Electron paramagnetic resonance detection of irradiated foodstuffs

Appl. Magn. Reson. 10, 357-373 (1996) Applied Magnetic Resonance ~ Springer-Verlag 1996 Printed in Austfia

Electron P a r a m a g n e t i c R e s o n a n c e Detec t ion o f

Irradiated Foodstuf fs

J. Raffi ~ and P. Stocker 2

I Laboratoire de Recherche sur la Qualit› des Aliments (LARQUA), Facult› de Saint-J› Marseille, France

2 Laboratoire de Chimie des Produits Naturels (LCPN), Facult› de Saint-J› Marseille, France

Received June 19, 1995

Abstract: This item deals with identification of irradiated foodstuffs by means of Electron Para- magnetic Resonance (EPR). EPR is the most accurate method for such routine applications since radicals are stabilized f o r a long time in all (part of) foods which are in solid and dry state; consequently, EPR can be applied to meat and fish bones, fruit and relative products (from vegetal origin), seafoods, etc.

1. General Introduction on Food Irradiation

1.1. Food Irradiation and Detection of Irradiated Foodstuffs

Food irradiation by X- and gamma-rays and by electron beam has been introduced recently, as a new technological process, in order to reduce losses and to improve hygienic quality [1-3]. Many investigations were devoted to possible health impacts. And, especially, in 1980, a Joint Food and Agriculture Organ- ization (FAO) / International Atomic Energy Agency (IAEA) / World Health Organization (WHO) Expert Committee meeting [4] concluded that "the irradiation of any food commodity up to an overall average dose of 10 kGy presents no toxicological hazard," hence, toxicological testing of foods so treated is no longer required". The Commission of the European Communities [5], as have many Health Authorities, concluded that these aspects were investigated to a degree that no other method of food processing has been examined so far. A s a consequence, more than 40 countries have legally accepted radiation treatment of different foods, although ir is still prohibited in others; moreover, a lot of countries have no specific regulations. To facilitate trade in irradiated foods, regulatory authorities in all countries appear to be interested in having simple

358 J. Raffi and P. Stocker:

and reliable methods to detect foods treated by irradiation [6, 7] and, consequently, to check on compliance with labelling regulations.

Effects caused by radiation treatment in foodstuffs are very small and quite similar to those produced by classic food treatment processes (heating, freezing) or natural evolution of the foodstuffs (autooxidation). To lay down a test for identification of irradiated foodstuffs, we have to check the presence of the following facts:

- a phenomena "characteristic" of the ionizing treatment, at least under certain conditions (to be well known);

- a phenomena measurable and relatively stable for at least the commercial shelf life of the foodstuff.

Severai detection methods, including Electron Paramagnetic Resonance (EPR), have been discussed previously [2, 6-25]. Considerable progress has been made recently, particularly due to the actions of the Reference Bureau of the Com- mission of the European Communities (CEC, Brussels) [12, 15, 26] and of the Joint Division of FAOI and IAEA in Vienna [27]. The BCR meeting held in Cadarache (France, 13-15 February 1990) decided to examine the potential methods of detection of irradiated food by setting up a concerted research action covering 4 areas, i.e., DNA methods, microbiological and biological methods, physical methods and chemical methods [15]. Afler another meeting in Ancona [12] and a three years concerted action, the main recommendations of the BCR [26] were:

a) to carry out further research on the different protocols, particularly in order to increase the speed of the determinations, to extrapolate the field of applications, and to try to fmd and develop new methodologies such as immuno- chemistry;

b) that the CEC follows the future of the protocols, asking that the present group of coordinators will be consulted by the European Committee of Nor- malization (CEN) during the discussion;

c) that the CEC could encourage training, via this group, of speeialists for EC countries not yet involved in detection work (Greece, Spain and Portugal) or for those from eastern European countries; exchanges of researchers (post-doctorate, etc.) should be encouraged;

d) that this working group informs Health and Food Control National Authori- ties in each country, and promotes the creation of national laboratory net- works.

Following the final report [26] of this Concerted Action, nine protocols were submitted to the European Committee of Normalization (CEN), in April 1993:

1) Electron Spin Resonance of irradiated meat bones. 2) Electron Spin Resonance of irradiated fish bones, teeth and scales. 3) and 4) Electron Spin Resonance of irradiated fruits (the two protoeols ap-

proximately correspond to fresh and dried fruits).

