 nm show the presence undoubtedly of chlorophyll compounds known as a green pigment group.44 Electron beam irradiation at doses higher than 1 kGy clearly had influence (P < 0.05) on these maxima expressed as a hypochromic effect. A small hypsochromic shift of 1 nm for both absorption peaks was also detected in samples irradiated with the highest applied dose. These observations suggest that the chlorophyll pigment level diminished by irradiation.
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Table 2. CIE L*, a*, b* parameters of sea buckthorn oil Irradiation dose (kGy) 0 1 3 6 8
L* 35.07 ± 0.01a 35.17 ± 0.11b 35.87 ± 0.01c 37.71 ± 0.06d 40.39 ± 0.02e
a* 54.62 ± 0.01a 54.73 ± 0.05b 54.89 ± 0.05c 55.22 ± 0.03d 56.78 ± 0.04e
b* 60.46 ± 0.02a 60.63 ± 0.18b 61.84 ± 0.02c 65.01 ± 0.11d 69.62 ± 0.03e
Values within each column with different superscripts are significantly different (P < 0.05).
a* values slightly increased (P < 0.05) as a function of the irradiation dose, and oil was less ‘green’, reflecting the possible loss of chlorophyll-derived pigments. An example to support this allegation is related to absorption peak at 670 nm assigned to chlorophyll compounds. Thus, the increase in the a* value with concomitant decrease in this peak intensity (inset graph of Fig. 2) were highly positive correlated (R2 = 0.998) confirming the reduction of chlorophyll pigment content. These findings are in agreement with those of other investigators45 who proved that chlorophyll content of the linoleic acid solution containing chlorophyll b affected greenness, resulting in an increase in the a*-value. Actually, according to Byun and co-workers,45 by its ability to breakdown chlorophyll, irradiation technology could be applied to reduce or eliminate the residual chlorophyll in oil processing without developing lipid oxidation during the irradiation process. The proportions of yellow had instead a greater increase (P < 0.05) with the irradiation dose, up to 69.62 ± 0.03 for 8 kGy, as a probable consequence of rearrangements of chemical structure, mainly of conjugated double bonds typical for carotenoid pigments. The lightness, L*, values had increasing evolution (P < 0.05) with the irradiation dose increase up to 40.39 ± 0.02 for 8 kGy in a similar manner as yellow coordinate. The positive variation in L* values indicates that the oil became lighter with increasing irradiation dose. Briefly, the greatest differences in L*, a* and b* between each irradiated sample and control one were in b* followed by L*, the colour of oil being more yellow and lighter as irradiation dose increased. These results suggest that oil suffered alteration of colour through bleaching caused by oxidation process because of irradiation. CIE L*, C*, h∘ coordinates For the CIE L*, C*, h∘ colour system, lightness, L*, is similar to that
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of CIE L*, a*, b* space, chroma, C*, is the quantitative attribute of colorfulness while hue h∘ is the qualitative attribute of colour being expressed in degrees: 0∘ (red), 90∘ (yellow), 180∘ (green), 270∘ (blue). In these terms, for raw sea buckthorn oil, the values of chroma and hue were 81.48 ± 0.01 and 47.91 ± 0.01∘ , respectively. This location is equivalent to reddish–orange colour on CIE L*, a*, b* space. Figure 3 illustrates a significant (P < 0.05) shift of chroma as the irradiation dose increases, showing a value of 89.84 ± 0.05 for irradiation dose of 8 kGy. The positive values of chroma differences for all irradiated samples show that e-beam irradiation led to greater intensity of oil colour. In a similar fashion, hue moved further away from the origin and in the upward direction with ∼3∘ (50.80 ± 0.01∘ ) at 8 kGy, indicating the movement of colour toward yellow as the irradiation dose increased. In this colour space, the greatest differences in chromatic parameters
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Figure 3. CIE L*, C* and h∘ parameters of sea buckthorn oil.
between each irradiated sample and control one were in chroma, C*, followed by L*, the colour of oil being more intense and lighter as irradiation dose increased. Therefore, the increase of the irradiation dose caused the colour bleach of investigated samples suggesting the modification of the colored compounds (i.e. carotenes, xanthophylls) as a probable combined result of isomerisation and oxidative reactions. As mentioned, carotenoids are susceptible to degradation during processing and storage. Their common degradation pathways are isomerisation, oxidation, and fragmentation of the carotenoid molecules promoting changes in colour as a result of the rearrangement or formation of cis-isomers, epoxides, short chain products and, in some cases, volatile compounds.46 – 48 Total colour difference 𝚫Eab Total colour difference is important when evaluating the relationship between the visual and the numerical analyses.42 Colour difference is an indicator of global changes and interaction among components of a material. According to several research groups,42,49,50 the perception of the colour difference, ΔEab , varies depending on the observed colour and the sensitivity of the human eye so that the following limits for human eye perception of ΔEab were nominated: • 3 – perceptible colour difference. Experimental data achieved in this study showed clearly that radiation-induced changes of oil chromatic parameters are irrefutably reflected in the total colour difference ΔEab , which increased (P < 0.05) with irradiation dose. The colour change involved two steps described by zero-order kinetics, having different rates in the range of tested irradiation doses (Fig. 4). Slow degradation of oil colour occurred with rate of 0.51 ± 0.06 kGy−1 (R2 = 0.971) up to 3 kGy, followed by a fast degradation with significant higher rate (1.79 ± 0.42 kGy−1 , R2 = 0.948) up to 8 kGy. In addition, colour differences distinguishable by the human eyes were noticed for samples irradiated with 6 kGy (ΔEab = 5.29 ± 0.12) and 8 kGy (ΔEab = 10.81 ± 0.05). These findings are in agreement with the imposed limits previously mentioned. Sea buckthorn oil is a complex compositional matrix in which the components
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Figure 4. Evolution of ΔEab of sea buckthorn oil after irradiation.
