lnternuiionul Journul of Food Science and Technology (1995) 29, 679-686

Evaluation of electron spin resonance technique for the detection of irradiated mango (Mangifera indica L.) fruits

B R I J B H U S H A N , ' " R A M A K A N T M . K A D A M , + P A U L T H O M A S & B A M B . S I N G H $ Food Technology and Enzyme Engineering Division, ' Radiochemistry Division and

Bombay, India. Radiobiology and Biochemistry Division, Bhabha Atomic Research Centre,

Summary

The electron spin resonance (ESR) technique was examined as a method for the detection of irradiated mango fruits. A symmetric ESR signal at g = 1.988 was detected in the hard seed cover (endocarp), the dry epidermal layer (testa) surrounding the kernel, and the soft kernel portions of the seed from four mango cultivars. The amplitude of the signal in the epidermal layer and seed cover showed a dose-dependent increase over control values. Qualitatively, however, no new signal was observed following irradiation, except that line width increased by 50%. Methyl cellosolve washing greatly reduced the intensity of the endogenous and radiation (1 .0 kGy)- induced ESR signals in the seed cover; results suggest phenolic substances as the source of free radicals. The similarity of naturally occurring ESR signals to that induced by irradiation seems to restrict the practical utility of this method in irradiated mangoes.

Keywords

endocarp, gamma irradiated food, kernel, phenolics, testa.

Introduction

In recent years irradiation has been increasingly accepted as a process to reduce food losses and improve the hygienic quality of foods. Although this method became legal in many countries fiollowing the conclusion of Joint FAO/IAEA/WHO Expert Committee on the safety and wholesomeness of irradiated foods (Anon., 1981), the technology remains unaccepted in others (Anon., 1991) mainly due to lack of suitable detection methods for irradiated foods. A relatively fast, simple and sensitive technique to distinguish lbetween irradiated and non-irradiated foods is required for law-enforcement authorities in control of the processing and trading of irradiated foods.

Several reviews have appeared on analytical detection methods for irradiated

Authors' addresses Food Technology & EnLyme Engineering Division. t Radiochemistry Division and & Biochemistry Division, Bhabha Atoinic Research Centre, Bombay 400085. India

679

680 B. Bhushan et al.

foods (DelincCe & Ehlermann, 1989; IAEA, 1991; Raffi & Belliardo, 1991; Leonardi et al., 1993). Results of a co-trial organized by the Community Bureau of Reference (BCR) on the use of electron spin resonance (ESR) spectroscopy for the identification of irradiated foods in 21 laboratories have also been published (Raffi et al., 1992).

Low doses of gamma radiation prolong the shelf-life of mangoes by delaying the ripening (Dharkar et af., 1966), and when combined with warm water-dip treatment, fungal diseases are also controlled (Padwal-Desai et al., 1973). In addition, irradiation offers an effective quarantine treatment against fruit flies and seed weevil infestation in mangoes (Thomas, 1986).

We have examined the use of ESR spectroscopy for detection of irradiated mangoes.

Materials and methods

Irradiation and sample preparation Mature, green, hard, unripe fruits of mango cultivars ‘Alphonso’, ‘Chausa’,

‘Langra’ and ‘Totapuri’ were procured from the local market. Whole fruits (0.2-0.38 kg) were irradiated at room temperature (27-30°C) with doses ranging from 0.1 to 1.0kGy in a Cobalt-60 Gamma Cell-220 (AECL, Ottawa, Canada) at a dose-rate of 0.047 kGy min-I.

As the water content of the fruit pulp (mesocarp) and skin (epicarp) is high (>70%), the gamma irradiation-induced radicals are not stable. We have examined the lower moisture content of the seeds for stable radicals produced by irradiation. Fruits were cut and each seed, free from pulp, was blotted on filter paper and separated into three portions (Fig. 1) i.e. the outer dry stony seed cover (endocarp), the interior soft portion (kernel) and the thin dry epidermal layer around the kernel (testa). These seed portions were cut separately into small pieces, frozen in liquid nitrogen and freeze-dried. The dried seed covers were passed through a pressure roller, and all samples were ground in a mortar and pestle and sieved through a single layer of muslin cloth to obtain a uniform sized powder. All samples were analysed in duplicate.

STONY SEED COVER

DRY THIN EPIDERMAL

Figure 1. Anatomy of mango fruit.

Rccnding vf ESH spectm An X-band Model V-4500 spectrometer (Varian Associates, Palo Alto, CA.