EPR Detection of Irradiated Foodstuffs 359

5) Thermoluminescence of irradiated herbs and spices. 6) Lipid method using volatile hydrocarbons, for irradiated poultry meat. 7) Lipid method using cyclobutanones, for irradiated poultry meat. 8) Direct Epifluorescence Filter Technique / Aerobic Plate Count for irradiated

herbs and spices. 9) Direct Epifluorescence Filter Technique / Aerobic Plate Count for irradiated

poultry mear.

In June 1994, the CEN created a working group (WGT8) who finally wrote and accepted five protocols: two by EPR (1 and 2, 4), two by chromatography (6, 7) and one by thermoluminescence (5). In June 1994, these five protocots (English text) have been voted and then transformed into "pre-norms" by the CEN. Further, in summer 1994, there were translated into French and German and sent in December 1994 to the different governments: Austria, Belgium, Den- mark, Eire, Germany, Finland, France, Greece, Island, Italy, Luxembourg, Neth- erlands, Norway, Portugal, Spain, Sweden, Switzerland and United Kingdom. Finally, these protocols will be approved soon as official ones.

In order to promote these protocols, the CEC decided to begin a new concerted action eoordinated by one of us (JR); in the fietd of the "CEC" COPERNICUS programme, this action will be devoted to the establishment of an Eastern Euro- pean network of laboratories for identification of irradiated foodstuffs (N~ - CT94-0134); the nine involved laboratories ate from Belgium, Bulgaria, France (2), Hungary (2), Poland (2) and Portugat.

1.2. Principtes of Food Irradiation

Finally, let us remember that, during the irradiation process, food is exposed to the action of gamma-rays, X-rays or accelerated electrons which ate capable to knock electrons out of their normal orbits in food atoms or molecutes. This effect which leads to changes in living materials is quantitatively small but very important from a qualitative point of view. The radiation dose, i.e., the quantity of energy absorbed by the food, is the most important factor which leads to the different effects, positive or negative, of the treatment: an irradiation dose of 1 gray corresponds to the absorption of an energy of 1 Joule per kilogram of foodstuff.

The dose used in food processing usually ranges from about 0.1 to 10 kGy and depends on the kind of food being processed and on the desired effect. If we are trying to identify irradiated foodstuffs, the minimal dose detectable by the studied protocol must be lower than the minimal dose required by the purpose of the irradiation, i.e., we will speak later of irradiations carried out for commercial purposes.

We shall only present in this paper the state of the identification of irradiated foods by EPR technique.

360 J. Raffi and E Stocker:

2. Application of the EPR Teehnique to Irradiated Foodstuffs

2.1. Basis Principles

EPR is a spectroscopic method permitting the detection of unpaired electrons, which can appear in different situations:

- defeets in semiconductors; - paramagnetic ions derived from transition or main group elements: these are

commonly observed in foods as many enzyme functions are mediated by transition metals;

- free radicals, including those induced by radiation.

The idea of studying foodstuffs by EPR is not used and not uniquely devoted to irradiated products. EPR was used for instance to study the free radicals induced throughout soda cooking, as well on models [28] as on milled wood and lignin (Pinus radiata) [29]. By measuring the concentration of free radicals induced in green and roasted coffee and by observing the change during storage, information has been obtained which concerns the influence of the various technological processes [30]. Some authors also thought that EPR study of free radicals may lead to accurate explanations concerning the differences in possible health hazards that are associated with coffee and tea [31 ]. However a lot of toxicological studies brought the proof of the wholesomeness of irmdiated foodstuffs [4], demonstrating well that the possible toxicity o f radicals is not directly linked with the presence of these radicals but with their reactivity and eventual formation of toxic products.

2.2. EPR Identification of Irradiated Foodstuffs

Ionizing radiation induces in food radicals which are generally very short lived and, therefore, cannot be used to identify irradiated foodstuffs. EPR can be used a s a detection test [32-40] only if the radicals are stable over the maxi- mum commercial storage life of food and if the corresponding signals are clearly distingu… from those of the unirradiated material, which is never available for commercial samples. This may only happen in the solid and dry components of food, where the rigid structure of the matrix inhibits radicals reacting with each other of with food components present in the wet portion of the food.

