THE EFFECT OF THE MATURATION PROCESS ON FERMENTED COCOA BEANS 11 : ACIDS , SUGARS , XANTHINES , PYRAZIMES AND ANTHOCYANINS

Absmact: This study describes the effect of the maturation process on selected flavour contributing components in the cocoa bean. I~Ton-volatile acids, sugars, xanthines, pyrazines and anthocyanins were studied. The study showed that holding fermented cocoa beans at ambient temperatures with some aeration for 48 hours (the maturation process) had an effect on the levels of flavours znd flavour precursors of cocoa. Although p H rises from 4.8 to 5.2 during maturation the content of the main non-volatile carboxylic acids (citric and oxalic acid) did not show any significant change. Maturation causes a loss of sucrose and an increase in reducing sugas:s (glucose, fructose and galactose). Xanthine content declines. Theobromine losses during maturation is approximately 30% and it is possible that part oE this loss is due t o migration from the cotyledon to the shell. While pyrazine content increased the profile of pyrazines formed during roasting appears unaffected by the maturation proceSs. Anthocyanin content declined by 40-45% during maturation.


Introduction
.Maturation is the teria used by 1,iau6 to describe the process of holding of fermented cocoa beans in thin Payers (for aeration) at ambient temperatures with little moisture loss for about two days.This was introduced primarily t o reduce acidity of the cocoa bean produced in Malaysia.

The studies of ~a c k i ~a s o t h ~~
showed that the maturation process in- creased the volatile carbonyl content changed its composition.The authors' previous studies2 sl~owed that there was an increase in volatile carbonyls and a decline in amino acid content and further that there appeared to be a relationship between the two processes.I t was therefore clear that the maturation process had some effect on flavours.
In this study the changes in acids, sugars, xanthines, pyrazines and anthocyanins in cocoa with the maturation process are reported.

Sampling, Fermentation and Maturation
Sampling, fermentation (basket method) and maturation were carried out as described previou~ly,~ samples being drawn during both the major and minor seasons from Wariyapola Estate, Matale.The samples contained, a mixed forester0 and criollo variety in the ratio of approximately 4: 1.The samples from major (September t o December) and minor (April to June) seasons could be taken as indicative of two possible extremes of sampling conditions.

Preparation of Sample for Analysis
Cocoa beans with the appropriate treatment were dried in the sun for 3-4 days to a moisture content of 10-15%.Analysis of acids and sugars were carried out on the entire bean (with shell) while analyses of xanthines and anthocyanins were carried out on deshelled cocoa bean.Analysis for pyrazines was carried out after roasting ( 1 4 0 ' ~ for 30 min.) and deshelling.Deshelling was carried out in certain instances as in these cases it is the procedure adopted by other workers -for anthocyanins presumably t o reduce interference.In the case of xanthines the procedure adopted has added significance since cocoa shell theobromine has also been measured.Pyrazines are measured after roasting as these components develop mainly after roasting.

~o n -k ~a t i l e acids
Non--volatile acids were extracted by the Paper Chromatographic method of packiyasothy7 and chromatographed on paper using n-butanol : formic acid : water (15 : 3 : 1) and aniline and furfural as the spraying reagent.Quantification was carried out by the method described by packiyasothy7 ping standards on the same paper.The coefficient of variation for the determina- . tion of citric and oxalic acid were 16.6 and 15.8 respectively.

Sugars
Sugars were separated by the method of Lato et U Z .~ and sprayed3 after chromatography.Quantification was carried out by TLC-UV densitometry (Camag model 76502 equipped with a strip chart recorder model 1107) using a Tungsten lamp at 436 nm (fructose) and 525 nm (glucose, galactose and sucrose) using techniques described previously.2Standard curves for glucose, fructose, sucrose and galactose gave gradients of 17.6, 20.0, 17.5 and 22.5 mm2 peak area per yg respectively and the coefficients of variations for the determination were 6.9, 3.0, 6.2 and 11.0 respectively.Standard curves were only used to confirm linearity of plots and standards on the same plates were employed for purposes of quantification.

Xanthines
Xanthiries were extracted by the method of ~enana$ake and ~ijesekera.' ' Xanthines .wereseparated by TLC as follows - A slurry of Kieselgel G2 (25g) in distilled water (50 ml) was applied on clean glass plates (20 x 20 cm) at a thickness of 5 0 0 ~.The plates were allowed t o stand for one hour at room.temperature and dried at 110'C for l h and determined by TLC-UV densitometry using a Deuterium lamp at 298 nm.Standard curves gave gradients of 11.5 and 21 mm2 peak area/pg theobromine and caffeine respectively.
Standard curves were used only t o ensure the peak area was proportional t o the theobromine and caffeine levels.As there was some doubt regarding consistency from plate to plate, standard spots were run along with the samples on the same plate for quantification purposes.The coefficient of variation for the technique was 7.2 and 3.7 for caffeine and theobromine respectively.

