electron spin resonance studies on γ-irradiated coffee bean parts

Electron spin resonance studies on c-irradiated coffee

bean parts

Brij Bhushan,1* Rajeev Bhat,2 Banavara Y. K. Rao,1 Rasheed Ahmad2 & Dilip R. Bongirwar1

1 Food Technology Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India

2 Department of Applied Botany, Mangalore University, Mangalagangotri, Mangalore 574 199, India

(Received 8 October 2001; Accepted in revised form 1 May 2002)

Summary Using electron spin resonance (ESR) free radicals, present naturally or formed after c-irradiation of parts of coffee bean, were examined by entrapping the sample in potassium

chloride powder in ESR quartz tubes. The ESR signal at g¼ 2.002 was more prominent in

the spermoderm than in the whole seed portion of the coffee beans. The c-irradiation of

coffee beans with doses of 5 or 10 kGy, normally used for decontamination, resulted in a

dose-dependent increase of a signal at g¼ 2.002 which was accompanied by a weak triplet

(aH c. 3.0 mT), and which was also more prominent in the spermoderm. While short-term

storage (24 h at 25 ± 0.5 �C) of irradiated beans resulted in a substantial loss of signal at

g¼ 2.002, annealing at 50 ± 0.5 �C for 16 h increased this signal intensity in greater

proportion than caused by irradiation alone, suggesting that generation of free radicals in

the two varieties of coffee beans is not unique to the irradiation processing alone.

Keywords Annealing, decontamination, food irradiation, free radicals.

Introduction

Recently, c-irradiation has emerged as a safe and

effective technique for microbiological decontam-

ination and extending the shelf-life of fresh and

processed foods (Thomas, 1986; Wilkinson &

Gould, 1998; FAO/IAEA/WHO Consultation,

1999). A dose of 250 Gy is adequate as an effective

quarantine treatment, but higher doses, of 10–

12 kGy, are recommended for total elimination of

fungi and yeast (Diehl, 1990; Ramakrishna et al.,

1991; Wilkinson & Gould, 1998). Codex General

Standard (Codex, 1984) provides guidelines and

handling procedures for irradiation of foods.

Although food irradiation has become legal in

forty-one countries, it remains unacceptable in

others (Anonymous, 1991).

In India, Arabica and Robusta coffee are the

two major plantation crops covering an area of

around 3.06 lakh ha with an annual output of

2.30 lakh tonne. About 80% of the coffee pro-

duced is exported, with an annual earning of c. 450

million US$ (Naidu, 1999). Coffee beans are prone

to spoilage by microbial contamination and insect

proliferation during processing, storage and

transportation. Fumigants like methyl bromide,

ethylene dibromide, etc. have been employed

commercially to control insect pests during stor-

age of coffee beans (Narasimhan, 1999). The

decision to ban the use of such ozone depleting

fumigants (Anonymous, 1993; Marcotte, 1993) to

protect the environment and for other safety

reasons, has created a renewed interest in the use

of physical processes, such as c-irradiation, as aneffective alternative for quarantine treatment

(Thomas et al., 1996; Bhushan & Thomas, 1998).

However, there is scarcity of data regarding the

status of free radicals in irradiated coffee beans.

Hence, the present studies were undertaken to

examine the presence of free radicals in coffee

bean parts and to ascertain the comparative

changes induced by conventional and c-radiationprocessing.

*Correspondent: Fax: +91 22 5505151;

e-mail: [email protected]

International Journal of Food Science and Technology 2003, 38, 11–16 11

� 2003 Blackwell Publishing Ltd

Materials and method

Coffee beans

Mature, freshly hulled, �A� grade, dry processed

coffee beans of 6–7 mm size, belonging to Arabica

(Coffea arabica L.) and Robusta (C. robusta ex.

Froehner) varieties, were procured from the coffee

curing works (Aspinwall and Co., Mangalore,

Karnataka, India). Healthy beans from the same

lot with no apparent physical damage or insect

infestation were used in the present studies. Cured

coffee beans had spermoderm (silver skin) embed-

ded in their central groove.

Irradiation

Coffee beans packed in biaxially oriented poly-

propylene (BOPP, 25 l) bags were exposed at

25 �C to c-radiation doses of 5 and 10 kGy in a cCell-220 60Co source (AECL, Ottawa, Canada),

having a dose-rate of 15 Gy min)1. Fricke dosi-

metry was used for measuring the absorbed dose

(Fricke & Hart, 1966). After irradiation, samples

were kept at )20 �C in a deep freezer for further

analysis at a later date. Non-irradiated coffee

beans packed similarly served as control.

Sample preparation

Coffee bean powder

Beans were powdered in an electrically operated

coffee grinder (Moulinex, Ballina, Ireland) and

the resulting powder was passed through a nylon

mesh to remove large broken pieces. Sieved

particles were subjected to re-grinding in a glass

pestle and mortar and passed through a single

layer of muslin cloth to obtain a fine powder of

median particle size of 67 lm, measured by

particle size analyzer model LA-500 (Horiba Ltd,

Kyoto, Japan).

