Journal of Stored Products Research 39 (2003) 149–157

Impact of gamma irradiation on the monsooningof coffee beans

Rasheed Ahmada,*, Babitha Tharappana, D.R. Bongirwarb

aDepartment of Applied Botany, Mangalore University, Mangalagangotri 574 199, IndiabFIPLY, BARC, Mumbai 400 085, India

Accepted 31 July 2001

Abstract

Monsooned coffee is a special coffee produced only in India. As the process is carried out during themonsoon period, the time taken for monsooning is dependent on rainfall and atmospheric humidity.Traditional methods employed in the monsooning process favour growth of microorganisms, which mayaffect the quality of the produce. The present study aimed to understand the role of microorganisms in themonsooning process. Coffee beans (both Arabica and Robusta variety) were subjected to irradiationtreatments of 5 kGy (kilo Gray) and 10 kGy doses in order to achieve the above objective. Irradiation led toreduction of natural mycoflora with the 10 kGy dose being more efficient than the 5 kGy dose. Aspergillusniger was found to be the dominant fungus colonising the beans prior to monsooning, whereas A. ochraceuswas found dominant during the course of monsooning. The other commonly encountered fungi werespecies of Aspergillus, Penicillium, Absidia, Syncephalastrum,Mucor and Rhizopus occuring at low numbers.At the start of the monsooning process, irradiated samples showed negligible numbers of microorganismsand also lesser number of fungal species during monsooning compared with the non-irradiated samples.Non-irradiated samples took five weeks for the completion of monsooning compared with two weeks forirradiated samples and hence it may be inferred that microorganisms may be playing a minimal role inmonsooning. r 2002 Published by Elsevier Science Ltd.

Keywords: Gamma irradiation; Coffee; Monsooning; Mycoflora

1. Introduction

Monsooning is a natural process of duplicating the taste and colour of coffee beans (Coffeaarabica L. and C. canephora Pierre ex Froehner), as they would have appeared out of the holds of

*Corresponding author. Tel: +91-824-742-272; fax: +91-824-742-367.

E-mail address: rasheeds 2k@yahoo.co.uk (R. Ahmad).

0022-474X/02/$ - see front matter r 2002 Published by Elsevier Science Ltd.

PII: S 0 0 2 2 - 4 7 4 X ( 0 1 ) 0 0 0 4 3 - 1

old sailing ships in years gone by. It is a process of curing of beans carried out only in India, wherethe beans undergo surface fermentation when exposed to the monsoon conditions. The endproduct is monsooned coffee which has special taste and quality including reduced acidity,mellowed syrupy cup and cheesy flavour. The practice of monsooning started as a result ofserendipity and to date the process is carried out traditionally with no clear cut quality parametersspecified. Nearly 70% of 205,000 tonnes of coffee produced annually in the country is processedfor export of which only 2.6% is reprocessed into monsooned coffee for export mainly to Italy andthe Scandinavian countries.Except for the work carried out on a few aspects of storage by Natarajan et al. (1961), Majumder

et al. (1961a-c), Subrahmanyan et al. (1961, 1963) and on chlorogenic acids by Balyaya andClifford (1995), very little information is available on the monsooning of coffee. The coffee beansbeing hygroscopic absorb moisture readily when exposed to high humidity conditions duringmonsoons, which is very conducive to the growth of microorganisms. In order to find out the roleof microbes and their possible effects on quality of monsooning, experiments were carried outwherein beans subjected to gamma irradiation were made to undergo the monsooning process.Radiation sterilization was used because of its suitability for processing heat-sensitive materials

such as biological tissues (Bradley, 1984). In coffee, gamma radiation has been used for insectdisinfestation (Manoto et al., 1991; Soemartaputra et al., 1991). According to Soemartaputra et al.(1991), a radiation dose in the range of 0.6–0.9 kGy gave 100% mortality of the coffee beanweevil, Araecerus fasciculatus (De Geer) and the treatment had no detectable effect on the caffeinecontent, fat, moisture content or pH of the irradiated beans. Irradiation at 0.5 kGy dose had noadverse effect on the flavour or aroma of coffee beans (Dias et al., 1978).The present study was undertaken (1) to elucidate the relation between traditional and gamma

irradiation-induced monsooning, (2) to determine the role of microorganisms in the monsooningof coffee, (3) to examine the possible effects of gamma irradiation on the fungal profile and (4) toinvestigate the effect of gamma irradiation on the time taken for monsooning. The results of theabove studies are discussed in this paper.

