Food Irradiation Principles 016

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Detection Methods for Irradiated FoodsE I L E E N M . S T E W A R TFood Science Division, Food Chemistry, The Department of Agriculture and RuralDevelopment for Northern Ireland and Department of Food Science,The Queen's University of Belfast, Belfast, Northern Ireland

14 .1 . INTRODUCTIONPreceding chapters have described in detail the principles and applications of thefood irradiation process. The uses of the technology are clearly wide-ranging andeffective, with minimum disruption to the functional or organoleptic qualities offood at the doses used on a com m ercial basis. Yet for years the technology has beenunderutilized by the food industry, treated with wariness by consumers and theirrepresentative organizations alike. However, the 1990s witnessed a significant ad-vancement in food irradiation processing; as a result, progress has been made incommercialization of the technology, culminating in greater international trade inirradiated foods and the implementation of differing regulations relating to its usein many countries. Consequently, this has led to the demand by consumers thatirradiated food should be clearly labeled as such and that methods capable ofdifferentiating between irradiated and nonirradiated products should be available.Thus a practical basis was sought to allow consum ers to exercise a free choice as towhich food they purchase. If a food is marketed as irradiated or if irradiated goodsare sold without the appropriate labeling, then detection tests should be able toprove the authenticity of the product.

Prior to the 1980s little progress was made in the development of reliabledetection methods for irradiated foods. Lack of emphasis in this area was duepartly to the fact that detection methods were deemed unnecessary because itwas considered that food products would be irradiated in licensed facilities andthat appropriate documentation would accompany irradiated foods throughout thefood chain. However, between 1985 and 1995 significant research took place, withthe result that in December 1996 the European Committee for Standardization(CEN) adopted five European Standards for the detection of irradiated foodsFood Irradiation: P rinciples and Applications, Edited by R. A. MolinsISBN 0-471-35634-4 2001 John Wiley & Sons, Inc.

CHAPTER 14


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(Table 14.1). Such progress was made possible because of the individual efforts ofresearch teams in many countries and the noteworthy international cooperation inthis field. The European Community (EC), through its Community Bureau ofReference (BCR), set up a collaborative program to develop methods to identifyirradiated food. On a worldwide basis, the Joint FAO/IAEA Division of NuclearTechniques in Food and Agriculture set up a coordination program on analyticaldetection methods in irradiation treatment of food (ADMIT) which promoted co-operation in this area.

14.2. CRITERIA FOR A RELIABLE DETECTION METHODThe criteria that an ideal detection method should meet were clearly documentedby the first research coordination meeting of ADMIT held in Poland in June 1990(Anonymous 1990), and were reconfirmed by the third and final ADMIT meetingheld in Belfast, Northern Ireland in June 1994 (McMurray et al. 1996). Two sets ofcriteria were elaborated; technical criteria, which are to be met if a qualitative orquantitative test is to be successfully developed; and practical criteria, which aredesirable if a method is to be applied widely by authorities concerned with thecontrol of labeling of irradiated food moving in trade. Table 14.2 lists both sets ofcriteria. It was determined that it is not necessary for each method to exhibit all thecharacteristics listed in order to be useful. However, the list was drawn up to serveas a standard for the "ideal" detection method against which proposed methodscould be evaluated.

The methods currently available for the detection of irradiated foods are basedon physical, chem ical, biological, and microbiological changes that, although m ini-mal, are induced in food during the irradiation process. This chapter reviews themost useful methods currently available for the detection of irradiated food. Forfurther information, the Royal Society of Chemistry publication by McMurray et al.(1996), which contains the proceedings of an International Meeting on Analytical

NumberEN1784EN1785EN1786EN1787EN1788

Topic of StandardFoodstuffs detection of irradiated food containing fat, gas

chromatographic analysis of hydrocarbonsFoodstuffs detection of irradiated food containing fat, gaschromatographic/mass spectrometric analysis of 2-alkylcyclobutanonesFoodstuffs detection of irradiated food containing bone, method byESR spectroscopy

Foodstuffs detection of irradiated food containing cellulose, methodby ESR spectroscopy

Foodstuffs detection of irradiated food from which silicate mineralscan be isolated, method by thermoluminescence

TABLE 14.1. CEN European Standards for the Detection of Irradiated Foodstuffs


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Technical CriteriaDiscrimination parameter measured in the irradiated food should be absent in the nparameter should be well characterized in the nonirradiated food and changes inducedand separableSpecificity other food processing methods and storage should not induce comparable cApplicability he test should apply throughout the dose range relevant to the irradiatiStability he parameter should be useful for at least the storage life of the irradiated Robustness he measurement should be insensitive to the following effects, or its reconfidence, e.g., dose rate, temperature at any stage of treatment or storage, other storfurther processing, admixture with other foodIndependence he method should not require samples of the nonirradiated food fromReproducibility and repeatabilityAccuracy and proper statistical validationSensitivity he method should be capable of detecting doses below the commerciallyDose dependence he method should be capable of generating a dose response curvemeasurement of dose applied to the food

Practical CriteriaSimplicity he method should not demand high levels of technical skills, data interprLow costSmall sample sizeSpeed of measurementThe method should apply to a wide range of food and food typesNon-destructive measurement of the parameterThe method should be capable of easy standardization and cross-calibrationConfidence that the method is resistant to fraud; It would desirable if the parameters to the associated packaging, e.g., mineral dusts

(a)

(b)(c)(d )(e)

(O(g)(h )(i)(J)

(a)(b)(c)(d)(e)< f )(g)(h)

Source: McMurray et al. (1996).

