Composting coffee wastes, a potential for ochratoxigenic fungi and ochratoxin A contamination

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World Mycotoxin Journal, November 2012; 5 (4): 373-376 Wageningen Academic P u b l i s h e r s

ISSN 1875-0710 print, ISSN 1875-0796 online, DOI 10.3920/WMJ2012.1386 373

1. Introduction

Ochratoxin A (OTA) is a nephrotoxic mycotoxin that also possesses carcinogenic, teratogenic, genotoxic, and immune-suppressive potential (Bhat et al., 2010). OTA has been classified as possibly carcinogenic to humans (Group 2B) by the International Agency for Research on Cancer (IARC, 1993). In view of its toxigenic nature, the European Commission has set maximum levels for OTA in various food products such as cereals, dried vine fruits, wine, coffee, and infant foods (EC, 2006). Occurrence of OTA in soil for growing coffee, green coffee beans and at various stages of coffee processing has been reported from various locations in the world (Batista et al., 2009; Ilic et al., 2007; Perrone et al., 2004; Taniwaki et al., 2003; Urbano et al., 2001; Velmourougane et al., 2010a,b, 2011a,b).

In India, nearly 80% of robusta and 20% of arabica coffee varieties are processed by a dry method, accounting for

approximately eighty thousand metric tonnes per year. As the husk produced during coffee processing is difficult to decompose, a major portion is used as fuel for tobacco curing or dumped as waste on farms. However, considering the current awareness about sustainable crop production and organic farming (Bernal et al., 2009; Hargreaves et al., 2008), coffee farmers increasingly use composting of husk for integrated nutrient management (Muralidhara et al., 2006). Composting is an effective way of recycling coffee processing wastes that are otherwise dumped leading to increased environmental pollution.

It has been demonstrated that the major OTA producing fungus Aspergillus ochraceus grows faster and produces more OTA in the husk than in the beans (Gopinandhan et al., 2006; Van der Stegen, 2004; Velmourougane et al., 2008a). Additionally, the presence of increased levels of A. ochraceus near the husk disposal in coffee curing works in India has been reported (Velmourougane et al., 2008a). Even

Composting coffee wastes, a potential source of ochratoxigenic fungi and ochratoxin A contamination

K. Velmourougane1,2, R. Bhat3 and T.N. Gopinandhan4

1Post Harvest Technology Lab, Coffee Research Sub Station, Coffee Board, Chettalli 571248, Coorg District, Karnataka, India ; 2Crop Production Division, Central Institute for Cotton Research, Indian Council of Agricultural Research, PB. No-2 Shankar nagar post, Nagpur 440010, Maharashtra, India; 3Food Technology Division, School of Industrial Technology, Universiti Sains, Malaysia, Penang 11800, Malaysia; 4Coffee Research Sub Station, Coffee Board, Chettalli-571 248, Coorg District, Karnataka, India; [email protected]

Received: 4 January 2012 / Accepted: 1 May 2012 © 2012 Wageningen Academic Publishers

Abstract

Ochratoxin A (OTA) has been extensively documented as a global contaminant of a wide variety of food commodities including the green coffee bean, but there is no clear information available on the spread of Aspergillus ochraceus in the coffee production chain. In this study, the growth of A. ochraceus and the fate of OTA during composting of solid coffee wastes, i.e. pulp and husk, were investigated. A trial was set up with pulp and husk alone or in combination, naturally and artificially contaminated with A. ochraceus. OTA was detected at levels up to 2.6 and 6.2 ng/g in naturally and artificially contaminated pulp, respectively. At the end of the composting process, 8.4 and 14.2 ng OTA per g were measured in naturally and artificially contaminated husk, respectively. Throughout the composting process, A. ochraceus counts did not show any clear increasing or decreasing trend.