2. Materials and methods

2.1. Irradiation of coffee beans

Freshly hulled dry green coffee beans of both the Arabica and Robusta varieties were collectedfrom Tata Coffee Limited, Mangalore, Karnataka in the month of May 1999. Seven kilogramseach of Arabica and Robusta samples were divided into batches of 500 g and sealed in biaxiallyoriented polypropylene (BOPP) packs, a widely used food packaging material and subjected to 5and 10 kGy doses of gamma rays in a package irradiator using a 60Co source at Bhabha AtomicResearch Centre, Mumbai.

2.2. Monsooning of irradiated coffee

During July to August, 1999, i.e. the monsoon season, the samples of irradiated Arabica andRobusta green coffee beans were spread on the floor in well ventilated cement floored godowns, in

R. Ahmad et al. / Journal of Stored Products Research 39 (2003) 149–157150

layers of 10–12 cm thickness to absorb moisture and bloat, along with the non-irradiated coffeebeans which served as the control. The spread coffee was raked at three hourly intervals usingwooden planks to ensure uniform absorption of moisture and prevention of mould growth. Oncethe beans had monsooned they were rebagged to equalise moisture and were checked formouldiness at frequent intervals.A portion of each sample was taken at weekly intervals and analysed for moisture content,

weight/volume ratio, mycoflora and colony forming units (c.f.u.) by following standard methodsexplained below. Before spreading of the beans, the samples were analysed for the sameparameters and these were considered the non-monsooned or zero day samples.For all these experiments, the non-irradiated samples served as control.

2.3. Analysis of the beans

2.3.1. Moisture content and water activityThe moisture content was estimated from the percentage loss in weight of 5 g of sample heated

in an oven at 1051C for 16 h (Clifford, 1985). The relationship between coffee moisture contentand water activity was determined by interpolation from moisture adsorption curves for Arabicaand Robusta coffee (Ahmad and Magan, unpublished data).

2.3.2. Weight/volume ratioWeight/volume ratio has been calculated by earlier workers (Natarajan et al., 1961) by the

water displacement method. In the present study, since the same coffee beans were routinelymonitored for their loss in density while monsooning, a modified method was employed as theformer method involves the contact of seeds with water thereby preventing the reuse of the sameseeds. In the current method, the moisture content of the beans was determined initially. Theweight/volume ratio was calculated by weighing seeds filling a volume of 100ml in a measuringcylinder. The seeds were tightly packed into this volume with the help of a metallic leveler. Theratio was calculated as

Weight=volume ratio ¼W1 � ðW1�m:c:Þ=100

V;

whereW1 is the fresh weight of beans (g); m:c: the moisture content (%); and V the volume (ml).The number of seeds occupying this particular volume was also noted.

2.3.3. Enumeration of microorganismsThe colony forming units (c.f.u.)/g were calculated as outlined by Aneja (1993). The percentage

incidence of microorganisms was determined by fungal counts from 100 seeds per treatment whichwere plated on potato dextrose agar and blotters (Neergaard, 1977). The percentage incidence ofthe fungi on the seeds was calculated by the formula

Percentage incidence ¼No: of seeds on which a species was present

Total number of seeds observed�100:

All the experiments were carried out under sterile conditions.