TABLE 14.2. Technical and Practical Criteria of a Detection Method for Irradiated F


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Detection Methods for Irradiation Treatments of Foods held in June 1994, as wellas reviews by Delincee (1995, 1998a), Desrosiers (1996), Glidewell et al. (1993),Raffi and Stocker (1996), and Schreiber et al. (1993a), are recommended.

14.3. PHYSICAL METHODS14 .3 .1 . ESR SpectroscopyElectron spin resonance (ESR) Spectroscopy is a physical technique that detectsspecies with unpaired electrons, for example, free radicals. As pointed out inChapter 3, ionizing radiation produces free radicals in food, and since ESR Spectro-scopy detects free radicals, it can be used to determine whether certain foods havebeen irradiated. In foodstuffs with a relatively high moisture content, such as meatand vegetables, the induced radicals disappear very rapidly. On the other hand, iffood contains components with a relatively high dry matter, for example, bone,seeds, or shells, the radicals may be trapped and remain sufficiently stable to bedetected by ESR (Desrosiers and Simic 1988). Experimental work has demon-strated that the technique can be employed for the detection of a wide variety ofirradiated foods, as detailed in reviews by Desrosiers (1996) and Raffi and Stocker(1996).

ESR has been utilized to detect the presence of radiation-induced free radicals inbone since the mid-1950s (Gordy et al., 1955). Archeologists use this technique todetermine the age of fossil bone and similar biological material from the ESRsignals induced by natural radiation. Furthermore, postirradiation dosimetry oftooth enamel by ESR has been used to determine the dose absorbed by humanssubjected to radiation exposure (Pass and Aldrich 1985). Onderdelinden andStrackee (1974) suggested that ESR Spectroscopy had potential as a method forthe detection of irradiated food containing bone, and this was confirmed by Doddet al. (1985). Research has shown that nonirradiated bone gives a weak, broadESR signal (Fig. 14.1) which increases in magnitude if the bone is ground into a

Irradiated

NonirradiatedFIGURE 14.1. ESR Spectra from irradiated and nonirradiated bone.


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pow der (M arino and Becke r 1968). The signal from irradiated bo ne is a large axiallyasymmetric singlet that can be easily distinguished from the endogenous signal(Desrosiers and Simic 1988, Stevenson and Gray 1989a).Two prevailing types of paramagnetic species have been observed following

irradiation of bone tissue. One is derived from bone collagen (Marino and Becker1968); the other is attributed to structural defects in the crystalline fraction of themineral constituent of bone, the hydroxyapatite [Ca10(PO4)6(OH)2] (Ostrowskiet al. 1974). The former decays slowly in contact with atmospheric oxygen, whilethe latter is extremely stable even after years of storage at room temperature in air(Ostrowski et al. 1974). It is surmised that the characteristic signal produced onirradiation of bone is due to CO^", CO^", or CO^" or ion radicals trapped in thehydroxyapaptite matrix (Cevec et al. 1972; Geoffroy and Tochon-Danguy 1982,Michalik 1975, Serway and Marshall 1967).The majority of work carried out using ESR has been concerned with chickenbone (Desrosiers and Simic 1988; Stevenson and Gray 1989a,b; Dodd et al. 1992;Onori et al. 1996) with bones from duck, turkey, goose, beef, pork, lamb, and froglegs also being studied to a limited extent (Dodd et al. 1985, Goodman et al. 1989,Raffi et al. 1989). The signal produced from all of these bones is essentially thesame; thus it is evident that ESR can be used for the qualitative detection ofirradiation in a wide range of meats containing bone. The reliability of the ESRmethod for identification of irradiated food containing bone has been validated in anumber of interlaboratory blind trials (Anonymous 1992, Desrosiers et al. 1990,1994, Raffi et al. 1992, Schreiber et al. 1993b, Scotter et al. 199Oa), and hassubsequently been endorsed by CEN as a European Standard (ENl786). The meth-od can, in addition, be used for the identification of irradiated mechanically recov-ered meat (MRM), a secondary food product from which small bone fragments canbe extracted (Gray and Stevenson 1989). Experimental work demonstrated that

even when the irradiated MRM is included as an ingredient in, for example, beefburgers or meat balls, it can be detected at inclusion levels as low as 3g/100g(Stevenson et al. 1996). A characteristic ESR signal similar to that obtained frombone has also been derived from irradiated eggshell (Desrosiers et al. 1996, Onoriand Pantaloni 1995). When tested by an interlaboratory blind trial (Desrosiers et al.1996), samples of irradiated egg shell we re identified with a 100% success rate evenwhen treated at doses as low as 0.3 kGy.As well as qualitative identification of irradiation, the use of ESR for the quan-

tification of irradiation dose has been investigated for chicken bone and eggshell.The review by Desrosiers (1996) lists the numerous references on this particularaspect of the ESR methodology. Onori and Pantaloni (1995) have also carried outwork on dose estimation.Preliminary studies by Dodd et al. (1985) demonstrated that ESR spectroscopyhad potential for the identification of irradiated Crustacea using the signal in-duced in the exoskeleton or shell, which has a relatively high dry matter content.This finding was later substantiated by workers such as Goodman et al. (1989),