The shape of the EPR signal must not depend on the foodstuff variety nor on the particular chemical composition of the sample. Once more we have to note that, in commercial conditions, we can never reach the true reference sample. Ir an estimation of the initial dose is also required, then the signal must be independent of storage time; in fact, we shall see that no means of quantifying the delivered dose on the food itself is available up to now [26, 41-46].

The principal food categories which may be studied by EPR in order to identify their irradiation treatment, are:

EPR Detect[on o f Irradiated Foodstuffs 361

- meat and fish species; - fi'uit and relative vegetal products; - shells and crustacea. Each one is separately detailed hereafter.

3. S t u d y o f I r r a d i a t e d M e a t a n d F i s h S p e c i e s

3.1. Meat

Studying the EPR signals induced in meat bones is not new as yet done for post-irradiation dosimetry [47] and for dating of bones and shells [48]. Details of the best recording conditions have been widely reported [41~15, 49-67] and checked by intercomparisons [49, 67-69]; the same conditions are now used by every expert laboratory, leading to the publication of a protocol by the Euro- pean Community [26].

There is quite no signal in the unirradiated samples (Fig. la), nevertheless a symmetric absorption (Fig. lb), with a g-factor of - 2.0044 and a peak-to-peak linewidth of A H - 0.6 t o t (6 G), due to the bone marrow can be observed.

The irradiated sample presents ah asymmetric absorption (Fig. lc) characterized by

g• - 2.0030-2.0033 and gll - 1.9969-t.9975 ,

which is due to the hydroxyapatite [Cat0(PQ)6(OH)2], main constituent of the bone [61]. I f marrow is not removed from the bone, the EPR spectrum is a mixture of these two last EPR signals (Fig. Id).

Generally, a small bone fragment is sufficient to record a good spectrttrn; as indicated above, ir is preferable to remove the marrow from the bone and to dry ir on a filter paper. Some authors prefer to grind the bone but taking care beforehand to remove the marrow cornpletely. Instead of using the bones, teeth

a b

c ~ d Fig. 1. Typical ESR sper of frog bones: a referear sample without marrow; b reference sample with marrow; e sample irradiated at 1.5 kGy and without marrow; d sample irradiated at 1.5 kGy

and with marrow.


Table 1. EPR Identification of irradiated mear or fish bones (including the parameter values finally fixed by the CEN experts).

Sampte preparation

Magnetic field

Microwave power

Signal channel

Temperature

Notes

Remove all traces of meat from a sect[on of bone. Split the bone and remove the marrow when poss[ble. Then slightly dry the bone, for instance in a usual drying apparatus, ir possible under reduced pressure. Take a small piece of borre (approx[mately 100 mg, 3.0-3.5 mm thick, 5.0-10.0 mm long) for the measurement.

342 mT centre field at 9.5 GHz mierowave frequency (g 2.00); 5 to 20 mT sweep width.

5 to 12.5 mW.

100 kHz modulation frequeney; 0.2 to 0.4 mT modulation amplitude. Sweep tate: 2.5 to 10 mT/min. Time constant: 20-50 ms.

Room temperature.

Ir there is an asymmetric signal with g of ~ 2.0031 and 1.9972, the bone has been irradiated.

can also be used. The complete protocol is publ ished elsewhere [26]: only the main details are put in Table 1.

EPR spectroscopy has been successful ly appl ied to the fol lowing bone containing meats: chicken, frog, lamb, turkey, pork, beef, goose, duck and rabbit. Regarding all publ i shed results the EPR detect ion method can very probably be used for all kinds o f mear bones. Ir should be pointed out that spectra which were treated with low doses up to approx imate ly 2-3 kGy often show a combination of unspecif ic and specific sigrml like in Fig. id i f the marrow is not removed.

Detect ion o f i r radiated samples is poss ible to a minimal dose o f 500 Gy, but in many cases (depending on the calc i f icat ion status o f the bone, which is nor- mally lower for small animals than for Iarger species), an ident i f icat ion is possible at a lower dose. The results o f this detect ion method are not s ignif icant ly influenced by heat ing the sample (e.g., boi l ing in water), i f no traces o f marrow remain on the sample.

