RELATIONSHIP OF FERMENTATION TIME TO THE CUT TEST AND ANTHOCYANIN CONTENT IN COCOA (THEOBROMA CACAO)

Cocoa collected from Wariyapola Estate, Matale,was fermented by the basket method. It was found that the;"cut test" can be applied to determine fermentation times of rpto two days provided the batch i~ a mixture of the criollo and forestero varieties. Although anthocyanin content declinea by nearly 70% within six days fermentation this parame~er cannot be generally used to determine fermentation time.


Introduction
Feirnentation, vrhich is an important step in the processing of cocoa in Sri Lanka has received insufficient attentior in the past and the process is largely uncontrolled.The duration of fermentation is a crucial aspect of primary processing of raw cocoa as it results in the formation of precursors of essential aroma compounds while bringing about a decline of astringency and bitterness.Therefore it ;s essential that cocoa beans are fermented for an optimum period in order to produce an acceptable flavour.
Fermentation trials on Sri Lankan cocoa have been reported by 0gutuga4 who monitored pH changes and temperature variation of the fermenting cocoa.However, this study does not report 2 specific period for fermentation and highlighted the tray method of fermentation for mixed varieties of beans.
The present study %was conducted on cocoa beans obtained from .Wariyapola Estate, Matale, 3ri i a n k a , where there is a mixed variety of forestero and cr,ollo beans.While it is known criollo beans generally require 2-3 days of fermentation1o and forestero beans 5-7 days, optimum fermentation time would also depend on the method of fermentation adopted.
Of the several methods of fermentation4 available the' basket method was used during this study as the target group of processors were the smallilolders.It is alsc noted that at the present time most smallholders limit fermentation time t o 1-1% days.
Raw material from the estate selected was subject to a basic study on selected fermentation parameters after which an attempt was made t o correlate fermentation and storage time with the cut test and anthocyanin content with a view t o aidlng in the grading of cocoa.This study is considered important as the present practice'of grading cocoa on external appearance is considered urjdesirable.

Plant Material
Mature pods of Theobroma cacao were collected from different divisions of Wariyapola Estate, Matale, Sri Lanka, pooled and a representive sample drawn.These pods were a mixture of the criollo and forester0 varieties in the ratio of approximately 7: 3.

2;2.1 Method.
The basket fermentation technique4 was employed.Total fermentation time was 6 days and samples were drawn daily.The beans were turned after 48 h fermentation.Fermented cocoa beans thus produced were sun-dried for 3 days after spreading on coir matting.

.Routine Measurements ' .
The following parameters were measured throughout fermentation:-(i).pH of water extract of beans and pulp6

Cut Test
The cut test was performed by cutting the cocoa beans (of varying fermentation and storage time) lengthwise and counting the number of lilac, cinnamon-brown, purple, purple-brown and brown beans which are easily distinguishable to the naked eye (modified from method of wood1 O).Approximately 1 kg. of cocoa beans was used for each experimental point.

Assay of Anthocyanin
Anthocyanins were extracted and assayed by two methodsthat of Petti-pher5 and Lees and F'rancis2 where absorbance is measured and converted to anthocyanin content by formulae given in the respective methods.

Method of Pettipher
Cocoa beans (100g) were deshelled and blended in 1% v/v HC1 (1 litre) using a Waring blender.The suspension was allowed to stand for 5 min.An aliquot (10 ml) was centrifuged for 1 0 min.
A 6 e p Pak C18 cartridge (Waters Associates, London) was prepared for extraction by passing methanol (2 ml) followed by (1% v/v) HC1 (5 ml) through the cartridge using a plastic syringe.
The supernatant (2 ml) of the acid extract was passed through the cartridge and the eluent discarded.The cartridge was then rinsed by passing through 1% v/v HCl(4 ml) and the eluent discarded.Air was passed through the cartridge to expel any remaining liquid.
The anthocyanins were eluted from the cartridge by passing through 50% (vlv) methanol (4.5 ml), followed by air, collecting the total eluent.The absorbance of the eluent was measured at 520 nm using a Perkin Elmer model 552 spectrophotometer.

Preliminary Results
As expected the pH of cocoa bean pulp increased (from 3.2) and finally stabilized a t about 4.5 and that of the bean decreased from pH 6.0 to 4.7 in 5-6 days.A final acetic acid content of 0.7% w/v was attained in the bean after 6 days of fermentation.A maximum temperature (41 -42OC) of the fermenting mass was attained around 4-5 days after fermentation beganthis was followed by a decline in temperature t o 38OC on the sixth day.
Total sugar content declined by about 15% during 6 days fermentation t o 1.7% w/w.While initially approximately 50% af the sugar was non-,reducing a t the end of 6 days fermentation approximately 95% of the sugar was reducing in nature.Cocoa fat content remained relatively steady between 40 and 44% w/w upto six days fermentation.criollo bean to turn from lilac to cinnamon-brown and the forester0 bean from purple to brown with fermentation.

Anthocyanin Content
Anthocyanin levels paralleled the results of the cut test (Table 2).

Discussion
The study defined the basic fermentation characteristics or mixed Sri Lankan criollo and forestero cocoa when fermented by the basket method.
Although the acidity of the fermented cocoa bean is low it is not as low as that reported by Liau in ~a l a ~s i a .~ The study revealed that fermentation time can be determined by the cut test soon after fermentation provided the plantation is a mixed forestero and criollo.The more comljlicated anthocyanin test is of less use especially as initial anthocyanin content may not be known.

I
Storage of beans results in a complex situation but still using the criolro beans one can distinguish between 0,l .and 2 days fermentation.The cut test is of less use at 3 or 4 days fermentation as for example, 3 day fermented beans after three months are difficult t o distinguish from a more fermented bean stored for a lesser time.
The anthocyanin test is .ofstill less use as it does not have the advantage " ' of ,evaluating the criollo and forestero, beans separately.Further it does not monitor the development of the brown pigment: As cocoa beans in Sri Lanka are almost never fermented beyond 2 days, the cut test has considerable value provided there is a significant proportion of criollo beans in .theparticular batch of cocoa.

5 -
Relationship o f Fermentation Time to the Cut Test

Table 1 .
The cut test with varying fermentation time

Table 4 .
Assay of cocoa bean duringsthree months of storage for anthocy-Absorbance was measured by the Method of Pettipher 5 100% anthocyanins represene approximately 19.5 p moles anthocyanins per 100 g cocoa