Desrosiers (1989), Raffi and Agnel (1990), Morehouse and Ku (1992), Helleet al. (1993a), and Stewart et al. (1992, 1994). However, it was evident from the


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variety of different ESR spectra published by these workers that the ESR signalderived from the shell of irradiated prawns or shrimp is species-dependent. It istherefore recommended that the analyzed species be identified by its scientificname to avoid discrepancies in experimental results (Anonymous 1992). The geo-graphical origin of a prawn or shrimp species has also been shown to influencesESR signal shape (Stewart and Gray 1996). Interlaboratory blind trials (Stewart andKilpatrick 1997) have demonstrated that irradiated Norway lobster (Nephrops nor-vegicus), brown shrimp (Crangon crangori), and Mediterranean crevette [Panaeus(Panaeus) semisulcatus] can be detected using ESR spectroscopy, but that irra-diated samples of a certain species of pink shrimp (Pandalus montagui) are noteasily identified. Further work is therefore needed to validate the method for addi-tional species. Research has further shown that ESR can be employed to detectirradiation treatment in shellfish such as mussels, oysters, and scallops using theattached shell (Raffi et al. 1996).

ESR spectroscopy can be a useful tool for the identification of irradiated foodcontaining cellulose and crystalline sugars. In most fruits, the water content variesfrom 80 to 95 % , and radicals induced in the pulp are not stable. How ever, the seeds,shells, or skins of fruits and also vegetables, could be used to detect irradiationtreatment, as their moisture content is low and the free radicals are, therefore,relatively stable. Raffi et al. (1988) first examined the ESR signal derived fromthe achenes (seeds) of strawberries, and derived a multicomponent signal that istypical of that from foodstuffs containing cellulose. A central single line is presentin both irradiated and nonirradiated samples (Fig. 14.2A,B), and is thought to arise

Magnetic field (m l) Magnetic field ( m l )FIGURE 14.2. ESR spectra of nonirradiated paprika (a) and paprika irradiated to 10kGy (b)(Source: Desrosiers et al. 1996; with permission).


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from a semiquinone radical. This signal increases with irradiation dose, but variesto a large extent with the water content. In the case of irradiated samples (Fig.14.2B), a pair of outlying lines, proposed to originate from cellulose, occurs to theleft and right of the central signal, the left one of which is more easily detected.These lines are not present in nonirradiated samples and therefore can be used as atest for irradiation treatment. In some foods, broad lines of low intensity due toM n 2 + can also be observed, although it should be noted that their position in themagnetic field is different from that of the radiation-induced signal.

The use of this identification test has been proposed for a wide range of fruits(Raffi et al. 1989, Desrosiers et al. 1989, Stachowicz et al. 1992, Deighton et al.1993, Glidewell et al. 1996). In addition, it has been successfully employed for thedetection of irradiated nuts, some aromatic herbs, and spices (Helle and Linke1992, Raffi 1996, Uchiyam a et al. 1990), as we ll as for certain packaging m aterialscontaining a high percentage of cellulose (Helle et al. 1993b, Stevenson and Gray1995). As stated previously (Table 14.1), the method has been implemented as aEuropean Standard (EN1987) following validation by a number of collaborativeblind trials (Desrosiers et al. 1996, Linke et al. 1996, Raffi et al. 1992, Schreiber etal. 1993b, 1996). Foods validated by interlaboratory blind trials include paprika,fresh strawberries, and pistachio nuts.Complex ESR signals (Fig. 14.3) have been derived from irradiated dried fruitssuch as dates, grapes, m ango, papaya , and pineap ple, wh ich are easily distinguish-able from nonirradiated samples. As the overall sugar content of fruits varies from60 to 75 % , and the m ain com ponents are D-fructose, D-glucose, and D-saccharose, itwas proposed that the signals induced in dried fruits by ionizing radiation are due tosugar radicals (Raffi et al. 19 91, H elle and Link e 1992, Stachow icz et al. 1992).Generally, these radiation-induced signals are sufficiently stable to allow identifica-tion of irradiated samples, even following several months of storage (Helle and

Irradiated

Nonirradiated

FIGURE 14.3. ESR spectra derived from irradiated (2kGy) and nonirradiated driedmango.


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Linke 1992, Raffi et al. 1991). Interlaboratory blind trials have indicated that thisESR method is useful for the identification of irradiated dried figs, mangoes, pa-payas, and raisins (Raffi et al. 1992, Linke et al. 1996). Following the successfuloutcome of these trials, the method is under consideration by CEN for adoption as aEuropean Standard.It is clear that ESR spectroscopy can be used for the detection of irradiationtreatment in a wide variety of foods. The technique is specific, rapid, and simple toperform and is nondestructive, thereby allowing samples to be reanalyzed.Although the cost is still substantial, the development of desktop ESR spectro-meters has significantly reduced the expenditure on necessary equipment, and themethod has become increasingly popular with food control laboratories.

14.3.2. Luminescence MeasurementLuminescence is the emission of light when trapped energy is liberated by theaddition of a chemical (chemiluminescence), heat (thermoluminescence), or light(photostimulated luminesence). Chemiluminescence (CL) and thermoluminescence(TL) were both studied initially as possible techniques for detection of irradiatedfoods. In CL, alkaline luminolhaemin solution is added to the dry substance and theCL response is measured with a light detector (Heide and Bogl 1987, 1988, Heideet al. 1989). However, as a number of problems were associated with the use of CL,and owing to the success of TL analysis, which was generally regarded as lessproblematic, the importance of CL declined and research on this technique wasdiscontinued.