USA) was used for recording the ESR signal in seed-cover samples from mangoes irradiated and stored for 1 month at 20°C. All other measurements were made with an advanced ESP 300 spectrometer (Brukcr Analytische Messtechnik, Karlsruhe, Germany). ESR spectra were recorded between 3400-3#U gauss at 9.74ti Hz and M m W , using 70-LHmg samples of powder packed 15mm deep in 3.5-mm i.d. Suprasil quartz tubes (Brukcr) fixed a t the same position in the ESK cavity. The first derivatives of absorption spectra were recorded using 1IH) kHz modulation frequency and 2.5 G amplitude. Microwave power, modulation amplitude and sweep width were varied Lo confirm fine structures and saturation of the signals. 1 ,l-Diphenyl-2- picryl hydrazyl (Dt’PH; FIuka. Ruchs, Switzerland) was employed as standard for Ci value determination and also to check calibratiun and sensitivity. The ESR signal height was measured in mm as the peak-to-pcak amplitude of the first derivative spectrum. ESR signal intensity was caiculated as the mtio of signal heightlsample weight. All ESK recordings were made at room temperature (25°C).

Washing wirh m ~ f l z y i cdkosoh~i~ Methyl cellosolve (ethylene glycol monomethyl ether; Sigma. St Louis, MU,

USA), an organic solvent for natural and synthetic resins, dyes and wood stains (Budavari, 1980) was used for washing the interfering compounds from mango seed cover powder a d microcrystalline cellulose (Mcrck, Darrnstadt, Ciermany). A weighed sample was stirred for 15 min with 20 volumes of solvent, centrifuged (IOmin, 1240 x g and the residue washed twice mnre as above; U . V . spectra of supernatants were recorded and the residues were vacuum dried for ESR analysis.

Results and discussion

The ESR signals at G=1.988 in three portions of the seed from 24-11 previously irradiated (1 .O kGy) and non-irradiated ‘Totapuri’ mangoes showed increased amplitude (Fig. Z), with line width increased by 50% from 7-9 gauss to 11-12.6 gauss in irradiated samples. The epidermal layer showed the maximum ESR signal, followed by seed cover and kernel. Their respective moisture contents were found to be 5.8, 13.4 and 25.6% (wet basis). The new type of ESR spectrum of irradiated mango keriiel reported by Desrosiers & McLaughlin (1Y89) was not detected in our studies, possibly due to differences in varieties and stability of ESR signals.

The ESR signal intensity in three portions of the seed from untreated and 1.0 kGy-irradiated mangoes of four cultivars recorded 24 h after irradiation (Fig. 3) showed the most incrcases in signal intensity upon irradiation in the epidermal layer and seed cover, with least in the kcmel. The higher radiation-induced signal in the seed cover than in the epidermal layer of ‘Langra’ matigoes may be due to composition a1 differences.

To study the stability of the ESR signal in irradiated stored mangoes, seed covers were selected for their moderate moisture content and sufficient sample size. The seed cover powder from ‘Alphonso’ mangoes irradiated with 0.35-0.75 kGy doses and then stored for I month at 20°C,, showed a persistence of ESK signal with their intensities varying in a dose-dependent manner (Fig. 4). The hard seed cover of mango, with a low moisturc content iind made up oi 3 crystal lattice nf cellulolignin molecules, could, on irradiation, provide trapped free radica! sites (Arthur, 1971;

682 B. Bhushan et al.

H 10 G -

Figure 2. ESR spectra of I .O kGy gamma-irradiated (-) and control (---) seed portions from 'Totapuri' mangoes. ESR spectra were taken 24 h after irradiation: peak-to-peak amplitudc (s) was used to compare signal intcnsity. The horizontal scale is in gauss (G) and thc arrow indicates the direction of increasing field.

MANGO VARIETY

TOTAPURI

LANGRA

CHAUSA

ALPHONSO

- 2.0s

w 1.0 i

2-0 ;

1-0 Y 9

1.0 y

I Q ln . '3

-1

2.0 2 > t

W I-

z

2'o# 1-0 Ji

a ln W

SEED COVER EPIDERMAL-LAVER K E R N E L

Figure 3. ESR signal intensity response to gamma irradiat~on in various portions of seed from four cultivars of mangoes. Control (0); irradiated, 1.0kGy (a).

ESR detection of irradiated mangoes 683

CONTROL

0.35 k G y

0.50 k G y

0.75 k G y

H 75 G -

h I

Figure 4. ESR spectra of seed covcr powder from gamma-irrdiated and stored mangoes of 'Alphonso' cultivar. Mangoes, irradiated to 0.35-0.75 kGy doscs and stored for 1 month at 2OoC, were used to prepare sccd cover powder. Thc horizontal scale is in gauss (G) and the arrow indicates the direction of increasing tield.

Raffi & Agnel, 1989). In addition, the free radicals in the more ordered regions of the fibrous cellulose would have a decreased accessibility to moisture vapour and oxygen and would therefore be long lived (Arthur, 1971). This was supported by our finding that, in comparison to the kernel, the hard dry seed cover showed higher ESR signals even up to 1 month after irradiation. Our observations also confirm earlier findings (Dodd et al., 1985; Stachowicz et al., 1992) that the radiation-induced ESR signal in fruits is non-specific and its rate of decay is moisture dependent.