interact among them exerting a certain protection to stress factors. Taking into account this aspect, it can be inferred that such protection could act against irradiation up to 3 kGy, explaining thus the slow rate of degradation of irradiated oil. One can consider the colour of sea buckthorn oil as a two-component system containing two principal groups of pigment compounds: yellow–red and green. As we have already seen, both chlorophyll pigments and carotenoid ones reduced by e-beam irradiation; the green pigments diminishing more than carotenoids. However, when the yellow–red pigments are present in considerable proportion, the removal of the green pigments does not have a large effect on the colour.44 Moreover, the intensity of the colour of sea buckthorn oil depends mainly upon the presence of carotenoids, which can explain the important variation of chroma simultaneously with degradation of carotenoid pigment as increasing of irradiation dose. Although from visual perception, which is a subjective way to assess color, no colour changes were perceptible for oil irradiated up to 3 kGy, the total colour difference indicated clearly considerable (P < 0.05) changes as increasing the irradiation dose. Changes in colour were previously reported for sunflower, soybean, and canola oils exposed to low or high levels of gamma radiation.28,34
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Pearson’s correlation analysis Pearson’s correlation test has been applied to analyse the relationship between investigated properties and irradiation dose. The correlation coefficients are shown in Table 3. The peroxide value, total colour difference and chromatic parameters such as L*, b*, C* and h∘ were strongly positive correlated (r = 0.95) with irradiation dose whereas the red–green coordinate a* had only good positive correlation (r ∼ 0.881) with irradiation dose. Conversely, the total carotenoid and phenolic contents as well as antioxidant activity were strongly negatively correlated (r = −0.93) with irradiation dose. Carotenoids and phenolic compounds have high contribution to the antioxidant activity indicated by the strong positive correlation coefficients (r = 0.987 and r = 0.925, respectively). According to Spiridon et al.,32 the synergism between the antioxidants in the mixture makes the antioxidant activity not only
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Fourier transform infrared spectroscopy Fourier transform infrared spectroscopy is an analytical tool often applied to qualitatively classify edible oil and fat products or to verify the authenticity of edible oils and fats, due to its simplicity, rapidity, and ease of sample preparation.51,52 The analytical evaluation of the FTIR spectrum of sea buckthorn oil showed bands typical to oils and fats according to existing literature.51 – 54 In the region of hydrogen stretching, the characteristic bands were identified around 3007 cm−1 (C—H stretching vibration of the cis-double bond —CH CH), 2953 and 2874 cm−1 (C—H asymmetric and symmetric stretching vibrations of the aliphatic —CH3 group), and 2924 and 2854 cm−1 (C—H asymmetric and symmetric stretching vibrations of the aliphatic —CH2 group). In the region of double bond stretching, the spectrum displayed a strong absorption band around 1744 cm−1 (ester carbonyl C O stretching) and a very weak peak around 1655 cm−1
(C C stretching vibration of cis-olefins). The spectral region of other bonds deformations and bendings showed bands at 1466 cm−1 (C—H bending (scissoring) vibrations of the —CH2 and —CH3 aliphatic groups), 1417 cm−1 ( C—H bending (rocking) vibration of the cis-disubstituted olefins) and 1378 cm−1 (C—H symmetric deformation of the aliphatic —CH3 group). In the fingerprint region, the peaks appeared around 1240 and 1166 cm−1 (stretching vibrations of the C—O ester groups and C—H bending vibrations of the aliphatic —CH2 groups), 1140 cm−1 , 1097 cm−1 and 1031 cm−1 (stretching vibrations of the C—O ester groups), 964 cm−1 and 912 cm−1 (out-of-plane bendings of trans- and cis-disubstituted olefinic groups), 722 cm−1 (overlapping of the C—H bending (rocking) vibration of the aliphatic —CH2 group and the out-of-plane vibration of the cis-disubstituted olefins). The spectral features of the e-beam irradiated oil samples were apparently similar to unirradiated oil (Fig. 5). No new functional groups were found in the FTIR spectra of irradiated samples. However, some differences related to the frequency and intensity of some bands have been found after irradiation. In the characteristic contour from 1300 to 900 cm−1 , a small peak shift of 1 cm−1 toward lower wavenumbers were detected for peaks centred at 1240, 1097 and 964 cm−1 even after the smallest irradiation dose applied. The weak peak at 1031 cm−1 assigned to the vibration of the O—C—C band of esters derived from primary alcohols suffered a larger shift to 1028 cm−1 after irradiation with 3 kGy. At the same time, the widening of the band (P < 0.05) took place with the increase of irradiation dose resulting in its disappearance at higher irradiation doses. The frequency of this band changes without specific pattern as the oxidation process advances.55 Conversely, the characteristic peak at 912 cm−1 related to the bending vibration of cis-disubstituted olefinic groups shifted to higher wavenumbers (915 cm−1 ) with broadening of band as increasing the irradiation dose. But according to Guillen and Cabo,55 the changes observed in the frequency of this band as the oxidation progresses are not relevant. On the other hand, decrease (P < 0.05) in the intensity (inset graph of Fig. 5) and width of the maximum absorption at 2924 cm−1 ascribed to the asymmetric stretching vibration of the aliphatic —CH2 functional group was also observed by increasing the irradiation dose. The changes identified in the spectral features of irradiated oil samples indicated that the structural integrity of sea buckthorn oil has been affected as consequence of oxidative degradation induced by irradiation. Although the alterations of spectral features occurred as irradiation dose increased, any specific pattern of changes could not be identified.
