Grading of fermented and dried cocoa beans using fungal contamination, ergosterol index and ochratoxin a production.

Sixty four samples of cocoa beans replicated in quadruplicates were collected from five warehouses from southwest Nigeria and examined for fungal loads, ergosterol and ochratoxin A The levels of all the variables obtained were further used as indices for cocoa grading into food quality, FoQ (erg < 5 mg/kg; OTA < 1 µg/kg), feed quality, FeQ (erg = 5~10 mg/kg; OTA in the range of 1.1~3.11 µg/kg), Screen for mycotoxin, SFM (erg = 10~20 mg/kg; OTA from 3.12 µg/kg and above) with fuel quality, FuQ having erg > 20 mg/kg and OTA > 6.12 µg/kg. Using these ergosterol indices, 18.75% of the cocoa beans examined was classified with the FoQ, 18.75% with the FuQ while 31.25% was classified with both the FeQ and the SFM, respectively. In conclusion, ergosterol can be used as a rapid index to grade fermented, dried cocoa beans meant for export.

Molds are common contaminants of agricultural commodities used for foods and feeds. Fungal development on the alimentary substrates can lead to different detrimental effects, alteration of technological properties, decrease of nutritive value and synthesis of mycotoxins (Pitt and Hocking, 1985). Evaluation of mold development is of interest to estimate global quality of raw materials and may be useful to take decision on their potential use. Ergosterol is considered as the principal sterol of fungi and it plays an important role as cell membrane component. Therefore, it has been proposed as a global indicator of mycological quality of foods and feeds (Bailly et al., 1999; Cahagnier, 1998; Schnurrer, 1993; Schwardorfk and Muller, 1980; Seitz et al., 1977, 1979). Ergosterol levels are commonly used as quality parameters in ecological (Sashdhar et al., 1989), industrial, (Hippelein and Rugermer, 2004), and agronomic environments (Kadakal and Artik, 2004; Sashdhar et al., 1989). Moreover, significant correlations were found between ergosterol and the major mycotoxins notably Fumonisin B1, Zearalenone, Deoxynivalenol, ochratoxin A, and patulin. Significant relationship has also been found between fungal development and ergosterol production in maize (Peitri et al., 2004), rice (Saxena et al., 2001), tomato (Kadakal and Ekincir, 2005) and wheat (Abramson et al., 2005). Therefore, ergosterol determination can be considered as a good index of fungal development on cereals and could be an early indicator of potential mycotoxin production. Its determination can be used in industry to screen productions, prior to mycotoxin analysis. On cereals, according to (Cahagnier, 1998), 3 µg of ergosterol per gram is considered as the maximum acceptable level for maize while for wheat, 8 µg of ergosterol per gram is the retained value for certifying correct quality of the grains. On the other hand, when the amounts of ergosterol are upper than 8 µg/g on maize and 12 µg/g on wheat, a quality of grains is suspected (Cahagnier, 1998).

Cocoa beans, a produce of commerce of Theobroma cacao is a principal raw material for the chocolate industry. Cocoa of commercial grade should conform to some criteria among which the absence of molds and mycotoxin production is one (Aroyeun et al., 2007). Grading of cocoa beans into acidity, slatiness, mouldiness are among the most prominent quality parameters for grading cocoa beans. All these methods are time-consuming and laborious. Using ergosterol index as to measure the possibility of mycotxin production in cocoa beans has not been reported. Since the method of ergosterol determination can be a faster and relatively precise method, this study was designed with the aim of grading contaminated cocoa beans using fungal determinations, erogsterol index and ochratoxin A formation.

Materials and Methods

Raw materials

Sixty four samples of cocoa beans replicated in quadruplicates were collected from five warehouses from southwest Nigeria.

Determination of fungal counts

Surface sterilized cocoa beans were plated on malt extract agar after serial dilution. Incubation was at 28 ± 2℃ for seven days. Thereafter, colonies growing on the plates were counted using a colony counter.

Extraction of ochratoxin A and quantification. After 7 days, isolates were grown in yeast extract sucrose broth containing (2% yeast extract, 15% sucrose). Incubation was done at 28 ± 2℃ for 7 days. The culture broths were later extracted using the method of (Varga et al., 2002).

