Technical note

EPR identi®cation of irradiated Monascus purpureus redpigment

Octavian G. Duliua,*, Mariana Ferdesb, Ovidiu S. Ferdesc

aUniversity of Bucharest, Department of Atomic and Nuclear Physics, MaÄgurele, P.O. Box MG-11, RO-76900, Bucharest, RomaniabInstitute for Food Chemistry, str. GaÃrlei nr. 1, RO-71576, Bucharest, Romania

cNational Institute for Lasers, Plasma and Radiation Instruments, MaÄgurele, P.O. Box MG-36, RO-76900, Bucharest, Romania

Received 25 March 1998; received in revised form 22 January 1999; accepted 24 January 1999

Abstract

Fresh red alimentary pigment extracted from Monascus purpureus fungus exhibits an intense EPR line consistingof a single, narrow line, attributed to a quinone radical. When irradiated with 7 MeV electrons or 60Co g-rays, theamplitude of this line increased with the absorbed dose following a saturation exponential dependency up to 10

kGy. During annealing treatment (isothermal heating at 1008C) the irradiation centers decay exponentially with ahalf-life time of 2.30 min. # 1999 Elsevier Science Ltd. All rights reserved.

Keywords: EPR; Irradiated food; Identi®cation; Quinone; Monascus

1. Introduction

Food irradiation with g-ray or high energy (7±10

MeV) electrons (Murrieta et al., 1996; Duliu et al.,1997; Desrosiers, 1996) at doses up to 10 kGy candestroy harmful bacteria and fungi with minor or even

without impairments of the organoleptic properties ofirradiated aliments. Due to the fact that in somecountries the sale for human consumption of irradiatedfoodstu�s is restricted or even prohibited, the positive

identi®cation of such kind of aliments represents anactual task of any state authority enabled to controlthe food quality.

To accomplish this task, several techniques basedupon the identi®cation of the various physico-chemicalchanges, which are induced during food irradiation,

are currently used (Delince e, 1991; Stevenson, 1993).

Among them, Electron Paramagnetic Resonance(EPR) suggested itself as one of the most adequatemethods due to its high sensitivity to unpaired elec-

trons present in all free radicals generated during ir-radiation. Generally, EPR have been used for theinvestigation of the hard part of foodstu�s, able to

retain for long time (up to 1 month) the irradiationfree radicals (Wieser and Regulla, 1988; Ra� andAgnel, 1989; Maloney et al., 1992; Ra� and Stacker,1996; Desrosiers, 1996).

In the last decades, the interest for new natural pig-ments to be used in food industry is continuouslygrowing. Among them there are red pigments pro-

duced by the fungus Monascus purpureus, used forlong time in di�erent zones of Asia as natural color-ants to traditional dishes. It is used in foodstu�s prep-

aration by replacing some traditional additives as E120 (cochineal), E 252 (potassium nitrate) and E 249(nitrites) (Berset, 1990, 1994; Fabre et al., 1993). TheMonascus purpureus dye, consists of at least six pig-

ments: anka¯avin, monascin, monascorubranine, mon-

Radiation Physics and Chemistry 57 (2000) 97±101

0969-806X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved.

PII: S0969-806X(99 )00305-9

www.elsevier.com/locate/radphyschem

* Corresponding author. Fax: (+) 40 1 420 8625.

E-mail address: odlu@scut.®zica.unibuc.ro (O. G. Duliu)

ascorubrin; rubropuctamine and rubropunctanin(Wong and Koehler, 1983), belonging to azaphiones

mould group which is included in to quinones com-pound. The azaphiones is an assembly of colored com-pounds, which have chromphores resembling those of

quinones. Some authors (Ra� and Agnel, 1989) haveattributed to quinone radicals an EPR line observed insome non-irradiated fruits (raspberry and strawberry

achenes).As a natural product, the Monascus purpureus red

pigment has to be treated to assure both hygienic and

quality requirements for microbial contamination with-out impairing color or other technological properties.One of the most appropriate treatments used for thispurpose is irradiation either by g-rays or accelerated

electrons. This treatment acts e�ectively on bacteria,fungi and moulds, keeping at the same time otherspeci®c culinary properties unchanged. Therefore, it is

desirable to identify a possible ionizing treatmentapplied to these kind of food ingredients.The present paper reports the results obtained by

EPR investigation of Monascus purpureus red pigmentirradiated with 60Co g-ray as well as with 7 MeV elec-trons in order to obtain new data regarding the ir-

radiation e�ects on this pigment and, at the same time,to use the EPR for identi®cation of a previous ir-radiation treatment.

