Food Sci. Biotechnol. 21(2): 573-579 (2012)

DOI 10.1007/s10068-012-0073-6

Applicability of Different Analytical Methods for the Identification of

γ-Irradiated Fresh Mushrooms During Storage

Kashif Akram, Jae-Jun Ahn, Gui-Ran Kim, and Joong-Ho Kwon

Received: 1 December 2011 / Revised: 3 January 2012 / Accepted: 11 January 2012 / Published Online: 30 April 2012

© KoSFoST and Springer 2012

Abstract Photostimulated luminescence (PSL), thermo-luminescence (TL), and electron spin resonance (ESR)analyses were performed to identify γ-irradiated (0, 1,2,and 3 kGy) fresh mushrooms (oyster, king oyster, andshiitake mushrooms) during storage at 5oC. PSL analysisgave negative results [<700 photon counts (PCs)] for thenonirradiated and intermediate (700-5,000 PCs) or positiveresults (>5,000 PCs) for the irradiated samples. The shape,intensity, and occurrence of TL glow curve in a typicaltemperature range (150-250oC) along with TL ratio (TL1/TL2) provided sufficient information to confirm the irradiationhistory of samples. Storage resulted in a negligible fadingeffect on PSL and TL characteristics. X-ray diffractionanalysis showed the abundance of feldspar and quartzminerals in the separated dust from mushrooms. In detailedESR analysis employing different sample pre-treatments,all samples were silent for radiation-specific ESR signalsgiving only a central signal (g=2.005) that showed anincrease in intensity upon irradiation.

Keywords: γ-irradiation, mushroom, identification,photostimulated luminescence, thermoluminescence, electronspin resonance

Introduction

Minimally processed fresh fruits and vegetables areincreasing in popularity because of their health benefits andconsumers’ lifestyle changes toward convenience foods (1).Mushrooms are being used extensively as health food all

over the world with a market value of over $45 billion(2,3). Button mushroom (Agaricus bisporus) has a capitalshare of 31.8%, whereas shiitake mushroom (Lentinula

edodes) and Pleurotus spp. follow with 5.4 and 14.0% ofthe world total mushroom production, respectively (4).However, the poor hygienic quality and short shelf-life ofmushrooms are main concerns towards safe consumptionand effective marketing around the globe (5).

Food irradiation for improving hygienic quality andshelf-life of different food products is one of the mostextensively studied technologies in the last century, whichhas demonstrated its technological applicability and safetyfor different irradiated food materials (6). Major healthauthorities have also endorsed the safety of food irradiationat any applied dose and its utility for public health (7).Several studies have reported the effectiveness of irradiationtechnology to improve hygienic and physical qualityattributes of fresh mushrooms (8,9). This technology isnow permitted in many countries for use on fresh (1-3kGy) and dried mushrooms (1-10 kGy) for differenttechnological objectives (5).

However, there is a lack of international consensus toconsider this technology as a general preservation method,and different national and international regulations enforcemandatory labeling, specific applied doses, and control ofirradiated food products in international trade (6).

Irradiated mushrooms, along with honey and otherfunctional foods items, have a 4% share in the internationaltrade of irradiated foods (10). Reliable identificationmethods are important to enhance the acceptability ofinternationally traded irradiated products but, unfortunately,none of available technique has the potential to identify allirradiated food items (11). Many studies have reported theidentification of different irradiated dried mushrooms,however very limited results are available in case of freshmushrooms (5). Dangl et al. (12) and Schreiber et al. (13)

Kashif Akram, Jae-Jun Ahn, Gui-Ran Kim, Joong-Ho Kwon ( )Department of Food Science & Technology, Kyungpook NationalUniversity, Daegu 702-701, KoreaTel: +82-53-950-5775; Fax: +82-53-950-677E-mail: jhkwon@knu.ac.kr

RESEARCH ARTICLE

574 Akram et al.

tried thermoluminescence (TL) method for detection ofirradiated fresh button mushrooms but poor results wereobtained because of low available minerals on themushroom surface. Similarly, in an inter-laboratory blindtrial, only 48% of mushroom samples could be identifiedas irradiated due to lack of contaminating minerals (5).With the quantity of contaminated minerals, the quality isalso very important as photostimulated luminescence(PSL) and TL response particularly depends upon mineralcomposition (14). Electron spin resonance (ESR) techniquehas been successfully applied for the identification ofdifferent irradiated dried mushrooms including buttonmushroom, Agaricus boletus, and Craterellus cornucopioides

(15,16). However, there is no published data explaining theeffectiveness of ESR technique to identify differentirradiated fresh mushrooms.