The physical process of TL is based on the fact that electrons in the excited statereturn to the ground state when thermally stimulated (Heide and Bogl 1987, 1988,Oduko and Spyrou 1990). When a substance exhibiting TL is exposed to ionizingradiation, electron-hole pairs are produced, and some electrons (or holes) may be-come trapped at certain sites in the material. They remain in these traps until theyacquire sufficient thermal energy to escape. As the material is heated, electrons arereleased from the traps andlight is emitted as they recom bine with holes. The intensityof the emitted light can be measured as a function of temperature, and the result is theso-called glow curve, which is characteristic of the examined substance. The TLphenomenon is not unique to irradiation, but if the TL response following irradiationis significantly greater than thebackground signal and the fading (i.e., decrease of theTL signal) is low over a period of weeks or months, then TL measurement may besuitable for determining whether foodstuffs have been irradiated. The initial workusing TL as a means of identifying irradiated food was carried out using wholesamples of herbs and spices (Sanderson et al. 1989a,b). However, a marked variationwas observed in the levels of light emitted by irradiated and nonirradiated samples ofthe same herb or spice, and problems were also encountered with the stability of thesignal during storage (Sanderson 1990). At first it was thought that the TL signal arosefrom the organic com ponent of the samples, but further research clearly showed thatthe signals from herbs and spices actually originated from adhering mineral grains,even though they account for less than 1 % of sample weight (Sanderson et al. 1989a,b,


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Autio and Pinnioja 1993, W agner et al. 1993). Th is was the mo st probab le explana tionfor the high va riation in results betw een different samp les of who le spices and herbs.Whereas measurements on whole samples may be appropriate for rapid screening(identifying irradiated samples with about 90% confidence), measurements on sepa-rated mineral grains offer a far higher level of confidence (Sanderson et al. 1989a).Unequivocal identification can be assured by incorporation of a reirradiation stepwhereby the ratio of the first and second glow curves are measured. These refine-ments, although time-consuming, give added confidence to the results obtained.

Following the identification of the source of the signal, the technique has beenapplied to other foodstuffs from which mineral grains can be obtained. The methodhas been validated by interlaboratory trials [see Sanderson et al. (1995, 1998) forreferences to interlaboratory trials] for herbs, spices, their mixtures, fresh fruits, andvegetables (strawberries, avocados, mushrooms, papayas, mangoes, potatoes), de-hydrated fruits and vegetables (sliced apples, carrots, leeks, onions, powderedasparagus) as well as shellfish, including shrimps and prawns. In the case ofshrimps and prawns, the mineral grains present in the intestinal gut are isolatedand analyzed. As a result of the success of these trials, the TL method was adoptedas European Standard EN1788.The standard TL method requires a physical separation of the minerals from the

food matrix. Thus, the need for careful laboratory preparation of TL samples andaccess to a calibrated source of ionizing radiation have limited the widespread useof TL for routine commercial or enforcement testing (Sanderson et al. 1996a). Inaddition, there are bioinorganic food components, such as bone and shells, wherethe intimate m ixture of inorganic and organic com ponents inhibit h igh-temperatureTL analysis. The development of photostimulated luminescence (PSL) has beenaimed at resolving the practical limitations of silicate TL methods. PSL employslight rather than heat as a stimulus for releasing the trapped energy induced byradiation in solid materials. The method has overcome the need for full mineralseparation and of providing radiation-specific stimulation schemes appropriate forbiogenic materials. A low-cost instrument has been developed (Sanderson et al.1994, 1996a) for high-sensitivity PSL measurements from food samples using thehighly radiation-specific UV-visible luminescence signals that can be stimulatedusing infrared sources. Samples are introduced in disposable petri dishes, withminimal preparation, and the instrument produces a qualitative screening measure-ment in 15 sec. Initial results showed that over 90% of irradiated herbs and spicescould be recognized without reirradiation (Fig. 14.4). There was a small overlapbetween high-sensitivity nonirradiated samples and low-intensity irradiated sam-ples. However, irradiating samples to a known dose and rereading the PSL signals(calibrated PSL) allows the sensitivity of the sample to be estimated. Thus, twomodes of operation may be employedithe "screening mode," where the lumines-cence intensity detected from the samples is used for preliminary classification intonegative, intermediate or positive bands; and "calibrated PSL" (CaIPSL), whichcan distinguish between low- and high-sensitivity samples, thus resolving ambig-uous or low-sensitivity cases. A positive screening result must be confirmed usingCaIPSL or another standardized method such as TL (EN1788).


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PSL / Photon countsFIGURE 14.4. PSL intensity data for 45 varieties of herbs, spices and seasonings measuredin duplicate over a 60 sec period (Source: Sanderson et al. 1995, 1996a; with permission).

The method has been tested by interlaboratory blind trials using shellfish, herbs,spices, and seasonings. In the case of shellfish, the signals from intestinally trappedsilicates can be stimulated through the membranes of dissected guts, and in somecases, through the whole body of the creature. The instrument is also capableof responding to signals from shells for at least several weeks after irradiation(Sanderson et al. 1996b). Because of the successful outcome of the collaborativeblind trials (Sanderson et al. 1995, 1998), PSL has been shown to be a reliablemethod for the identification of irradiated food and is currently being considered byCEN for adoption as a European Standard.