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Figure 5. FTIR spectra of unirradiated and irradiated samples of sea buckthorn oil (inner window: variation of peak intensity at 2924 cm−1 with irradiation dose). Table 3. Pearson’s correlation coefficients Parameter Irradiation dose (D) Total carotenoid content (TCC) Total phenolic content (TPC) Antioxidant activity (AA) Peroxide value (PV) Lightness (L*) Red–green coordinate (a*) Yellow–blue coordinate (b*) Chroma (C*) Hue (h∘ ) Total colour difference (ΔEab )
D
TCC
TPC
– −0.998 −0.993 −0.930 0.987 0.959 0.881 0.959 0.949 0.978 0.957
– – 0.987 0.937 −0.977 −0.964 −0.894 −0.964 −0.956 −0.980 −0.963
– – – 0.925 −0.990 −0.933 −0.829 −0.933 −0.919 −0.964 −0.930
dependent on the concentration, but also on the structure and the interaction between the antioxidants. Antioxidant activity had also negative correlations with peroxide value, chromatic parameters, and total colour difference. On the other hand, peroxide value was positive correlated with chromatic parameters and total colour difference. Total colour difference showed negatively correlation with carotenoid and phenolic contents and consequently with antioxidant activity while it was positively correlated with peroxide value.
CONCLUSIONS
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The influence of e-beam irradiation up to 8 kGy on antioxidant chemical compounds, radical scavenging potential, oxidative status, as well as colorimetric and spectral features of sea buckthorn oil was evaluated and discussed in correlation with irradiation dose. Irradiation exerted no considerable effect on phenolic
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AA – – – – −0.949 −0.820 −0.732 −0.820 −0.807 −0.848 −0.818
PV – – – – – 0.903 0.801 0.903 0.888 0.934 0.900
L* – – – – – – 0.971 1.000 0.999 0.994 1.000
a*
b*
C*
– – – – – – – 0.971 0.980 0.939 0.973
– – – – – – – – 0.999 0.994 1.000
– – – – – – – – – 0.988 0.999
h∘ – – – – – – – – – – 0.993
content. Degradation of total content of carotenoids followed zero-order kinetics with rate constant of 13.5 ± 0.4 mg kg−1 kGy−1 . Antioxidant activity was also negatively affected especially at higher irradiation doses (≥6 kGy). The peroxide value was below the limit indicating the oxidative rancidity though it had an increasing trend with the irradiation dose. Chromatic parameters changed by irradiation, the colour of oil being more yellow, intense and lighter as irradiation dose increased. No colour changes were distinguishable by the human eye for oil irradiated up to 3 kGy, but the total colour difference indicated significant changes involving a two-step pattern associated with slow degradation of oil colour (rate of 0.51 ± 0.06 kGy−1 ) up to 3 kGy, followed by a fast degradation (rate of 1.79 ± 0.42 kGy−1 ) up to 8 kGy. Therefore, colour measurement that is non-destructive and requires only a small sample can be suitable tool to evaluate the aesthetic aspects and to monitor the quality in industrial processing of sea buckthorn oil.
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Although the spectral features of the irradiated oil samples were apparently similar to control, some differences related to the frequency and intensity of some bands have been found after irradiation indicating radiation-induced alteration of the oil structural integrity. In conclusion, electron beam irradiation up to 3 kGy can be an effective eco-friendly mean of ensuring oil safety with minimal undesirable changes in its quality. Therefore, an appropriate balance between favourable and side effects of oil irradiation might be achieved by responsible selection of processing parameters. In addition, it is important to accept that some potential loss of chemical compounds with bioactive role can be compensated by the improved hygiene of the irradiated food.
ACKNOWLEDGEMENTS This work was supported by project Nucleu (2014) and IIN (Electron Accelerators Laboratory of the National Institute for Lasers, Plasma and Radiation Physics).
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