The objective of this study was to explore the effectivenessof physical detection techniques (PSL, TL, and ESR) toconfirm irradiation status of fresh mushrooms during 4weeks of storage. PSL and TL characteristics of dependsupon quantity and quality of contaminated silicate minerals.Hence, the presence and composition of minerals presenton the mushroom surface was also confirmed by SEM andX-ray diffraction techniques, respectively.

Materials and Methods

Samples, irradiation, and storage Oyster (Pleurotus

ostreatus), king oyster (Pleurotus eryngii), and shiitake

(Lentinula edodes) mushrooms were purchased from alocal market in Daegu, South Korea. The samples werepacked in polystyrene trays, covered with plastic film, andirradiated (0, 1, 2, and 3 kGy) using a Co-60 γ-ray source(AECL, IR-79; MDS Nordion International Co. Ltd.,Ottawa, Canada) at the Korean Atomic Energy ResearchInstitute in Jeongeup, Korea. Absorbed doses were confirmedby alanine dosimeters with a diameter of 5 mm (EMS 104EPR analyzer; Bruker BioSpin, Rheinstetten, Germany),and the free-radical signals were analyzed using a BrukerEMS 104 EPR analyzer (Bruker BioSpin). All sampleswere stored at 5±1oC for 4 weeks.

PSL analysis PSL measurements were performed asdescribed by EN 13751 (17) using a SURRC PPSLIrradiated Food Screening System (serial #0021; ScottishUniversities Research and Reactor Center, Glasgow, UK).The outer skin of the mushroom samples was carefullyseparated using a sterile blade and placed in a disposable50-mm diameter petri dish (sterlin type 122; Bibby, Glasgow,UK) with the outer surface in an upward direction. ThePSL photon counts (PCs) were measured in subdued lightconditions, and the results were interpreted in accordance

with protocol EN 13751 (17) using a lower threshold(<700 PCs/60 s=negative) and an upper threshold (>5,000PCs/60 s=positive) values. The signals between the 2thresholds were reported as intermediate, which requiredfurther confirmatory analysis to obtain valid results.

TL analysis The TL analyses were conducted as describedin the protocol EN 1788 (18), in which the inorganic dustminerals (≥0.2 mg) were separated from the mushroomsamples by rinsing the surface with distilled water (Grade3). The TL measurements were performed using a TLreader (Harshaw 4500; Thermo Fisher Scientific Inc.,Waltham, MA, USA) from an initial temperature of 50oCto a final temperature of 400oC at a rate of 6oC/s. The firstmeasurement was made on the extracted minerals to obtainthe first glow curve, TL1. To normalize the TL response,the samples were re-irradiated at 1 kGy and re-measured toobtain the second glow curve, TL. Full-process blankswere also prepared through all steps and were measuredwith sample discs to define minimum detection limits(MDL). Samples with a TL intensity 10 times more thanthe MDL were used for the TL ratio calculation. TL1

shape, intensity, and TL ratio (TL1/TL) were used tointerpret the results by following the protocol EN 1788(18).

ESR analysis Different parts of the mushrooms (stemskin and internal flesh, cap skin, and internal flesh, andgills) were separated carefully with a sterile blade, and allsamples were dried with 2 different methods: in a freezedryer (Model SFDSF1; Sewon Freezing Engineering Co.,Seoul, Korea) or using an alcoholic extraction technique(18). Approximately 0.1 g of dried sample was placed in aquartz ESR tube (5-mm diameter). The protocol EN 1787(20) was used to measure ESR signals, targeting theradiation-induced cellulose radicals, using an X-band ESRspectrometer (JES-TE 300; Jeol Co., Tokyo, Japan) atroom temperature under the following conditions: power,0.4-5 mW; frequency, 9.18-9.21 GHz; center field, 327±2mT; sweep width, 10-25 mT; modulation frequency, 100kHz; modulation width, 1-2 mT; amplitude, 50-400; sweeptime, 30 s; and time constant, 0.03 s.

SEM and X-ray diffraction (XRD) measurements SEMand XRD measurements were performed to determine thepresence of mineral dust on the mushroom surface and tocharacterize the composition of the isolated minerals fromthe mushroom samples, respectively. The cross-sections ofthe outer surface (skin) of the mushroom cap wereseparated and used for SEM analysis (S4300; Hitachi Co.,Tokyo, Japan) after freeze-drying. Acceleration voltagewas set to 15 kV. The samples were coated with goldbefore microscopy.

Identification of Irradiated Fresh Mushrooms 575

Inorganic dust (about 0.5 g) was separated from themushroom samples by washing with distilled water andcharacterized using a multipurpose X-ray diffractometer(X’ Pert Pro; PANalytical, Almelo, Netherlands). The X-raydiffractometer was calibrated using silicon powder(corundum) as a standard reference material. The analysiswas conducted under the following conditions: X celerator(Ultra fast) detector; 0-60o scan angle; 11.9o/s scan rate;Gonio of scan axis; continuous scan mode; and Cu Kα

radiation with a wavelength of 1.540598 Å. The spectrawere obtained between 0 and 60oθ at a 2oθ scan. The peakswere analyzed by comparison with reference data.