14.3.3. Viscosity MeasurementWhen polymers are subjected to ionizing radiation, their molecular structure ischanged; as a result, there is a change in viscosity. Dwight and Kersten (1938)observed that relative to nonirradiated samples, the gelling capacity of irradiatedapple pectin was depressed; that is, the structural viscosity decreased. Subsequentstudies by Kertesz et al. (1956) revealed that changes in viscosity occurred at0.2 kGy in the case of pectin and at 10 kGy in cellulose. This effect of viscosityvariation was first used by Mohr and Wichmann (1985) to detect irradiation treat-ment of spices. Using a radiation dose of 8 kGy, a marked decrease in viscosity wasfound in suspensions of black pepper, mustard seed, savory, and caraway, with aless pronounced effect being observed for green pepper. Similar results were laterobtained by workers such as Farkas et al. (1990) and Barabassy et al. (1996) forheat-treated suspensions of white and black pepper, nutmeg, ginger, and marjoram

Irradiated (6 kGy)Unirradiated

Nmboo

vo


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after irradiation at a dose of 8 kGy, with no viscosity changes observed in samplesof allspice, garlic powder, and onion powder. The identification limit by the visco-metric method depends on the spices analyzed; it is 4 kGy for peppers and 2 kGyfor cinnamon. At 8 kGy or higher doses, the method is applicable for detection ofirradiated spices having a considerable starch content.The method, however, has been applied largely for the detection of irradiatedpeppers. According to Hayashi et al. (1996a), viscosity rates are influenced by theconditions for viscosity measurement such as shear rate, temperature, and type ofviscometer, and it is not easy for laboratories that do not possess an irradiationsource to establish criteria for judging whether peppers have been irradiated. Con-sequently, a new normalized parameter was incorporated into the methodology formore reliable detection of irradiated pepper samples (Hayashi et al. 1996a). Acollaborative blind trial carried out by Hayashi (1996) concluded that the viscositymeasurement can be used, at least as a screening method, for detecting irradiatedblack and white pepper. It was noted that procedures for gelatinizing pepper sus-pensions, the viscosity measurement, and the detection parameters, need to beestablished for each viscometric system. The results of the collaborative trial alsosuggest that the pH of the suspensions should be adjusted to a value as high aspossible for better differentiation of irradiated and nonirradiated peppers.

14.3.4. Electrical Impedance MeasurementThe membranes of living tissue, whether of plant or animal origin, play a vital rolein the selective transport of ions; consequently, a change in membrane propertiesmay be traceable by a change in ion transport. Therefore, it was suggested thatelectrical impedance and conductivity may be useful for the identification of irra-diated tissue and cells. Measuring the impedance was found to be a reliable andpractical technique for detecting irradiation treatment of potatoes (Schertz, 1970,1991, Hayashi et al. 1982). The conductivity is measured by puncturing a potatotuber with an electrode and passing alternating current through it. The technique isquite simple, takes only 3 min to perform, and does not require a trained operator.Schertz (1970) showed that the electrical conductivity or impedance of irradiatedpotatoes at a low frequency of alternating current increased for about 6 h postirra-diation and then remained at a level higher than that of the nonirradiated potatoes.Subsequent studies determined that the ratio of impedance magnitude at 50 kHz tothat at 5 kHz, measured immediately after puncturing the potato tuber, is dependenton the radiation dose received, independent of storage temperature, and stableduring storage for at least 6 months after irradiation (Hayashi et al. 1982, Hayashi1988).

Later work by Hayashi et al. (1996b) led to the conclusion that the 5:50-kHzimpedance magnitude ratio measured at 22-250C at the apical region of the potatotuber with 1-mA alternating current, results in the best detection of irradiationtreatment. It should be noted that the 5 :50-kHz ratio appears to be dependent onpotato cultivar, and hence information is required about the cultivar if irradiated


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potatoes are to be identified. If the potato variety is known, irradiation treatmentcan be detected, since the 5:50-kHz ratio is not influenced by planting locality.

14.3.5. Other Physical MethodsA number of other physical methods have been suggested as possible irradiationdetection tests, but they have limited application at present (Delincee 1998a,McMurray et al. 1996). For example, degradation caused by ionizing radiation inthe volatile oils, lipids, carotenoids, and starch of dry-food ingredients is indicatedin the near-infrared (near-IR) wavelength region by changes in the reflectancespectrum (Barabassy et al. 1996). Small but relatively permanent changes in thenear-IR reflectance spectra of several spices, including black and white pepper,paprika, cinnamon, and allspice, can be observed after irradiation, as a result ofexcited molecules in the absorption peaks, that is, changes in amplitude and spec-tral shifts. The changes observed are dependent on the radiation dose applied andthe elapsed time following irradiation. The identification limit by the near-IRmethod for the spices examined was approximately 3-4 kGy.