Detection o f i r radiat ion treatment is not s ignif icant ly influenced by storage t imes up to 12 months. EPR can also be appl ied for the identif icat ion o f i rradiat ion o f a secondary product such as mechanica l ly recovered meat (MRM); it is possibte to detect the characterist ic rad ia t ion- induced EPR signal in bone fragments extracted from irradiated M R M [54].

As for post- i r radiat ion dosimetry [47], it is possibte to use the fact that the signal intensity is proport ional to the dose, to obtain an es t imate o f it in the case o f meat bones. But the chemical composi t ion and the crystal l ini ty o f the

EPR Detect[on of Irradiated Foodstuffs 363

bone influence the number of radicals present [56-58] and, consequently, the signal intensity. "Dose response curves" were proposed [41, 43], apptying inere- mental doses of irradiation to bones. The procedure requires access to an irra- diator and is not so promising as initially thought [47]; despite dosimetric corrections between the 14 laborato¡ involved in the European Intercomparison, there is an overlap between the different results [44, 45]. The induced signal, with two different symmetries (axial and orthorhombic), is more complieated that initially thought [63]. Furthermore the different chemical and physical states, may complicate the results, leading to different limits of dose linearity: up to 14 kGy for frog legs [62], only - 9 kGy for poultry bones. Thus the BCR decided that we cannot use EPR for quantitative measurements on meat bones. This is not so surprising as quantitative EPR spectrometry requires special conditions and, in particular, a good knowledge of the origin and structure of the species giving the spectrum [46].

However, for a unique laboratory, there should be generally no difficulty in determining whether medium (1-3 kGy) or high doses (7-10 kGy) of irradiation have been applied. We should notice that ir is at present the unique case where it is possible and interesting to obtain an estimate of the initial applied dose whatever would be the irradiation temperature (in commercial conditions).

3.2. Fish

A s a reminder, there are two main fish categories: Osteichthyens and Chondrich- thyens. If we have quite no information on the second category (i.e., more fundamental research must be carried out), EPR signals of irradiated or unirradiated fish bones, i.e., Osteichthyens, are the same as those of mear bones: fish bones, scales and jaw may be used also as made out of hydroxyapatite. However, measurements on fish bones and scales require much more experience than those conducted on meat bones as the rigidity and crystallinity of the fish bones are less important that the ones of meat bone; moreover, it is quite impossible to remove the marrow.

The types of EPR spectra for unirradiated and irradiated samples are shown in Fig. 2. Ir should be pointed out that spectra of samples which were treated with low doses up to approximately 2-3 kGy often show a combination of unspecific (Fig. 2a) and specific signal (Fig. 2b).

The EPR protocol (Table 1) has been successfully applied to the foUowing bone containing fish [44, 45, 62, 68-73]: trout, sardine, salmon, whiting, halibut, cod and maekerel. The EPR detection method can very probably be used for other bone-containing species but requires kinetical studies to ensure that the lifetime of the EPR signal is longer than the commercial lifetime of the product.

Detection of irradiated teeth is possible to a minimal dose of 500 Gy; for bones the minimal dose is 1 kGy. Detection of irradiation treatments is not signifi-


Fig. 2. ESR spectra of trout bones. Unirradiated sample (a) and sample irradiated at 2 kGy (b). Field: 350_+2.5 mT (3500+25 G).

cantly influenced by storage times of up to 5 months [71], in case of fish stored at 4-5~ i.e., the maximal commercial storage temperature o f those fish.

4. S tudy o f Irradiated Fruit and Relat ive Vegetal Products

This paragraph covers fresh and dried fruits, nuts and probably certain spices and vegetables, i.e., vegetal foodstuffs. However the method was first applied to fruits and gives consequently a special focus upon them.

4.1. EPR of Fruits

As always, radicals are not stable in an aqueous medium and, consequently, EPR signals can onty be observed in solid parts of fiaait (seeds, pips, stones, peels) of in dried fruits. As the chemical composition of this foodstuff is more complicated than in bone (one main constituant), the signals induced by irradiation are more complex and more dependent from the fruit species and from the observed part of the foodstuff; moreover, there is generally a strong signal in the unirradiated sample, due to the lines of Mn 2+, a transition metal ion linked to enzymes present in the fruit. This is why two types of protocols were checked by intercomparison [44] and finally given to the BCR [26]. We shall first focus on fruits and only discuss later the case of other relative vegetal producta.