Keywords: coffee processing wastes, composting, Aspergillus ochraceus, ochratoxin A

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374 World Mycotoxin Journal 5 (4)

though studies have been conducted to assess the possible sources of OTA in coffee beans, no systematic study has been carried to assess the extent of OTA contamination in compost prepared using coffee processing wastes. In the present study, we investigated the growth of the ochratoxigenic fungus A. ochraceus) and the extent of OTA contamination during composting of solid coffee wastes, i.e. pulp and husk. It was envisaged that the results would be useful for the implementation of good agricultural and good manufacturing practices along with a HACCP approach in the coffee growing regions of the world. To our knowledge, this is the first systematic study on these aspects in India.

2. Material and methods

Composting process

Trials were conducted at the Coffee Research Sub-Station (North-Coorg, Karnataka, India) to study the survival of A. ochraceus as well as the fate of ochratoxin A in naturally and artificially inoculated compost of coffee solid wastes. To this end, pulp and husk and a mixture of both at one tonne/heap (in triplicate) were used as basic composting material along with weed biomass and other green materials available at the on-farm level. The compost heaps were turned once a month and analysed for changes in A. ochraceus counts and OTA concentration. In addition, composting trials were conducted with compost heaps artificially inoculated with broth cultures of a one-week old A. ochraceus spore suspension at approximately 107 spores/ml per 100 kg of substrate. The strain of A. ochraceus used was a native virulent strain previously isolated from coffee beans during the ‘Mould Survey Programme’ in India under the ‘Global Mould Project’ and tested for in vitro OTA production on sterilised coffee beans as well as by the agar plug method.

Enumeration of Aspergillus ochraceus

Samples for mycological enumeration were collected every 30 days during the turning process of compost. Three samples were randomly taken from a single heap and pooled together to form a representative sample. 0.5% peptone water was used as diluent (Anonymous, 2006). A. ochraceus was enumerated using selective DG-18 medium (Hocking and Pitt, 1980). The plates were incubated in triplicate at 30±1 °C. A. ochraceus colonies appearing after 5-7 days of incubation were counted and expressed as colony forming units (cfu)/g sample, and identified according to Klich and Pitt (1988).

Ochratoxin A analysis

OTA analysis was performed in compost samples collected during monthly intervals until the end of composting (120 days). Extraction and purification of OTA in compost samples were carried out following the procedure described

by Gopinandhan et al. (2008). In brief, OTA was extracted from 25 g finely milled compost sample with 500 ml of methanol:3% aqueous sodium hydrogen carbonate (1:1, v/v) in a 500 ml glass blender jar using a blender at high speed for 3 min. A 50 ml aliquot of sample extract was centrifuged at 5,000 rpm for 10 min and supernatant was filtered through GF/B microfibre filter (Whatmann, Maidstone, UK) under vacuum. Four ml of filtrate was transferred to an amber coloured, graduated cylinder and diluted to 100 ml with phosphate buffered saline. The whole diluted extract was applied to an immunoaffinity column (OchraTest; Vicam, Milford, MA, USA) at a flow rate of 2-3 ml/min. After washing the column with 10 ml of Milli-Q water (Millipore, Billerica, MA, USA), OTA was eluted with 4 ml of HPLC grade methanol. To ensure the complete removal of bound toxin, methanol was left in the column for at least 3 min by reversing the flow of methanol (back flushing) 2-3 times. The eluate was then evaporated to dryness under a stream of nitrogen at 40 °C and the residue was redissolved in 500 μl of HPLC mobile phase.

OTA was quantified by reverse-phase HPLC with fluorescence detection (Shimadzu-LC 10A Series; Shimadzu, Kyoto, Japan). HPLC analysis was performed using 45% acetonitrile/55% 4mM sodium acetate:acetic acid (19:1, v/v) on a Spherisorb ODS II 5 micron 250×4.6 mm i.d. (Waters, Milford, MA, USA) connected to a precolumn ODS Hypersil 5 micron 25×4.6 mm i.d. (Thermo Scientific, Waltham, MA, USA). Separation was performed at ambient temperature at a flow rate of 1 ml/min. Excitation and emission maximum were 330 and 470 nm, respectively. The calibration curve was obtained using the linear squares regression procedure of the peak area vs. the concentration. The linearity of the OTA (Sigma-Aldrich Chemical Private Limited, Bangalore, India) standard curve at concentration of 0.1, 1, 5, 10, 20 and 30 ng/g was good, as shown by a correlation coefficient (r2) value of 0.9913.

