Materials and methods Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol149.Issue2.2000:

information is, however, an essential biochemical base for the elucidation of mechanisms of trans- port and metabolism in the grains and should provide targets for the improvement of the technological and agronomic character of the species. In this study we describe changes in concentra- tions during grain maturation of a limited number of components free sugars, sugar alcohols, car- boxylic acids and inorganic anions likely to be implicated in final beverage quality in the two main commercial coffee species, Coffea arabica Arabica and C. canephora Robusta. Sucrose, while being significantly degraded during roasting, remains in roasted grains at concentrations of 0.4 – 2.8 dry weight DW and is likely to con- tribute to beverage sweetness [16]. It is also the main contributor of reducing sugars which are implicated in Maillard reactions occurring during roasting. Inorganic, chlorogenic and carboxylic acids contribute to the final beverage acidity [3], acidity being associated with better flavour and aroma [17]. The principal interest of this study is the rela- tionship between concentration profiles in the young grain and the more mature stages in an attempt to begin to identify biochemical mecha- nisms of transport and accumulation for a range of components. The grain of Coffea species is dominated by a well developed maternal perisperm tissue up to approximately the halfway stage of maturation from the ‘pinhead’ stage until approximately 15 weeks after flowering WAF, following which the locular space is progressively filled with endosperm up to full grain maturity at between 20 and 30 WAF. The mature coffee grain has been convincingly identified as endosperm [18] and not perisperm as suggested previously [19]. This identification has been supported by more recent studies [20 – 22]. Wormer [23] also observed the role of the perisperm, during the expansion occurring during the first half of the maturation period, in defining the final size of the locular space. Although virtu- ally nothing is known about the characteristics of metabolism and transport in the fruits and grains of coffee species, the relatively large size of the two principal tissues and the duration of the matura- tion period provide an interesting model for the study of relationships between them during grain development.

2. Materials and methods

2 . 1 . Plant material Fruit from varieties of Robusta Dormilon and Rom and varieties of Arabica CRM, Caturra Commercial and Caturra 2308 were obtained from trees cultivated on the experimental farm of Nestle´ RD Centre Quito, Ecuador. Trees were cultivated on the same site at an altitude of ap- proximately 80 m above sea level MAS. Fruit were harvested during 1996 at defined stages fol- lowing flowering, frozen immediately in liquid ni- trogen and packaged in dry ice for transport. Samples normally consisted of ten fruit 20 grains. 2 . 2 . Extraction Grain tissues were separated from pericarp and hulls locules. For a detailed analysis of free sugars in some cases the perisperm in the young fruit was separated entirely from the endosperm, in other cases the entire contents of the grain were used without separation. The tissue was homoge- nized in a mortar with liquid nitrogen and the powder obtained was lyophilized for 24 h Lyolab bII, Secfroid. The sample was stored at − 20°C where necessary before being weighed and sus- pended in 70 ml of double-distilled water previ- ously pre-heated to 70°C, shaken vigorously and incubated for 30 min at 70°C. After cooling to room temperature, the sample was made to 100 ml by adding double-distilled water and then filtered on paper Schleicher and Schuell filter paper 597.5. An aliquot 3 ml of the filtrate was filtered a second time on a C 18 cartridge SEP PAK which had been equilibrated beforehand with 3 ml methanol followed by 3 ml of water. 2 . 3 . Separation by HPAE-PED Sugars of extracted coffee grain tissues were separated by HPAE-PED according to Ref. [24] using a Dionex PA 100 4 × 250 mm column. Dionex HPAE-PED was also used to determine the concentrations of oligosaccharides column PA 1, 4 × 250 mm, sugar alcohols column MA 1, 4 × 250 mm and carboxylic acids and inorganic anions column AS 11, 4 × 250 mm. Oligosaccha- rides and sugar alcohols were separated isocrati- cally with 81 mM NaOH and 480 mM NaOH, respectively. Carboxylic acids and inorganic an- ions were separated by a gradient of 0.5 – 38.25 mM NaOH for 18 min. Phytic acid was eluted with a gradient of 23 – 60 mM NaOH for 1 min. The injected sample volume was 20 ml and the flow rate usually 1 mlmin. Standards of sugars, oligosaccharides, sugar alcohols, carboxylic acids and inorganic anions were from Sigma St. Louis, MO except stachyose tetrahydrate which was from Extrasynthe`se Genay, France. All results represent the mean of three repetitions.

3. Results