In unirradiated samples, two different kinds of EPR signals may be observed [32, 53, 74-82]:

- a six line signal (Fig. 3, lines A) due to Mn 2+, which is not influenced by irradiation [32, 80]; each line is separated from the closer one by - 8.5 toT (85 G);

- a central single Iine (Fig. 3, line B and Fig. 4a), which increases with the irradiation dose, but varies to a large extent with the water content; it may be related to a quinone radical [79].


C

I I I

B

I I I A

Fig. 3. ESR spectra of strawberries irradiated at 2 kGy. A and B lines are respectively due to Mn 2+ and quinone radicals both present in irradiated and unirradiated achenes. Only C signal is typical

from irradiated samples.

The EPR spectrum of irradiated samples may present the two signals of unirradiated samples and two other kinds of signal depending on the composition of the studied food part:

- if low weight sugar (i.e., glucose, fructose, saccharose, etc.) are present, the induced signal is very complex and large (Fig. 4b), but very predominant and then allowing easy identifications;

- in other cases, the "characteristic" signal is a triplet whose only satellite lines can be found (in Fig. 3, only the left line C can be observed); it is due to a cellulose radical, but these lines are generally very weak with regard to the other ones, and then lead to a more difficult identification.

According to these results two protocols were first proposed for fruit identification, and then submitted to European authorities [26]. But, after discussions

-%_

Fig. 4. ESR spectra of dried grapes. Unirradiated (a) and 2 kGy irradiated (b) samples. Field: 349+5 mT.


Table 2. EPR identification of irradiated food containing cellulose (ineluding the parameter values finally fixed by the CEN experts).

Sample preparation

Magnetic field

Microwave power

Signal channel

Temperature

Notes

Take about 100 mg of seeds, pips or cristalline parts of food; if necessary, wipe them on filter paper and place in a standard EPR tube.

348 mT centre field at 9.5 GHz mierowave frequency (g ~ 2.00); 20 mT sweep width.

0.4 mW.

100kHz modulation frequeney, 0.4 to 1 toT modulation amplitude. Sweep rate: 5 to 10mT/min. Time constant: 100-200 ms. Increase the gain to saturate the central Iine and thus amplify the minor peaks.

Room temperature.

If there is a peak or a shoulder at ~ 3 mT (30 G) on the left o f the central saturated line, the foodstuff has been irradiated (Fig. 3, line B). Note that, generally, a weaker line may be observed on the right, degending of the presence or not o f other lines such as those of Mn :+ (Fig. 3, fines A). I f the left line is not apparem, vai3~' the modulation amplitude in ordr to ensure its detection.

between the experts o f the CEN working group WG8, only the protocol relative to "cellulose" products was proposed to be transformed into a CEN norm, only the main details of which ate put in Table 2.

EPR spectroscopy has been successfully applied to the following fruits: berries stored at 4-5~ [80], frozen berries [79, 80], French prunes [25], shells of coconuts, hazelnuts, pistachio nuts, peanuts, walnuts and almonds.

Detection of irradiated samples is possible to a minimal dose of 500 Gy. De- tection of irradiation treatments is not significantly influenced by storage times up to 10 months for frozen berries and fruit nuts and stones, and only 25 days for berries stored at 4-5~

4.2. Other Fruits

In case of other fruits, in particular dried ones, another protocol (Table 3) was initially proposed, but finally not accepted by the working group as the "multicomponent signal" is not very well defined and show very large variations from one fruit to another; more fundamental research is required before such a tentative protocol will be accepted.

However, we can note that EPR spectroscopy has been tentatively applied to the following fruits: dried grapes, banana and papaya, date stones, coconuts,

EPR Detection of Irradiated Foodstuffs

Table 3. Tentative protocol for EPR identification of dried fruits.

367

Sample preparation

Magnetic field

Microwave power

Signal channel

Temperature

Notes

Take about 100 mg of seeds, pips or fragments of stones or of dried fruits; if necessary, wipe them on filter paper and place in a standard EPR tube.

345-350 mT centre field at 9.5 GHz microwave frequency (g ~ 2.00); 10 mT sweep width.

2 mW.

100 kHz modulation frequency, 0.2 mT modulation amplitude. Sweep rate: 2 mT/min. Time constant: 100-200 ms.

Room temperature.