Recovery tests were performed in triplicate by spiking OTA-free samples with OTA standard solution at 5 and 10 ng/g, followed by the same procedure as described above. The average recovery was 89 and 82%, respectively. The limit of detection based on a signal-to-noise ratio of three was 0.1 ng/g.

3. Results and discussion

At the start of the composting process, more colony forming units of A. ochraceus were present in husk than in pulp of naturally contaminated compost (Table 1). This may reflect differences in moisture content and water activity between husk and pulp (Panneerselvam et al., 2000). Moreover, pulp has been reported to contain higher amounts of polyphenols and tannins, conferring resistance to microbial invasion of coffee fruits (Velmourougane et al., 2008b). Throughout the composting process, A. ochraceus counts did not show

Composting coffee processing wastes and ochratoxin A contamination

World Mycotoxin Journal 5 (4) 375

any clear increasing or decreasing trend either in pulp or husk, alone or in combination.

Data on OTA concentrations during composting of coffee waste are presented in Table 2. OTA contamination appeared to be a natural phenomenon in both pulp and husk. OTA was observed to increase steadily with the progress of the composting process of pulp and husk, alone or in combination, in both naturally and artificially contaminated compost heaps. The increase appeared to be greater for husk than for pulp. As A. ochraceus did not show any visible increasing or decreasing trend during the composting process, the rise in OTA levels might be due to the presence of other OTA-producing Aspergillus spp. such as section Nigri (Astoreca et al., 2010a,b; Esteban et al., 2006).

In conclusion, the present study indicates that processing wastes could be a potential source of toxigenic fungi and

OTA contamination during coffee production. Awareness should be created among coffee farmers regarding the risks of composting these materials without any adequate care. The simple practice of maintaining a minimum distance between the drying yard and composting area may prevent the spread of A. ochraceus spores and other mycotoxin-producing fungi to coffee.

Acknowledgments

The present work was carried out under the ICO-CFC-FAO Global Mould Project GCP/INT/743/CFC Enhancement of Coffee Quality through Prevention of Mould Formation at the Coffee Research Substation Chettalli (Coffee Board, India). The authors gratefully thank the director of research (Coffee Board, India) and Dr. Y. Raghuramulu, coffee scientist (ICO-CFC-FAO Project), Coffee Board and Ministry of Commerce (government of India) for their keen interest and support during this study.

Table 1. Aspergillus ochraceus counts (103 cfu/g) during composting of coffee processing wastes.

Days after composting

0 30 60 90 120

Coffee waste A1 B A B A B A B

Pulp 0 1.0 4.7 0.7 2.8 0.5 2.8 0.8 3.3Husk 2.4 2.3 2.3 2.5 6.1 0.6 3.1 1.2 3.6Pulp + husk ND2 2.5 4.6 1.8 5.2 1.5 4.3 2.2 4.7

1 A = naturally contaminated compost; B = compost artificially contaminated with A. ochraceus. All values are the mean of three replicates.2 ND = not determined.

Table 2. Fate of ochratoxin A (ng/g) during composting of coffee processing wastes.

Days after composting

0 30 60 90 120

Coffee wastes A1 B A B A B A B

Pulp 0.37 0.94 2.46 1.23 3.50 2.13 4.10 2.60 6.20Husk 1.66 3.51 5.00 5.26 7.35 6.24 9.26 8.36 14.24Pulp + Husk ND2 1.58 3.00 4.20 4.28 5.50 6.90 6.75 8.45

1 A = naturally contaminated compost; B = compost artificially contaminated with Aspergillus ochraceus. All values are the mean of three replicates.2 ND = not determined.

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376 World Mycotoxin Journal 5 (4)

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