In order to examine the possible source of the natural and radiation-induced signals, ESR spectra of seed cover were recorded before and after washing with methyl cellosolve. Washing considerably reduced the intensity of both natural and radiation-induced signails. Thus, the reduction in the natural signal intensity of 'Alphonso' and 'Langra' seed cover was 75.5% and 71.5%, respectively. In 1 .O kGy-irradiated 'Totapuri' mangoes the reduction in signal intensity of the seed cover was 87.5%, compared to 20% for the kernel which possessed a low natural signal. The U.V. absorption spectra (Fig. 5 ) of mango seed cover washings showed a major peak at 303 nm and a minor peak at 263 nm in the first washing, suggesting the phenolic nature of the extract. The second washing showed peaks at 277 and 241 nm, while the third washing did not show any well-defined peaks. Melanin-type pigments naturally occurring in seeds of fruits (Goodman et al., 1989) and membrane-bound semiquinones (Harborne, 1980; Raffi & Agnel, 1989) are reported to give an ESR signal similar to that produced upon irradiation. Mango seed coat (testa) contains catechin, epicatechin, ethyl gallate and leucocyanidin (Subramanian & Nair, 1971),

684

WAVELENGTH, nm

Figure 5. U.V. absorption spcctra of three successive washings of mango seed covcr powder in methyl ccllosolvc. First washing (-): second washing (----): third washing (.---...........) .

the enzymatic oxidation of which gives rise to o-quinone, with intermediate transient species possessing paramagnetic properties. In woody plants, besides cellulose and sterols, lignin comprises 20-30% of biomass and the mechanism of lignification also involves free radical formation by enzyme-catalysed reactions on precursors (Hwang et al . , 1991; Lewis & Yamamoto, 1990; Steelink, 1972).

With a view to finding out the contribution of compounds extracted by methyl cellosolve to the natural and radiation-induced ESR signals, the response of washed and unwashed seed cover to irradiation was studied. For this purpose vacuum-dried seed cover powder from ‘Langra’ mango was exposed to varying dosed of gamma radiation before and after three successive solvent washings. The ESR signal intensity of unwashed samples plotted on a semi-log paper against radiation dose showed a straight line relationship (Fig. 6). It did not pass through the origin, as non-irradiated control samples also showed an ESR signal. Washed and then irradiated powder did not show a linear dose-response curve primarily due to a considerable reduction (70%) in the natural signal caused by washing. Seed cover powder irradiated at 0.5 and 1.0 kGy doses showed, after washing, 87% and 90% reduction in signal intensity, resulting in levels similar to that observed in non-irradiated control samples (Fig. 6). The results also suggest an increased extractability of phenolic compounds following irradiation. Methyl cellosolve extract of seed cover failed to show an ESR signal at room temperature. This may be attributed to free radical decay in the solvent, favouring radical-radical interaction.

Pure microcrystalline cellulose did not show any natural ESR signal. Upon irradiation, it showed a triplet resulting from a free radical at C5 (Arthur, 1971) and a straight line relationship between radiation dose and ESR signal intensity (Fig. 6). Washing with methyl cellosolve resulted in only partial decay of the triplet signal.

ESR detection of irradiated mangoes 685

Figure 6. Influence of washings with methyl cellosolvc on ESR signal intensity response and decay in irradiated cellulose and mango sced cover powder. Seed cover: irradiated (-o-): washed and irradiated (PA-): irradiated and washed (PO---). Ccllulosc: irradiated (-=-); irradiated and washed (--Up).

Washed seed cover, upon irradiation with the same dose of 1.0 kGy, showed a similar signal but with a lower relative intensity of 1.21 compared with 1.6 for the unwashed seed cover, without the appearance of the triplet due to cellulose radicals; this suggests that the phenolics were not completely extracted by solvent washings. These results therefore suggest that phenolics with high G (number of free radicals 100eV-') values (Tabata et af., 1991) are the major contributor to the ESR signal in natural samples of mango seed.

Conclusions

The similarity of the endogenous and radiation-induced ESR signals appears to restrict the practical utility of the ESR technique for the detection of irradiation treatment in mango fruits. Phenolic substances seem to be the major source of ESR signals in mango seed. It is likely that post-harvest metabolic changes occurring during ripening of mango fruits result in the accumulation of free radicals similar to that produced by irradiation.

Acknowledgments

We wish to thank Drs M.D. Sastry and A.G. Page of the Radiochemistry Division for generously allowing us to use the ESR spectrometer.

686 B. Bhushan et al.

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Received 12 February 199.7. revised and accepted 30 August 1994