R. Ahmad et al. / Journal of Stored Products Research 39 (2003) 149–157 151

3. Results

When the samples were analysed to assess the effect of irradiation, a total of 7 species of mouldsbelonging to five genera were isolated from the non-irradiated coffee beans (Table 1). Analysisshowed that in the non-irradiated coffee, Aspergillus niger Van Tieghem was the dominant funguswith an occurrence of up to 100% on both blotters as well as on PDA in Arabica samples whereasin Robusta it was 67% on blotters and 95% on potato dextrose agar. Upon irradiation at 5 kGydose, there was a drastic reduction in the occurrence of this fungus to 3% and a further reductionto 1% at 10 kGy dose in both varieties of coffees. The rest of the 6 fungi also showed a reductionin their occurrence upon irradiation.There was a reduction in the c.f.u./g in the sample irradiated at 5 kGy dose while those

irradiated at 10 kGy dose showed complete absence of microorganisms (Table 2).During monsooning, Arabica variety irradiated at 10 kGy absorbed the highest moisture of

22.7% from an initial level of 12% within the first week (Table 3). In irradiated Robusta, however,both doses showed equal uptake of moisture of around 19.3%. There was a very slow uptake ofmoisture (18.2%) in the non-irradiated Arabica and a moisture content of 18.4% in non-irradiated Robusta sample.

Table 2

Number of colony forming units/g in Arabica and Robusta coffee beans after gamma irradiation at 5 and 10 kGy

Arabica Robusta

Control 5 kGy 10 kGy Control 5 kGy 10 kGy

Bacterial count 16,036 132 106 9558 28 0

Fungal count 120 4.0 0.33 762.3 0.41 0

Table 1

Percentage occurrence of fungi in varieties of coffee after gamma irradiation at 5 and 10 kGy

Fungus Arabica Robusta

Standard blotter method Potato dextrose agar Standard blotter method Potato dextrose agar

Control 5

kGy

10

kGy

Control 5

kGy

10

kGy

Control 5

kGy

10

kGy

Control 5

kGy

10

kGy

Aspergillus

ochraceous

1 0 0 9 1 0 0 1 3 4 1 0

A. flavus 9 6 0 9 0 0 2 0 0 2 0 0

A. niger 100 3 3 100 2 1 67 3 0 96 4 1

Mucor sp. 0 0 0 26 0 0 0 0 0 2 0 0

Penicillium

rugulosum

2 0 0 0 0 0 0 0 0 0 0 0

Penicillium sp. 2 0 0 0 1 0 0 0 0 0 0 0

Syncephalastrum sp. 0 0 0 0 0 0 0 0 0 1 0 0

Total No. of species 4 2 1 4 3 1 2 2 1 5 2 1

R. Ahmad et al. / Journal of Stored Products Research 39 (2003) 149–157152

The weight/volume ratio was found to decrease rapidly in the case of irradiated Arabicasamples from an initial 0.6 to 0.3 in a span of two weeks, whereas in the non-irradiated samples itdecreased from 0.6 to 0.4 only at the end of four weeks (Table 4). For Robusta samples however,there was no difference between non-irradiated and irradiated samples. The number of seedsoccupying a fixed volume decreased considerably indicating the swelling of beans. The number ofirradiated Arabica seeds decreased from 421 to 223/100ml indicating that the beans had swollento double their size. Swelling in Robusta was less than in Arabica. The non-irradiated samplestook five weeks to absorb the required level of moisture and swell after which they weretransferred to gunny bags, whereas the irradiated samples swelled within one week and weretransferred to gunny bags.The irradiated samples of both varieties before monsooning irradiated at both 5 kGy and

10 kGy showed two species belonging to a single genus, i.e Aspergillus. However, the non-irradiated, non-monsooned zero day samples showed 5 species of fungi belonging to 2 genera inArabica while Robusta showed 6 species belonging to 2 genera. Aspergillus niger was the

Table 3

Physical and mycofloral changes in irradiated coffee kept for monsooning

Variety Arabica Robusta

Weeks of monsooning 0 1 2 3 4 5 0 1 2 3 4 5

Control

Moisture

content (%)