14.4. CHEMICAL METHODS14 .4 .1 . HydrocarbonsMost of the volatile products formed in food by irradiation originate from the lipidfraction; hence it was proposed by Nawar (1988) that measurement of radiolyticproducts from food lipids could form the basis for chemical methods to identifyirradiated foods. Both the quantitative and qualitative patterns of the radiolyticproducts have been found to depend largely on the fatty acid composition of thelipid. According to LeTellier and Nawar (1972), the composition of the productsformed by irradiation of a lipid, or a lipid-containing food, can be predicted to acertain degree if the fatty acid composition of the lipid is known. Hydrocarbons,aldehydes, methyl and ethyl esters, and free fatty acids are the major classes ofvolatile compounds produced by irradiation (see Chapter 3, Table 3.2).

Of the hydrocarbons produced by each fatty acid on irradiation, only two areformed in relatively large quantities (Nawar and Balboni 1970). One has a carbonatom less than the parent fatty acid, and results from cleavage at the carb on -carbo nbond alpha to the carbonyl group ; the other has two carbon atoms less and one extradouble bond, and results from cleavage beta to the carbonyl (Nawar et al. 1990).Nawar and Balboni (1970) reported on the feasibility of detecting irradiated porkmeat (Fig. 14.5) by analysis of six "key hydrocarbons," namely, tetradecene(Ci4:i), pentadecane ( C i 5 : 0 ) , hexadecene (Ci 6 . O, heptadecan e (C 1 7 : 0 ) , hexadeca-diene ( C i 6 : 2 ) , and heptadecene ( C n . O, which are typically produced fromthe three major fatty acids of pork fat, that is, palmitic, stearic, and oleic. Workcarried out by Nawar et al. (1990) on irradiated chicken indicated thattetradecene, hexadecadiene, and heptadecene were considered to be the most


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FIGURE 14.5. Gas chromatographic analyses of the volatile hydrocarbons from the lipidfraction of irradiated chicken, beef and pork (Source: Nawar et al. 1990; with permission).

promising hydrocarbons, since they are found in the highest concentrations and areabsent or present at a low level in nonirradiated samples. A linear relationshipbetween irradiation dose and each of these compounds has also been demonstrated(Mo rehouse et al. 199 1, H annisdal 1993). Storage studies demonstrated thatalthough there was a decrease in the concentration of the hydrocarbons when meatsamples were stored for 16 weeks at -2O 0 C, the reduction was small comparedto the amount of these compounds formed during irradiation and would notcompromise reliable detection of irradiation treatment (Nawar et al. 1990).For detection of hydrocarbons, the fat is firstly extracted from the samples bymelting out, which is suitable for high-fat foods such as chicken and pork, orisolated using solvents such as a mixture of n-pentane and 2-propanol or usingrc-hexan e. Th e hydrocarbon fraction is obtained from the fat ex tract by absorptionchromatography (Florisil), prior to separation by gas chromatography (GC) anddetection with a flame ionization detector (FID) or mass spectrometer (MS). Sig-nificant research has demonstrated that the hydrocarbon method can be widelyapplied to irradiated fat-containing foods, including meat and meat products, froglegs, fish, shrimp, Brazilian beans, Camembert cheese, and sponge cake prepared

Pork

Beef

Chicken


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with irradiated liquid egg (Bergaentzle et al. 1994a, Morehouse et al. 1991,Morehouse and Ku 1992, Schulzki et al. 1995, Villavicencio et al. 1997). Followingthe success of a number of interlaboratory blind trials that tested for raw chicken,pork, beef, Camembert cheese, avocado, mango, and papaya (Schreiber et al.1993b, 1994, 1995, 1996, Stevenson 1994) the method was adopted by CEN asEuropean Standard en!784.

The use of on-line coupled liquid chromatography-gas chromatography (LC-GC) and LC-LC-GC for detection of hydrocarbons produced on irradia-tion in complex lipid matrices has been investigated. This is a highly efficienttechnique for sample preparation and enrichment that allows the determination oftrace components at a lower detection limit, providing a higher sep aration efficiencyand more accurate selection of certain fractions. Schulzki et al. (1995, 1997) havesuccessfully applied the technique to difficult samples such as fish oil, the fat frommango kernels, and avocado flesh, as well as to fat extracted from sponge cakecontaining irradiated liquid egg.

14.4 .2 . 2-AlkylcyclobutanonesBesides the major classes of compounds, LeTellier and Nawar (1972) isolated aseries of cyclic compounds from simple triglycerides irradiated in vacuum at6OkGy. These compounds are known to be substituted cyclobutanones having thesame number of carbon atoms as the parent fatty acid from which they are formedand an alkyl group located at ring position 2. In addition, 2-dodecylcyclobutanone(2-DCB) was isolated by Handel and Nawar (1981) from a synthetic phospholipidirradiated at 500 kGy. This research by Nawar and colleagues provided the theore-tical basis for the work of Stevenson et al. (1990) and Boyd et al. (1991), whoexamined the feasibility of using the 2-alkylcyclobutanones as markers for irra-diated, fat-containing foods. They concentrated on using 2-DCB, which is formedfrom palmitic acid on irradiation, as an indicator of irradiation treatment of chickenmeat. As cyclobutanone standards were not available commercially, 2-DCB had tobe synthesized prior to com m encem ent of the work. A solvent extraction techniqueusing hexane was subsequently developed for the extraction of the lipid fractionthat contained the cyclobutanone. This extract was fractionated by adsorptionchromatography (Florisil) and analyzed by gas chromatography-mass spectrometry(GC-MS), with the MS employed in the selective-ion monitoring mode.