Statistical analysis All measurements were conductedfor 3 different packs (n=3), and the results are reported asmeans standard deviations (SD). The data analysis wasconducted using Origin 6.0 (Microcal Software Inc.,Northampton, MA, USA).

Results and Discussion

PSL properties Various studies have demonstrated thatPSL is an effective technique for rapid and easy screeningof irradiated samples (17). Table 1 shows the PSL photoncounts (PCs) for the control and irradiated mushroomsamples before and after storage. All nonirradiated samplesproduced negative counts (<700 PCs) before and after

storage. One kGy irradiated king oyster mushroomsamples were intermediate (700-5,000 PCs), but withpretty high PCs (4,442), whereas the 2 and 3 kGy treatedsamples were positive (>5,000 PCs). All irradiated oystermushroom samples yielded intermediate results; however,an increase in PCs was observed with an increase inirradiation dose. The highest PSL count was obtained from3 kGy irradiated king oyster mushroom samples (690,800).However, the most promising results were provided byirradiated shiitake mushroom samples, which were positivewith good PCs (12,397) even for 1 kGy irradiated samples(1 kGy irradiated king oyster mushroom samples gaveintermediate results). The inconsistency of the PSL resultsfor different samples may be attributed to variations inquantity and quality of inorganic dust on the surfaces ofdifferent mushroom samples (21). A slight decrease in PSLcount was observed with storage, which was clearer in thecase of all 3 kGy irradiated samples. These results were ingood agreement with those reported for γ-irradiated freshkiwifruit (22).

TL characteristics The TL analysis was employed as aconfirmatory analysis to identify the irradiated mushroomsin accordance with EN 1788 (18) after obtaining themineral fraction, which was enriched with silicate minerals,using a density separation method. All nonirradiatedsamples produced a low-intensity TL glow curve with amaximum peak at about 300oC (Fig. 1), providing

Table 1. Accumulated photon count (PCs/60 s) from fresh mushrooms samples during 4 weeks of storage at 5oC

SampleStorage(day)

Irradiation dose (kGy)

0 1 3

King oystermushroom

0 00371±231)(-)2) 000444±1548(M) 11,544±3665(+) 690,800±86642(+)

8 53±68(-) 003,178±2008(M) 05,865±1550(+) 061,925±16479(+)

Shiitake

mushroom

0 311±96(-) 12,397±2906(+) 14,778±5529(+) 38,711±6013(+)

8 69±36(-) 00736±3285(+) 10,583±3273(+) 14,746±3316(+)

Oystermushroom

0 81±36(-) 01,150±233(M) 01,852±371(M) 03,519±911(M)

8 09±38(-) 01,086±372(M) 01,621±253(M) ,0 929±696(M)

1)Mean±SD (n=3)2)Threshold value: T1=700 (non-irradiated), T=5,000 (irradiated), (-)<T1, T1<(M)<T, (+)>T

Table 2. TL ratio (TL1/TL ) of the minerals separated from fresh mushrooms during 4 weeks of storage at 5oC

Sample Storage (day)Irradiation dose (kGy)

0 1 3

King oyster mushroom

0 0.015±0.0011) 0.527±0.023 1.075±0.063 1.232±0.144

8 0.014±0.007 0.590±0.055 0.973±0.052 1.126±0.156

Shiitake

mushroom

0 0.005±0.001 0.859±0.028 1.240±0.037 1.867±0.084

8 0.003±0.001 0.633±0.02 0.947±0.100 1.202±0.037

Oystermushroom

0 0.012±0.006 1.002±0.203 2.035±0.141 1.882±0.234

8 0.003±0.001 0.407±0.119 0.633±0.321 1.300±0.131

1)Mean±SD (n=3)

576 Akram et al.

sufficient information to confirm the absence of anyirradiation history. The reliability of the minerals used toobtain these results was further confirmed by a TL ratio<0.1. All irradiated mushroom samples revealed typical TLglow curves with maximum peaks of 150-200oC. The TLratios of all irradiated samples were also >0.1, confirmingthe properties of minerals on the TL discs to obtain thevalid results. Figure 1 illustrates that the results werereproducible for discriminating irradiated from nonirradiatedsamples even after 4 weeks of storage with negligiblechanges in TL intensity. Similar results were also reportedby Jo et al. (22) for irradiated fresh kiwifruits. After 4weeks of storage, the 1 kGy-irradiated oyster mushroomsample produced a TL ratio of 0.4. The protocol EN 1788(18) recommends that the TL glow curve shape should beconsidered in case of a TL ratio between 0.1 and 0.5 toobtain conclusive information. Lower TL ratio was alsoobserved by other scientists when identifying irradiatedsesame seeds (23). Poor TL results from irradiated buttonmushroom samples have been reported due to a lack ofsufficient minerals on the mushroom surface. In this case,increased sample weight for washing can effectivelyaddress the problem (5).