12.2 18.2 17.1 14.3 15.7 16.0 12.8 18.4 17.3 17.0 14.6 15.8

Wt/vol. (g/ml) 0.62 0.48 0.45 0.45 0.45 0.43 0.60 0.49 0.46 0.44 0.46 0.47

No. of

seeds/100ml

416 345 308 287 264 281 420 361 319 300 308 322

Fungal count

(c.f.u./g)

108 250 338 255 345 286 112 189 211 249 246 212

IrradiatedF5 kGy

Moisture

content (%)

11.6 21.5 21.0 19.6 20.2 17.4 12.4 19.2 18.9 16.3 17.1 16.8

Wt/vol. (g/ml) 0.61 0.47 0.34 0.33 0.34 0.34 0.63 0.47 0.48 0.46 0.46 0.46

No. of

seeds/100ml

421 286 239 234 248 222 434 333 333 321 312 321

Fungal count

(c.f.u./g)

8 81 112 139 152 164 2 62 105 129 112 86

IrradiatedF10 kGy

Moisture

content (%)

12.0 22.7 20.8 18.6 20.9 18.2 12.9 19.3 19.2 18.2 19.2 18.3

Wt/vol. (g/ml) 0.62 0.46 0.38 0.33 0.32 0.33 0.62 0.48 0.47 0.44 0.45 0.47

No. of

seeds/100ml

424 289 241 220 242 212 424 343 325 314 322 330

Fungal count

(c.f.u./g)

2 73 127 216 236 219 0 75 74 116 122 113

R. Ahmad et al. / Journal of Stored Products Research 39 (2003) 149–157 153

dominant fungus before monsooning with 100% occurrence in non-irradiated samples and o4%occurrence in irradiated non-monsooned samples. During monsooning, in the irradiated beans,the dominant fungus was A. ochraceusWilhelm with occurrence up to 100% and to a considerableextent, A. niger in both Arabica and Robusta samples. Fungi such as A. candidus Link, A. wentiiWehmer, Penicillium rugulosum Thom and Penicillium sp. were found to occur in both the non-irradiated as well as irradiated samples undergoing the monsooning process. Aspergillus tamariiKita, Absidia sp., Syncephalastrum sp., Rhizopus sp., and Fusarium sp. were present duringmonsooning in the non-irradiated samples but were completely absent in the irradiatedmonsooned samples although 2% in the Robusta sample irradiated at 10 kGy dose containedAbsidia sp. The percentage occurrence of Aspergillus wentii andMucor sp. was low. Fungi such asPhialomyces sp., Acremonium strictumW. Gams, Aureobasidium sp., Penicillium spinulosum Thomand P. chermesinum Biourge were found during the monsooning process of irradiated samplesonly. Penicillium spinulosum was common but Acremonium strictum and Aureobasidium sp.

Table 4

Mycofloral changes (percentage infection) during monsooning of irradiated Arabica and Robusta coffee beans

Arabica Control 0 100 6 1 4 26 0 0 0 5

3 72 86 44 0 18 0 12 0 5

5 52 100 76 0 22 0 8 12 6

5 kGy 0 4 2 0 0 0 0 0 0 2

3 0 100 0 0 20 100 0 60 4

5 0 100 0 52 18 80 12 16 6

10 kGy 0 1 2 0 0 0 0 0 0 2

3 0 100 0 0 0 100 0 60 3

5 0 98 0 100 0 64 12 36 5

Robusta Control 0 100 4 1 10 20 0 16 0 6

3 70 92 56 0 16 0 16 0 5

5 62 100 48 0 24 0 20 8 6

5 kGy 0 2 4 0 0 0 0 0 0 2

3 0 100 0 0 18 72 0 12 4

5 0 100 0 60 18 42 0 16 5

10 kGy 0 1 1 0 0 0 0 0 0 2

3 0 100 0 0 56 56 0 16 4

5 0 100 0 20 42 32 0 20 5

aAbsidia sp., Acremonium strictum, Aspergillus wentii, Aureobasidium sp., Fusarium sp., Mucor sp., Penicillium.

chermesinum, Phialomyces sp., Rhizopus sp. and Syncephalastrum sp. occurred at low numbers.