The specificity of 2-DCB as a marker for irradiation treatment was confirmedbecause it was not detected in either raw or cooked, nonirradiated minced chickenm eat but its presence w as confirmed in irradiated samples (Boyd et al. 199 1). It wasalso reported that 2-DCB persisted for at least 20 days in irradiated chicken meatstored at 4 0C , and that the concentration increased linearly with radiation dosewithin the range 0.5-10.0 kGy. Further evidence of the stability of the compoundwas provided by the fact that it was detectable in chicken meat that had beenirradiated with gamma rays and electron beams 12-13 years earlier (Crone et al.1992a). Cooking chicken in a convection oven either before or after irradiation didnot eliminate the dose-response relationship that was apparent in noncooked,


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irradiated chicken, and although the amount of 2-DCB was reduced, it was stillpossible to confirm that the products had been treated with ionizing radiation(Crone et al. 1992b).While initial studies focused on detection of 2-DCB, the synthesis of an authen-

tic standard of 2-tetradecylcyclobutanone (2-TCB) (Crone et al. 1993) allowed themethod to be modified so that both these cyclobutanones could be employed asmarkers for irradiated, fat-containing food. As with 2-DCB, the amount of 2-TCBformed in chicken meat increased with increasing dose, although the concentrationpresent was less than that of 2-DCB, thereby reflecting the lower percentage ofstearic acid in chicken meat relative to palmitic acid. As well as serving as areliable postirradiation markers for chicken meat, 2-DCB and 2-TCB have beendetected in irradiated liquid whole egg (Stevenson et al. 1993), beef, lamb, (Crone1992, Stevenson 1994), ground beef (Stewart, 2000 personal communication),mango, papaya (Stewart et al. 1998, 2000) salmon meat and Camembert cheese(Stewart et al. 2000) as well as prawn meat (McMurray et al. 1995) and mechani-cally recovered meat (Crone 1992). Recent studies have demonstrated that theseradiation markers can also be identified in foods containing irradiated ingredients,for example, cakes containing irradiated liquid whole egg and beef burgers contain-ing irradiated ground beef (Fig. 14.6) (Stewart, 2000, personal communication).The 2-alkylcyclobutanone has been validated by interlaboratory trials for chickenmeat, pork, and liquid whole egg (Stevenson et al. 1994a,b), and was adopted asEuropean Standard EN1785 in 1996. Similar to the hydrocarbon method, the cy-clobutanones can potentially be used as markers for any fat-containing food that hasbeen irradiated.

Time (min.)FIGURE 14.6. Selected ion monitoring of the sum of ions m/z 98 and 112 from(a) standards of 2-dodecylcyclobutanone (2-DCB) and 2-tetradecylcyclobutanone (2-TCB);(b) beef burgers containing irradiated (1 kGy) ground beef; and (c) beef burgers containingnon-irradiated ground beef.

Internal standard


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An authentic standard of 2-tetradecenylcyclobutanone (2-TDCB), producedfrom oleic acid on irradiation, has been synthesized and its presence confirmedin irradiated chicken meat, papaya, and mango (Hamilton et al. 1995, Stewart et al.1998, 200 0). However, as this cyclobutanon e is m ore difficult to detect and quantifyin comparison to 2-DCB and 2-TCB, it is not used routinely for detection ofirradiation treatment.

Other studies (Lembke et al. 1995, Rahman et al. 1996, Twefik et al. 1998,Stewart, 2000, personal communication) has also indicated that supercritical fluidextraction (SFE) is a useful techniqu e for the isolation of the 2-alkylcyclobutanonesfrom irradiated, fat-containing foods. Stewart and co-workers (Stewart, 2000, per-sonal communication) developed a method whereby the cyclobutanones are selec-tively extracted from an irradiated sample into hexane, and the extract passedthrough a Florisil solid-phase extraction (SPE) cartridge prior to analysis by G C -M S. The latter method has been successfully used to identify irradiated chickenmeat, pork, liquid whole egg, ground beef, and mango, as well as irradiated groundbeef and liquid whole egg contained within beef burgers and cakes, respectively.Work has been conducted to develop an enzyme-linked immunosorbent assay(ELISA) for detection of the 2-alkylcyclobutanones (Elliott et al. 1995, Hamilton etal. 1996, Nolan et al. 1998). Initially, polyclonal antibodies were raised to a cyclo-butanon e derivative with a side-chain length of 10 carbon s, and incorporated into anELISA that was shown to be capable of detecting cyclobutanones in chicken meatirradiated at commercial doses (Elliott et al. 1995). Antibodies were also raisedagainst a C i 2 cyclobutanone derivative and incorporated into an ELISA thatwas used to detect irradiated liquid whole egg (Nolan et al. 1998). As the validated

2-alkylcyclobutanone method is time-consuming and requires the use of relativ-ely specialized equipment, the availability of such ELISAs would be useful forrapid and simple, on-site screening of irradiated foods. However, more research isneeded before these can be used on a routine basis.14.4 .3. O/f/7o-TyrosineAromatic compounds are prone to attack by hydroxyl radicals. Phenylalanine, anaromatic amino acid found in most food proteins, reacts with hydroxyl radicalsgenerated from radiolysis of water to form ortho-, meta-, and para- tyrosine. Initi-ally, it was presumed that ortho- and ra