Coffee bean parts

The cross-sectional view of the anatomy of a

coffee berry is shown in Fig. 1a, such that an

inside-up view of a seed in Fig. 1b reveals some

spermoderm remaining embedded in the central

groove of the coffee bean. This silver skin

(spermoderm) part of the coffee bean, constituting

c. 1.4% (w/w), was separated physically with the

help of a stainless steel spatula and a sharp needle.

It was chopped into fine pieces in a Borosil glass

beaker (25 mL). The beans devoid of silver skin,

served as a whole seed portion and were cut into

small pieces (2–3 mm) by using a nail cutter. These

two parts were entrapped separately in pre-pow-

dered potassium chloride (KCl) columns of height

2.5 cm in electron spin resonance (ESR) tubes of

Suprasil quartz (Wilmad, NJ, USA).

Electron spin resonance measurements

An X-band Bruker EMX 6/1 spectrometer (Bru-

ker Analytische Messtechnik, GMBH, Karlsruhe,

Germany) was used for recording the ESR spectra

of powdered samples packed 25 mm deep in ESR

tubes (Wilmad, 3.5 mm i.d.) and fixed at the same

position in the ESR cavity. The first derivatives of

the absorption spectra were recorded using instru-

mental settings depicted in Table 1. The calibra-

tion and sensitivity of the instrument were checked

Figure 1 Anatomy of coffee berry. (a) Cross-sectional

view of a coffee berry, (b) an inside-up view of a coffee bean

seed showing central groove-embedded with spermoderm

(silver skin).

ESR studies on c-irradiated coffee bean parts B. Bhushan et al.12

International Journal of Food Science and Technology 2003, 38, 11–16 � 2003 Blackwell Publishing Ltd

routinely by using 1,1-diphenyl-2-picryl hydrazyl

(DPPH; Bruker No. 9702 D 153) as the standard

for determination of the g-value. To visualize

the presence of free radicals in silver skin and

in the whole seed part of the coffee bean, the

samples were weighed (10–25 mg) in a Sartorius

Basic BA61 analytical balance (D-37075; Gottin-

gen, Germany) and entrapped in KCl powder in an

ESR tube for scanning. The ESR signal height was

computed (using offset correction) as the peak-to-

peak amplitude of the first derivative spectrum and

the signal intensity was calculated as arbitrary

units per unit sample weight (AU mg)1).

Annealing

Powdered samples of irradiated and control coffee

bean were placed separately in Borosil Petri plates

(50 mm diameter · 15 mm height) and heated in a

Thermostat Vacuum Oven (Townson and Mercer

Ltd, Croydon, England) set at 50 ± 0.5 �C for

16 h. After annealing, samples were allowed to

attain room temperature and placed in a dessica-

tor until they were used for further analysis.

Moisture measurement

The moisture content of irradiated, non-irradiated

and stored coffee beans was measured gravimet-

rically as per the standard procedure described by

Clifford (1985).

Results and discussion

Non-irradiated coffee bean powder gave a sharp

and a clear signal at g¼ 2.002 (Fig. 2). This has

been attributed to quinone-like products present

in almost all vegetable products and exhibiting

pale yellow to dark black colour (Clifford, 1985;

Raffi & Agnel, 1989). Samples irradiated at a

10-kGy dose, however, exhibited enhanced

amplitude of this signal accompanied by new

signals of low amplitude separated by 6 mT. Raffi

& Agnel (1989) have suggested that these new lines

with an intensity ratio of 1:2:1 are part of a triplet

with a real hyperfine constant value of 3 mT. The

central part of the triplet and the natural line

seems to have almost the same g-factor and are

therefore not distinguishable. However, as the

central line is a sum of the �left� and �right� lines,both contributed to the increased signal at

g¼ 2.002 in irradiated coffee beans. The left line

(lower field) is part of a cellulosic radical triplet

and is being tested to evolve a suitable detection

method for irradiated foods of plant origin (Raffi

& Agnel, 1989; Maloney et al., 1992; Desrosiers

et al., 1995; De Jesus et al., 1999).

Figure 3 shows the comparative influence of

various physical treatments (irradiation, short-

term storage and annealing) on free radical

content of coffee bean varieties. The c-radiationexposure caused a dose-dependent increase in the

prominent signal at g¼ 2.002, which was more

pronounced in powdered samples of Arabica than

in samples of the Robusta variety. As a result of

annealing, both the varieties showed a similar

increase in signal intensity to that caused by

"Cellulosic radical"

"Left g"

2.0204 2.002 1.9948 g factor

"Central g" "Right g"

Irradiated,10kGy

Non-irradiated

aHAH=6 mT

=3 mT

Figure 2 The ESR spectra of non-irradiated and irradiated

Arabica coffee bean powder.