Variety

Treatm

ents

Weeks

Aspergillusniger

A.ochraceus

A.tamarii

A.candidus

Penicilliumrugulosum

Penicilliumsp.

Penicilliumspinulosum.

Syncephalissp.

Totalno.ofspeciesa

R. Ahmad et al. / Journal of Stored Products Research 39 (2003) 149–157154

occurred in low numbers. Penicillium chermesinum and A. strictum were found inthe irradiated Arabica samples only. The incubated beans were fully colonised by A. ochraceus.The irradiated monsooned beans showed the presence of Syncephalis sp., A. candidus andPenicillium sp. in the second week while the non-irradiated beans, however, were clean inappearance and showed the presence of Syncephalis sp. and Penicillium sp. only towards thefourth and fifth week.The species of Fusarium, which are field fungi, were isolated from the freshly hulled as well as

stored non-irradiated samples but were soon replaced by storage fungi such as species ofAspergillus and Penicillium. These observations are in line with that of Emayavaramban andRamabadran (1985).

4. Discussion

Earlier studies on irradiation of coffee have employed a dosage up to 1 kGy and at this doseno appreciable change in flavour and taste of coffee was observed (Dias et al., 1978). It hasbeen reported that a radiation dose of 12 kGy is necessary to eliminate all filamentous fungi andyeasts (Cuero et al., 1986; Ramakrishna et al., 1991). In the present study, however, beanswere irradiated at high doses of 5 and 10 kGy mainly to eliminate the mycoflora present on thebeans in order to establish the role of fungi in the monsooning process. From the study, it isevident that the 5 kGy dose is effective for the elimination of fungi but not bacteria, as bacteriawere found in both varieties of coffee irradiated at this dose, but at much lower levels than in thenon-irradiated samples (Table 2). However, A. niger was found even in samples irradiated at10 kGy dose which suggests that it may be more resistant to irradiation than other microbes(Anonymous, 1999). The results reported here show that a dose of 10 kGy gamma rays may beuseful for achieving faster monsooning as this took place in one week compared with five weeksfor non-irradiated beans. At this stage it may be inferred that the fungi may not have a role in themonsooning process as there is no initial inoculum and there are less fungi associated with gammairradiation-induced monsooning. Moreover, the chances of production of undesirable secondarymetabolites is greatly reduced. As monsooning is considered an aging process, it is possible thatgamma irradiation brings about quick aging of the bean thereby shortening the time taken formonsooning. The role of gamma irradiation in inducing aging has been reported by Sur and Basu(1985, 1993) and Bagchi (1974) wherein the by-products of aging and gamma irradiation werefound to be similar.Aspergillus ochraceus is responsible for the production of ochratoxin A (OTA)

causing nephrotoxicity in humans (Bhat and Vasanthi, 1999) and the natural occurrenceof OTA in coffee was first reported by Levi et al. (1974). OTA is not destroyed during roastingand brewing and is fully extracted into the coffee brew. A. ochraceus grows optimally at 25–301Cover the water activity range of 0.995–0.85. However, ochratoxin production onagricultural substrate is restricted to > 0:88aw at optimum temperature (Magan andLacey, 1989). Such studies have not been carried out on coffee. Because of highermoisture content and the occurrence of A. ochraceus in monsooned coffee, work is nowbeing initiated to examine the relationship between gamma irradiation, monsooning andproduction of OTA.

R. Ahmad et al. / Journal of Stored Products Research 39 (2003) 149–157 155

Acknowledgements

The authors thank the Department of Biotechnology, Government of India and the Council ofScientific and Industrial Research for financial assistance.

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