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its formation from phenylalanine during sample preparation (Meier et al. 1990,Chaqui-Offermanns and McDougall, 1992).Ortho-Tyrosine may be measured using capillary gas chromatography and massselective detection (Karam and Simic 1988a) with high-performance liquid chro-

matography and UV detection (Karam and Simic 1988b, Zoller et al. 1991), or byhigh-performance liquid chromatography and fluorescence detection (Chaqui-Offermanns and McDougall 1992, Meier et al. 1996). To date, extensive collabo-rative testing has not been carried out for this method.14.4.4. Gas EvolutionThe detection of irradiated foods by determination of evolved gases such as carbonm onoxide, hydrogen, hydrogen sulfide, and am m onia has been investigated. Furutaet al. (1992) estimated the amount of carbon monoxide in irradiated frozen chickenmeat, beef, and pork by expelling the trapped gas using microwave heating anddetecting released carbon monoxide in the headspace using GC. Investigations byRoberts et al. (1996) using frozen chicken meat and shrimp meat also yieldedpromising results for detection of carbon monoxide evolution. In an attempt tospeed up the time of analysis, Delincee (1993) used an electrochemical sensor toestimate the carbon monoxide content in the gas released from irradiated food.

The value of hydrogen as a marker for irradiated food is nullified by its rapiddiffusion, which leads to considerable loss unless its escape is impeded. Dohmaruet al. (1989) analyzed green pepper by gas-liquid chromatography and observedthe partial retention of hydrogen in the solid. A new hydrogen-specific electronicsensor for screening irradiated food was demonstrated by Hitchcock (1993), whocarried out experiments based on aqueous solutions of hydrogen and on irradiatedwater. Hitchcock (1995, 1996, 1998) later showed that the specific sensor could beapplied to irradiated frozen chicken mince and chicken legs for up to 76 days and141 day s postirradiation, respectively, at doses as low as 0.5 kGy. Results for frozenprawns (Hitchcock 1998) were not as promising as those from chicken meat be-cause the signals fell more rapidly after storage, and lower doses (0.5 kGy) couldnot be detected after 25 days at - 1 8 0 C . Hitchcock (1998) also adapted the methodfor the detection of irradiation treatment in eggshells given 0.1-0.8 kGy, in whichcase the signals were stable for at least 100 days. It was, therefore, concluded thatdetermination of hydrogen in thawed samples of frozen foods offers a reliable,rapid, and robust screening method for irradiation treatment. As this method isbased on an electronic sensor incorporated into a simple headspace analysis, it isparticularly useful as an inexpensive, on-site screening procedure. False positivesare not observed, but it should be noted that failure to detect hydrogen provides noproof of a nonirradiated sample. The technique is limited to frozen foods such aschicken that can be thawed inside the analyzer.

The use of multiple gas sensors has been proposed by Delincee (1996a) toincrease the reliability of the gas evolution method in detecting irradiated foodssuch as frozen chicken, mechanically deboned poultry meat, and shrimps.By applying several gas sensors, a "gas fingerprint" for different foods can be


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established, but more work is needed to establish background values for variousfoods and to determine the influence of validation parameters. As for other screen-ing methods, samples suspected of having been irradiated should be analyzed againusing validated methods such as EN1784 to EN1788.

14.4.5. Other Chemical MethodsA num ber of other methods based on chem ical changes in food due to the irradiationprocess have been evaluated (Delincee 1998a). The methods that show the mostprom ise are the lipid hydroperoxide test and an immunochemical method for detec-tion of irradiated egg. Radiolysis of foods containing fat in the presence of oxygenleads to the production of organic peroxides that can be simply determined by, forexample, iodometric titration (Qi et al. 1993). The method has been used for theidentification of foods such as fresh pork, minced meat, braised chicken, and liquors(Qi et al. 1993, Qi and Wu 1996), and a blind trial has been successfully condu cted(Qi and Wu 1996) using pork. This method is poten tially of benefit to labora tories thatcannot afford the sophisticated equipment required by the standardized methods.However, there are a num ber of disadvantages in using organic peroxides as radiationmarkers. They are produced by autooxidation and are unstable, and the presence ofanti-oxidants and oxygen levels in the food, as well as the composition of the food,influence yields (Roberts 1996). Therefore, as recommended for other screeningtests, combining this method with measuring, for example, the ESR signal fromirradiated bone, would increase the possibility of detecting irradiated foods.

Irradiated eggs can be identified immunochemically by the specific detection ofradiation-induced degraded fragments of egg white proteins. Protein and peptideantigens can generally be detected by im m unoblotting using specific antibodies, evenin the presence of other coexisting proteins and peptides (Kume et al. 1994, Kumeand Matsuda 1996). It has been suggested that this immunochemical method wouldbe applicable not only to detection of irradiated egg but also of irradiated egg w hiteproteins added to foods. The m ethod has been app lied to chicken m eat and shrimp butwith limited success; thus further research is required (Kume and Matsuda 1996).

14.5. DNA METHODS14.5.1 . DNA "Comet Assay"It is now widely accepted that DNA is the major cellular target for ionizing radia-tion. It is the radiation-induced DN A dam age that is responsible for inactivation ofmicroorganisms, destruction of insects, inhibition of sprouting in bulbs and tubers,and for the delay of ripening in some fruits. Therefore, it was logical to investigatewhether radiation dam age to DNA in food could be utilized as a m eans of detectingthe irradiation treatment. Ionizing radiation has been shown to induce three major

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