Samples have been irradiated, when there is a multicomponent signal such as Fig. 4b. The absence of a signal indi- cates an unirradiated sample. If there is a single line present in the spectra, such as line B in Fig. 3, they might be irradiated ir the fruits are of "eellulose type". In order to avoid any mistake, please use the proper method.

etc. Detection o f irradiated samples is possible to a minimal dose o f 500 Gy. Detection o f irradiation treatments is not significantly influenced by storage times up to 10 months.

4.3. EPR Identification o f Relative Irradiated Vegetal Products

For a tong time, research on identification o f other fruits and vegetal products such as spices did not lead to favourable conclusions [53, 76-78, 82-87]; the main radio-induced EPR signal decreases too fastly with storage time and dis- appears before the maximal usual commercial storage time. In fact, the truly studied peak was the B signal (Fig. 3) and not the "characteristic'" signal C (Fig. 3) observed for these fruits. Consequently, a lot o f new studies have to be carried out but, examining the yet published literature, ir is evident that this "cellulose" signal is present for a lot o f products [53, 76-78, 85, 86]; the unique problem, but not the smallest, being to check if the lifetime o f the characteristic EPR signal is longer than the usual commercial storage time o f the product [87] and that other treatments do not change the signal shape [88]. We have to note for instance that the last IAEA ADMIT intercomparison orga- nized on "cellulose" products by M. Desrosiers (NIST, Gaithersburg, USA) was also carried out in beginning 1993 on black pepper and paprika; but a lot o f other products (fruits, spices, dried vegetables, etc.; see Table 4) must be studied and may be identified as irradiated by EPR spectroscopy.

For instance, in case o f aromatic herbs [21], the lifetime o f the EPR signal is not generally long enough to allow the identification o f such irradiated plants


Table 4. Some products where kinetical informations are known.

Produet Type of speetrum Storage Reference Referenee 3 Irradiated 4 time 5

Apricot 1 B M, C < 100 d. [68] Banana j B M > 2 y. [68] Btlberry 2 B C > 19 d. [79] Ctnnamon ~ B C > 1 y. [86] ChiLIP - - B? > 3 m. [87] Chilli pepper I B C > 1 y. [86] Coconuts ] B M, C > 2 y. [68] Dates 1 B M > 2 y. [68] Dried grapes j B M > 2 y. [68] Figs 1 B C < 50 d. [68] French prunes ~ B C > 2 y. [68] Grapes 2 B M, C > 30 d. [78] L[me 2 B C > 30 d. [781 Mushroom j - - M > 1 m. [87] On[on j B C 60-150 d. [68] Paprika t - - of B C > 3 m. [87] Pistachio t B C > 2 y. [68] Raspberry 2 B C > 20 d. [79] Red currant 2 B C > 18 d. [79] Red pepper ~ B C > 1 y. [86] Strawberry 2 B C > 23 d. [79] White mustard t - - B? > 3 y. [87]

t Study earried out on dried product. 2 Product stored in fresh state: the ESR study was carr/ed out on the dry part of this product. 3 B: central [hae (quinone radical 7), see [20, 60--62, 73, 103]; M: multicomponent s/gnat (due to

numerous radieals from irradiated fructose, glueose, saceharose, etc.), see [20, 60~52, L03, 1051; C: Iriplet whose central line [s hindered by "B" (due to cellulose), see [23, 26, 44, 45, 68, 79, 80].

+ In addition to "referente" signal. 5 y. = year, m. = month, d. = day.

d u r i n g t h e i r c o m m e r c i a l s t o r a g e ; i n t h i s c a s e , E P R c a n b e u s e d as a p r e s u m p -

t i o n b u t m e m u s t u s e t h e r m o l u m i n e s c e n c e t o g e t a n i r r a d i a t i o n p r o o f .

5. Study of Food Containing Shells

C o m p l e x s i g n a l w e r e o b s e r v e d i n i r r a d i a t e d s h e l l s , r e f l e c t i n g a n o v e r l a p o f

s p e c t r a c a u s e d b y v a r i o u s r a d i c a l s [35 , 4 8 , 71 , 72 , 89 ] ; t h e y m u s t b e r e l a t e d to

t h e c h e m i c a l s t r u c t u r e o f t h e she I l , i .e . , c h i t i n a n d M n 2- s i g n a l s .