2. Materials and methods

2.1. Red pigment

The red pigment was obtained by surface biosyn-thesis of a Monascus purpureus strain from the collec-tion of Institute of Alimentary Chemistry, Bucharest.

The fungus had been grown on solid natural culturemedia represented by coarsely ground rice (24% r.h.;pH 6.0±6.5) inoculated with 20 ml of spore suspension.

The biosynthesis has been carried out by aerobic incu-bation (Koji culture) for 7 days at 308C and stirreddaily. After this time the material was air-dried for 2 hat 808C and ground to obtain the ®nal alimentary red

dye and ®nally used for further irradiation and EPRmeasurements. In order to check the red pigment foroptical density, it was ethanol extracted from the ®nal

dye by using, diluted up to 1:2000 and measured forthe characteristic wavelengths of 400 and 510 nm. Therelative humidity of the dye, measured by an IR moist-

ure analyzer, usually varied in the range of 8±10%. Itremained almost constant during irradiation, EPRmeasurements and 3-month storage.

2.2. Irradiation

500 mg samples were wrapped into paper envelopes

and irradiated at room temperature with 60Co g-ray at

the Irradiation Facilities of the Institute of Physics and

Nuclear Engineering in Bucharest at a dose rate of 5

kGy hÿ1 as well as with 7 MeV electrons produced by

the linear accelerator of the National Institute for

Lasers, Plasma and Radiation Physics in Bucharest. In

the case of g-ray, the absorbed dose has been deter-

mined by means of a Super Fricke chemical dosimeter

Fig. 1. (I) Experimental EPR spectrum of 10 kGy gamma

irradiated Monascus purpureus red pigment. This spectrum

consists of a central line (C+D) and two small satellites (A

and B). The central line represents a superposition of two

EPR spectra: line D of the unirradiated sample and a singlet

related to quinone radicals generated during irradiation. (II)

The same spectrum after 15 min of isothermal annealing at

1008C. This spectrum (line D) is, within the experimental

errors, identical with those, recorded before irradiation.

External lines belong the IIIrd and the IVth CaO:Mn2+ while

the vertical doted one display the position of the DPPH eta-

lon.

O.G. Duliu et al. / Radiation Physics and Chemistry 57 (2000) 97±10198

while in the case of electrons it has been monitored by

an ionization chamber. The absorbed doses were, forboth kinds of irradiation treatments, equal to 0.1, 0.25,0.5, 1, 2.5, 5, 7.5 and 10 kGy respectively.

2.3. EPR measurements

All EPR measurements have been performed at

room temperature by using an X band JEOL JES ME3X spectrometer provided with a cylindrical TE011 res-onance cavity with 100 kHz magnetic ®eld modulation.

All EPR spectra have been recorded in the same con-ditions i.e., microwave power of 6 mW, modulationamplitude of 0.2 mT, gain of 4000, sweep time of

25 min. Red pigment sample, weighting about 200 mg,have been sealed into quartz tubes and tightly boundinto a special holder, so that at each measurement they

were ®xed in the same position. Thus, the errors intro-duced by their misalignment have been avoided.The magnetic ®eld sweep has been calibrated by

using CaO:Mn2+ (Shuskus, 1962) and DPPH (2,2-

diphenyl-1-picryl-hydrazil) standard samples. TheCaO:Mn2+ standard sample have been introduced lat-erally into resonant cavity through a special hole

designed for this purpose by the manufacturer andkept in the same position for the entire time ofmeasurements.

2.4. Thermal treatment

Isothermal annealing was performed by immersing

sealed sample tubes (6 cm height, about 200 mg red

dye) into boiling water (1008C) for di�erent periods oftime varying between 1 min and 6 h. After each ther-mal treatment, the EPR spectrum was recorded im-

mediately.

3. Results and discussion

3.1. EPR spectrum of non-irradiated and irradiatedpigment

Non-irradiated pigment presents, at room tempera-ture, an intense EPR spectrum (see Fig. 1) consisting

of an asymmetric singlet (line D) characterized by a g-factor ge�,D=2.0043 2 0.0004 and a line widthDB=0.7220.05 mT. By separating the benzene soluble

fraction from the natural mixture of all six pigments,we observed that both insoluble fraction and driedsupernatant present similar EPR spectrum. Takinginto account both gyromagnetic factor and line-width,

this line can be attributed to a stable quinone radical,previously observed in a large category of vegetalsample as hard part of fruits or thermal degraded

wood (Ra� and Agnel, 1989).Irradiated red pigment presents a relatively complex

EPR spectrum, consisting of a broad asymmetrical line

(line C+D) with an e�ective gyromagnetic factorge�=2.004920.0004 and a line width DB=0.5420.05mT. Superposed on this broad line we have noticedanother two narrow satellite lines, each of them

Fig. 2. The dependency of the amplitude of the EPR central line, expressed in relative units, on the absorbed dose: *Ðline ampli-

tude of the irradiated samples; QÐline amplitude of the same samples after 15 min of isothermal annealing at 1008C.