Pyrazines
Pyrazines were extracted by the method described by ~eineccius* and subject to gas liquid chromatographic analysis using.aVarian equipped with a Model 9176 strip chart recorder and a computing integrator.
The stainless steel column (length 3m diameter 3mm) used contained 10% carbowax 20m supported on chromosorb W (80 to 100 mesh).The GLC analysis was carried out using following conditions.Injector temperature, 210°C; detector temperature, 230°C; temperature programme, 60-120'C at 2Oc/min; He (carrier gas) flow rate, 25 mllmin; H2 (detector gas) flow rate, 25mlJmin; and 0, (detector gas) flow rate, 50 ml/min.Peak area normalization method was used for quantification.Peak identification was carried out by retention time data and peak enrichment only.

Anthocyanins
Anthocyanins were extracted.and determined by the method of Lees and Francis.

Acids
Whereas fermentation over from 4 t o 6 days caused a slight decline in pH (from 4.8 t o 4.7), the maturation process resulted in an increase of pH t o 5.2.However, nonvolatile acids which were mainly citric acid and oxalic acid showed no significant change (Table 1 ) (the differences were well within the standard errois of the estimation techniques).

Sugars
The reducing sugars identified were glucose, galactose and fructose.It was observed that reducing sugar content increased during the maturation proa cess (Table 1 ) .

Xanthines 3
There were two main xanthines in cocoa bean (deshel1ed)-theobromine and caffeine.Maturation caused a decline in xanthine content (Table 2) while theobromine content declined in the cotyledon,its levels in the shell of cocoa bean increased. .wazines content increased from 5.7 pg/g by -60% to 9.3 pglg on fermeni tation and roasting.Maturation process had no great effect on the profile.ofdifferent pyrazines after roasting (Table 3).Maturation had a marked effect on anthocyanin content (Table 4).

Discussion
The original concept of maturation process of Liau6 was to reduce acidity.This reduction of acidity also encountered with Sri Lankan cocoa where acidity rose from pH 4.8 to 5.2 with maturation.However, these studies reveal that non--volatile acids do not change significantly with maturation.This is not unexpected as Liau's6 original hypothesis was that increased aeration would cause loss of acetic acid by oxidation via the Krebs cycle.This is also borne out by the results of ~b e ~~u n a s e k e r a l which showed that the percentage acetic acid in total acid volatiles declined from 55 t o 45% while the percentage of the other major volatile acids (propionic and butyric) remain relatively unchanged.
" The ,results also showed' that although sucrose content declined the increase in reducing sugar far outweighed this decline.This indicated that reducing sugars were also being formed from other sources, possibly through the hydrolysis of other glycosides.The increase in reducing sugar content is significant as i t is one of the important flavour precursors which subsequently undergo reaction during roasting of cocoa.
The decline in xanthines appears to be due in part to the migration of theobromine from the cotyledons to the shell (although this may not have a " marked effect on cocoa flavour it could be significant in the process for extracting theobromine from cocoa shell waste).The migration itself is not surprising as during fermentation the cellular organization of the cocoa cotyledon is disrupted and permeability enhanced.Therefore, diffusion along a concentration gradient is plausible especially as theobromine is moderately soluble in acid medium.
The increase in pyrazines with maturation is expected as maturation causes an increase in reducing.sugarswhich reacts with nitrogenous compounds t o form pyrazinesl during the roasting prdcess.The pyrazine profile does not appear t o change much with maturation.More detailed studies on this aspect using reliable internal standard for quantification will need to be conducted if further conclusions are t o be drawn.
Anthocyanin content declines much more in maturation than iq extended fermentation.This is understandable as conditions in maturation are more aerobic.The reduction in anthocyanin content is of importance as it would result in a reduction in astringency and development ~f the cha+acteristic colour of chocolate.
The study indicates that maturation, in additioh to the well known effect in reducing acidity, can also play a part in flavour development by affecting the level of flavour precursors in cocoa bean.

Table 2 .
Effect of the maturation process on xanthines samplea Xanthine Content (%) Results expressed as a. percentage of cocoa bean weight a Major and minor season samples : data for minor season sample in parenthesis -Not detected Coefficient of variation of assay technique 3.7 and 7.2 for theobromine and caffeine respectively.

0
All samples have been roasted (140 C, 30 min.) and deshelled.Assay by Gas Liquid Chromatography -individual pyrazines results expressed s a percentage of total basic volatiles (Note : pyrazines have not been expressed on the basis of cocoa bean weight due to problems encountered with the internal stancixrd), a Major and minor season samples : data, for minor season sample in parenthesis 3.5 Anthocyanins

a
Anthocyanin content determined after drying by method of Lees and Francis and expressed as p moIes/lOOg cocoa Major and minor season samples : data for minor season in parenthesis.

Table 4 .
Effect of maturation process on the anthocyanin content