ESR properties The ESR analysis on radiation-inducedradicals in food samples with higher moisture content ispossible by employing an effective drying technique (24).A detailed ESR investigation on the freeze-dried samplesin different tissues (stem skin and internal flesh, cap skinand internal flesh, and gills) of all studied mushrooms wasconducted to identify radiation-induced signals. A singlepeak with a g-value of 2.005 was observed in all nonirradiatedsamples, which was associated with organic radicals, asreported by others in a plant food matrix (25). Uponirradiation, most of the samples showed a clear increase inthe central signal (g=2.005) with the influence of Mn2+ oneither side of the main peak. All samples were silent forradiation-induced signals with the exception of the oystermushroom flesh sample, which produced a weak peak onthe right side of the main signal. There was the possibilityof a radiation-induced cellulose signal, as described by EN1787 (20), but the left peak was absent. Deighton et al. (26)concluded that the left peak is a radiation-specific cellulosesignal, whereas the right peak because of nonradiationspecific lignin signals, which may also overlap by Mn2+

signals (27). An alcoholic extraction technique (19) wasemployed instead of freeze drying to confirm our results.Comparable results were found for all the samples exceptthe flesh of oyster mushroom, in which the right peak wasabsent, confirming the effect of Mn2+ in the sample.Different scientists reported radiation-induced ESR signals,especially from crystalline sugar radicals, for the identificationirradiated dried mushrooms (15,16). However, in this study

of irradiated fresh mushrooms, identification was notpossible using ESR technique.

Mineral existence and composition It is well establishedthat luminescence characteristics, which are the basis of TLand PSL identification techniques, depend upon the qualityand quantity of inorganic dust adhering to sample surfaces(17,18). SEM image is showing the presence of inorganic

Fig. 1. TL glow curves and intensity of the minerals separatedfrom irradiated fresh mushrooms during 4 weeks of storage at5oC.

Identification of Irradiated Fresh Mushrooms 577

dust on the mushroom surface in Fig. 3A. Large quantitiesof mushroom samples were washed to get the inorganicdust for XRD analysis, but only shiitake mushroom samplesprovided a sufficient amount of inorganic dust (about0.5 g). A lack of minerals on the mushroom surface hasalso reported by others (5,17). This also explains the leastPSL counts in the case of irradiated oyster mushroomsamples. Although the highest PSL count was obtained in3 kGy irradiated king oyster mushroom samples; however,the results from shiitake mushroom samples were moredose dependent and promising (positive at 1 kGy irradiation),demonstrating the uniform availability of minerals onmushroom surfaces. Figure 3B shows the mineral composition

of the inorganic dust separated from shiitake mushroom.Quartz and sodium-feldspar were found as major mineralcomponents on mushrooms during the XRD analysis,which defined the main luminescence characteristics (PSLand TL) of the samples (11,21).

In summary, PSL analysis revealed its potential as arapid screening method providing negative counts fornonirradiated samples. The 1 kGy-irradiated king oystermushroom samples were intermediate, whereas the 2 and 3kGy-treated samples were positive. All irradiated oystermushroom samples produced intermediate results. Themost promising results (positive) were obtained for irradiatedshiitake mushroom samples. Storage showed little effect on

Fig. 2. ESR spectra of the irradiated fresh mushrooms after employing different drying techniques.

578 Akram et al.

PSL count, but was prominent in 3 kGy-irradiated samples.In the TL analysis, all the samples provided sufficientinformation of TL intensity, TL glow curve temperaturerange, and TL ratios to clearly discriminate irradiated andnonirradiated samples, whereas the effect of storage wasnegligible. The detailed investigation showed the inappro-priateness of the ESR method to characterize irradiatedmushroom samples. XRD analysis revealed the presence ofquartz and sodium-feldspar as dominant mineral componentson the mushroom surface. PSL analysis may be useful forscreening of irradiated fresh mushrooms, whereas TLanalysis provides confirmatory results after separatingsilicate minerals.

Acknowledgments This research was supported byTechnology Development Program for Food, Ministry forFood, Agriculture, Forestry and Fisheries, Republic of Korea.

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Fig. 3. SEM images showing the presence of mineral dust(enclosed within the circles) on the fresh mushroom surface(A) and XRD spectra marked with major minerals components(Q, Quartz; F, Na- Feldspar) of mineral dust separated fromshiitake mushroom samples (B).

Identification of Irradiated Fresh Mushrooms 579

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