Table 1 Operating conditions for ESR scanning

Microwave frequency 9.731 GHz

Microwave power 1 mW

Modulation frequency 100 KHz

Modulation amplitude 0.4 mT

Scan field 342–352 mT

Receiver gain 1 · 105

Time constant (TC) 163.84 m s

Sweet time 41.94 s

Number of scans 1

Temperature 23 ± 0.5 �C

ESR studies on c-irradiated coffee bean parts B. Bhushan et al. 13

� 2003 Blackwell Publishing Ltd International Journal of Food Science and Technology 2003, 38, 11–16

irradiation alone, indicating a cumulative effect of

both irradiation and heating. It was interesting to

note that short-term storage (24 h at 25 �C)resulted in a significant decline in the level of free

radicals in irradiated samples (Fig. 3) but not in

non-irradiated samples. Our observation corro-

borates the data of De Jesus et al. (1999) showing

an increase in free radical signal in crushed and

oven-dried Kiwi fruit. Thus, it is clear that heating

tends to generate quantitatively similar or even

more free radicals than produced by irradiation

alone. Also, the induced radioactivity has never

been observed in foods irradiated as per the Codex

convention. Therefore, public concern about the

safety aspect of irradiated foods has no scientific

basis (Diehl, 1990).

The stack plot in Fig. 4 provides a comparison

of the ESR signal profile in cut pieces of non-

irradiated and irradiated coffee bean parts. In both

these varieties, a dose-dependent increase in the

signal intensity was observed. Silver skin respon-

ded more favourably to radiation treatment than

the whole seed part. For example, a prominent

signal of intensity 374 AU mg)1 was observed at

g¼ 2.002 along with weak satellite peaks in the

silver skin portion of Arabica coffee beans irradi-

ated with a total dose of 10 kGy. Under similar

experimental conditions, the whole seed portion

gave a six times lower signal value of

61.16 AU mg)1. The values recorded for Robusta

were slightly higher but showed a similar trend as

that for Arabica. However, in the non-irradiated

whole seed portions, Robusta showed a signal

intensity of 20.05 AU mg)1 while no such signal

could be detected in the Arabica variety. The

present results on intense signals in the silver skin

are in conformity with earlier observation by

Ikeya et al. (1989) on coffee beans and our own

findings on thin epidermal layer of irradiated

mango seeds (Bhushan et al., 1995).

During radiation processing of fruits and

vegetables, the moisture content is of great signi-

ficance as it controls the net radiochemical chan-

ges on irradiation as well as on storage (Thomas,

1986; Bhushan et al., 1995; Wilkinson & Gould,

1998). The moisture content of non-irradiated

Arabica coffee beans was observed to be 12.26%

(w/w) as against 10% (w/w) shown by the other

variety. Irradiation with 5 and 10 kGy dose,

however, resulted in insignificant moisture loss of

0.2% and 0.4%, respectively, in the Robusta

variety, however the losses were much higher (i.e.

0.81% and 2.12%, respectively) in the Arabica

variety.

Conclusions

Present studies on two prime varieties of coffee

beans, irradiated for decontamination purposes

have shown no adverse effects on its safety, as

commonly used processing parameters, like

annealing (heating), also produced free radicals,

which decayed on storage. Also, silver skin

(spermoderm) constituting only about 1.4% of

the total seed material, proved to be a richer

source of free radicals for ESR studies. The ESR

examination of cut pieces embedded in KCl

powder, provided data on the status of free

radicals, even in samples of very minute size.

C. arabica

C. robusta

Radiation dose (kGy)

Sig

nal I

nten

sity

(A

u m

g–1

Sam

ple

wt.)

200

150

100

50

0

200

150

100

00 5 10

50

Figure 3 Effect of c-irradiation, storage and annealing on

the level of free radicals in coffee bean varieties. Whole

coffee beans, after the treatment were powdered separately

as described in the text and examined for prominent ESR

signal intensity at g¼ 2.002. The data was normalized for

unit sample weight and values computed as mean ± s.d. of

three independent experiments were plotted as histogram

against the radiation dose – fresh, stored, 24 h,

annealed, 50 �C, 16 h.



These observations could help in evolving suitable

ESR-based detection methods, not only for coffee,

but also for irradiated spices and stone fruits

possessing thin epidermal layers.

Acknowledgments

Authors are thankful to Dr (Mrs) A.M. Samuel,

Director, Bio-Medical Group, BARC, for her

keen interest and constant encouragement during

the course of this study. Also, authors from

Mangalore University, thank Board of Research

in Nuclear Sciences (BRNS), Department of

Atomic Energy, India, for financial assistance.

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electron spin resonance studies on γ-irradiated coffee bean parts

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