O.G. Duliu et al. / Radiation Physics and Chemistry 57 (2000) 97±101 99

characterized by greater e�ective gyromagnetic factorsi.e., ge�,A=2.016820.0004 and ge�,B=2.012420.0004(line A and B). The amplitude of the EPR line corre-sponding to the irradiated sample was, for an absorbed

dose of 10 kGy, about ®ve times more intense than forthe same sample before irradiation.Following the thermal treatment, the line shape

changed, turning to those observed for non-irradiatedsamples (line D on Fig. 1). These changes occurred inthe ®rst 10 min of isothermal annealing, later the EPR

spectrum presenting a remarkable thermal stability, itsamplitude remaining unchanged after 6 h of continu-ous thermal treatment (see next paragraph).

It is clear that the more intense line is due to a rad-ical that appears as a radiation damage, possible someother thermolabile specie of quinone radicals. For thisassumption pledge the gyromagnetic factor as well as

the line-width (Ra� and Agnel, 1989). For the othertwo small satellites, it would be necessary for more in-vestigation to clarify their origin.

No notable di�erences concerning EPR spectrabetween g and electrons irradiated samples have beenobserved.

3.2. Dose dependency and thermal annealing of the EPRsignal

In the range of absorbed dose of 0.1±10 kGy, bothfor g and electron irradiated samples we noticed thesame kind of dose dependency of the amplitude the

EPR signal, better described by a saturation type re-lation:

A�D� � A0 � AD

�1ÿ exp�ÿD=D0�

� �1�

where: A0 is the amplitude corresponding to annealedspectrum; AD is the amplitude corresponding satur-

ation; D is absorbed dose; D0 is the dose correspond-ing to the 0.63 (1ÿ1/e ) of the maximum amplitude(saturation factor).

The dependency of the EPR line amplitude by theabsorbed dose is reproduced in Fig. 2. From thiscurve, we obtained for the D0 dose the value: 2.9920.27kGy. Once irradiated, the EPR spectrum of red pig-

ment shown to be very stable at room temperature, itsamplitude remaining, within experimental errors, thesame for more than 3 months of storage.

During the thermal annealing, di�erent centers haveshown distinct responses. The most thermolabile cen-ters were A and B whose EPR lines vanished after

1 min of isothermal heating. For the main line wehave noticed an exponential decay with the annealingtime, better described by the equation:

A�t� � A0 � �Aÿ A0� exp�ÿt=TD� �2�where: A0 is the amplitude corresponding to annealedspectrum; A is the amplitude corresponding to

absorbed dose D; TD is the half-life time at 1008C; t isthe annealing time.In Fig. 3 is reproduced the experimental plot of the

Fig. 3. The dependency of the amplitude of the EPR central line on the annealing time (at 1008C) in the case of a 5 kGy g-ray irra-

diated sample.

O.G. Duliu et al. / Radiation Physics and Chemistry 57 (2000) 97±101100

EPR central line amplitude of the g-ray 5 kGy irra-diated red as a function of annealing time at 1008C.For this case, Td=2.4120.14 minÿ1.It must be pointed out that following 15 min of ther-

mal treatment, the amplitude of the EPR line regains

its initial value. This characteristic is in our opinionvery important because, in this way, the absorbed dosecan be simply calculated by comparison the amplitudes

of the EPR line for the same sample before and afterthermal annealing.

4. Concluding remarks

From the results presented in this work concerningthe dependence of the amplitude of the EPR line withthe absorbed dose, storage time and thermal treatment

it can be concluded that the red pigment extractedfrom Monascus purpureus fungus presents in theabsence of any irradiation treatment an intrinsic signal,possible a stable quinone radical, over which at last

three other types of radicals are superposed followingthe irradiation. The most intense of them presents apositive correlation with the absorbed dose, a fact that

makes this signal a good indicator for identi®cation ofirradiated pigment.

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