T h e c a s e o f s e a f o o d s u c h as m u s s e l s , c l a r o s o r s e a ] ] o p s is r e l a t i v e l y s i m p l e

(F ig . 5 ) ; t h e E P R s i g n a l i n t e n s i t y i s v e r y h i g h , t h e u n i r r a d i a t e d s a m p l e s i g n a l

b e i n g v e r y l o w o r a b s e n t [35 , 71] , a l l o w i n g d e t e c t i o n l i m i t s a r o u n d 5 G y [72 ,

9 0 ] ; t h e r e is q u i t e n o d e c r e a s e o f t h e E P R s i g n a l i n t e n s i t y w i t h t i m e , E P R

b e i n g u s e d fo r d a t i n g o f f o s s i l s h e l l s [47 , 48 ] .


(Gauss)

i I I I I 3440 3460 3480 3500 3520

Eg3

g4 . . . , - - ~ ~ . ~ . ~

I I I I 3440 3460 3480 3500

(Gauss)

I 3520

Fig. 5. ESR speetra of referente (a) and irradiated (b) at 5 kGy oyster shells. Field: 348+5 toT.

j~ q i-

p

Fig. 6. ESR spectra of nippers from reference (dotted line) and ionized at 5 kGy (solid tine) Norway lobster. Field: 350--50 mT. The two speetra are shifted by 0.4 mT for an easier reading.

370 J. Raffi attd P. Stocker:

On the opposite, the signals detected in crustacea cuticles, such as Norway lobster (also called Dubtin Bay prawn or scampi), is yet complex in unirradiated samples, due to a large Mn 2-- signal: consequently, the differences between irradiated and unirradiated samples are very weak (Fig. 6); moreover the signal intensity, which increases with irradiation dose, decreases more or less slowly during storage, teading to difficulties in identification of irradiated samples [91-93]. Some proposals have been made [71, 91, 92] but more research is required before writing a protocol.

6. Other Potential Applications of EPR and General Conclusions

Some other studies have been carried out on other dried products such as milk protein concentrate powders [94]; using our knowledge on the nature of radio- induced radicals in starches [95] we have tried to apply EPR to the detection of irradiated cereals at usual water contents (10-13%); but among the two main induced radicals, the more stable is the less characteristic one, leading to limits of detection time varying from several months to one year, according to the cereal varieª [96].

Some authors also tried to use other EPR techniques such as spin trapping which may be used on foods containing low molecular weight molecules. Sug- ars such as glucose irradiated in powder state can be easily studied by spin trapping [97]; if this method is too complicated for routine control, it can be used in some cases to distinguish the different induced radicals.

As shown above, the EPR technique is a promising physical method, i.e., non destructive one, which can be introduced presently for routine control. Even if limited to dried products or dry parts of foodstuffs, the method is rapid with trained personnel and can detect very low doses: the sample preparation takes from 2 to 15 minutes and recording from 3 to 20. The usual basic research equipment is costty (about 1 million French francs or more) but new routine apparatus are now sold, easier to use and less expensive (about the third).

Concerning the protocols described above, a systematic work has to be carried out in order to extrapolate the present results to the other foodsª belonging to the same class. New categories have yet to be explored and it could be noticed that the same technique can be applied to detection of other irradiated products such as cosmetics and pharmaceuticals [98].

The international organizations such as BCR and IAEA have allowed, particularly during the four last years, a very large devetopment of identification methods of irradiated foods [26, 27]: as already said, two EPR protocols have been recently presented to the European Committee of Normalization in order to become official in a short delay, i.e., probably 1995 or 1994. They will be surety transformed further in worldly recognized protocots (ISO, etc.).


Acknowledgements

We h a v e to t h a n k the B C R for s u p p l y i n g the c o n c e r t e d a c t i o n and a l l o w i n g the

use o f t ex t s a n d f igures f rom B C R repor t s , and B r u k e r for l e n d i n g a n E M S 1 0 4

spec t rome te r .

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Author's address: Prof. Dr. Jar Raffi, Laboratoire de Recherche sur la Qualit› des Aliments (LARQUA), Facult› des Sciences de Saint-J› Avenue Escadrille Normandie-Ni› 13397 Marseille cedex 20, Franee

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Electron paramagnetic resonance detection of irradiated foodstuffs