Quantification of ochratoxin A by high performance liquid chromatography (HPLC)

This was done in accordance with (Teren et al., 1996).

Determination of ergosterol content in cocoa beans

Extraction, quantification of ergosterol was done using HPLC apparatus in accordance with (Lamper et al., 2000).

Results

In Table 1, the higher the fungal counts and the ergosterol values are, in most cases the higher the ochratoxin A values are. This finding was in agreement with previous reports on the direct relationship between ergosterol, fungal counts and mycotoxin production (Lamper et al., 1999; Varga et al., 2002; Czaczyk et al., 2002). (Reid et al., 1999) observed an interaction between Fusarium graminearum and Fusarium moniliforme and disease progress, fungal biomass, mycotoxin accumulation and ergosterol formation. (Ng et al., 2008) estimated fungal growth using ergosterol assay as a rapid tool in assessing the microbiological status of grains and feeds. Ergosterol has been determined in grains with different levels of fungal contamination by (Maria et al., 2001). Samples CB1 with fungi counts of 62.10 × 102 cfu/g had 6.33 µg/kg of ochratoxin A and 24.23 mg/kg of ergosterol. High fungal counts of 33.02 × 102 cfu/g in CB4 and ergosterol content of 20.41 mg/kg corresponded with the OTA observed (3.12 µg/kg). Sample CB6 with fungal counts of 30 × 102 cfu/g, had a corresponding ergosterol content of 17.21 mg/kg and a low content of OTA (1.86 µg/kg). In this case, there is a possibility that OTA producing fungi might be present in the cocoa samples but maybe the OTA production capacity of the fungi was low. Samples CB1, CB6, CB8 had high fungal counts, high ergosterol and high OTA values. In sample C14, fungal counts, ergosterol and OTA did not correlate. The high OTA production, which does not correlate with either ergosterol or OTA, might indicate presence of high OTA producing fungi even though it might be present in low counts. Samples CB12, and CB13 had low fungal counts; low ergosterol and low OTA in agreement with the hypothesis of (Schnurrer, 1995) on the direct relationship of fungal counts, ergosterol and deoxynivalenol. CB10 had fungal counts of 11.10 × 102 cfu/g, ergosterol of 10.47 mg/kg but a correspondingly lower OTA of 0.09 µg/kg indicating the presence of OTA producing fungi with low production capacity. Samples CB15, CB16 fell in the category where the ergosterol, fungal counts and OTA have a direct relationship.

Table 1

Fungal contamination, Ergosterol and OTA of cocoa bean samples

*Values recorded were averages of separate experiments; ±-standard deviations; FOQ- Food Quality; FUQ- Fuel Quality; SFM- Screen for Mycotoxin- FEQ- Feed Quality

The results obtained in this study supported the usefulness of the quality grading system described by (Schnurrer, 1995). Based upon ergosterol and OTA content of the samples, only 18.25% of the samples reached FOQ grade (< 5 mg/kg ergosterol), OTA of this class is < 1 µg/kg and 18.25% fell in the FEQ grade with the ergosterol of 50~10 mg/kg and OTA > 1 µg/kg. 31.25% fell in the category of Screen for mycotoxin (SFM) grade having ergosterol of 10~20 mg/kg and OTA in the range of 1.68~2.31 µg/kg while 31.25% also fell in the last grade Fuel quality grade (FuQ) with ergosterol > 20 mg/kg and OTA from 3.12 µg/kg and above. Based on the grading system, the cocoa beans obtained form the warehouses in the food quality grade (FOQ) included CB10, CB12, CB13. Samples in the feed quality grade (FeQ) were CB5, CB7, CB9, CB11 and CB14. Those for screen for mycotoxin (SFM) included CB2, CB3, CB6, CB15 and CB16. Those in the fuel quality grade (FuQ) category were CB1 and CB4 respectively (Table 2).

Table 2

Cocoa Bean grading based on ergosterol values

In conclusion, the use of ergosterol can be used as an indicator of good cocoa bean quality and to predict the possibility of ochratoxin A formation by mycotoxigenic fungi in cocoa beans. In conclusion, this index can be relevant as a rapid test for screening cocoa beans samples meant for export.