EFFECT OF LIPASE ACTIVITIES DURING FERMENTATION ON AROMA FORMATION IN CACAO SEEDS (Theobroma cacao)

• Main Author: Widowaty, Windy
• Format: Theses
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/35524
• Institution: Institut Teknologi Bandung

Cocoa beans are seeds from Theobroma cacao plant. During processing, cocoa beans will experience physical, chemical and biological changes. Characteristic aroma and flavor of chocolate will be formed. Fermentation of cocoa beans (Theobroma Cacao L var. Forastero) was conducted to establish an ideal-typical chocolate sense and taste while reducing bitter taste existing in cacao beans. Fat is a major component found in cocoa beans that is as much as 50% of its weight. Lipase (triacylglicerol acylhydrolase, EC 3.1.1.3) is an enzyme that catalyses hydrolysis of glycerol ester bond and synthesis of glycerol esters. Lipase has been widely used as a catalyst for the formation of esters, both in the laboratory scale and industrial scale. The advantage in the use of enzymes as catalysts are highly selective. In food industry, lipase is used as the aroma forming agent in the process of cheese and chocolate crumb making. It has not been known how the activity of lipase from cocoa beans mainly from existing varieties in Indonesia (Forastero; Cocoa lindak) without any microbes or fungi during the fermentation process and how they affect the resulting aroma. The purpose of this study was to determine the effect on the lipase activity of cocoa beans during the fermentation and its impact on the resulting aroma. In this study, isolation of lipase enzyme was conducted from cocoa beans that have been fermented for 0, 3, 5 and 7 days (sample code H0, H3, H5 and H7). As a comparison, fermentation of endogenous enzyme deactivated-cacao beans by crude extract of lipase enzyme were also conducted at 0, 3, 5 and 7 days (sample code HE0, HE3, HE5 and HE7) as well as by the addition of Rhizopus oligosporus as lipase, cellulose, glucanase and other exogenous hydrolase enzymes-producing microbes (sample code HR0, HR3, HR5 and HR7). Cocoa fermentation was conducted in a closed erlenmeyer flask with a sterile cotton and the fermentation temperature was maintained at 37oC. Isolation of enzymes was made by acetone precipitation and lipase activity assay was performed using the substrate p- nitrofenil palmitic acid (pNPP). Fermented beans was dried at 50oC for 15 hours and blended. Isolation of the aroma compound was conducted with soxlet extraction method and the extraction results were analyzed using GC-MS method. Results showed that endogenous lipase spesific activity of cacao beans increased during fermentation process and reach its highest activity at day 5 (0.0446 µg pNP/µg protein/min (H0), 1.1811 µg pNP/µg protein/min (H3), 1.3242 µg pNP/µg protein/min (H5) and 1.2493 µg pNP/µg protein/min (H7)). While the assay results from lipase activity of cocoa beans that have been deactivated and fermented with lipase crude extract was 0.1590 (HE0), 0.2262 (HE3), 0.1530 (HE5) and 0.2396 µg pNP/µg protein/min (HE7). Assay results from lipase activity of cocoa beans that have been deactivated and fermented with Rhizopus oligosporus were 0.0371 (HR0), 0.1680 (HR3), 0.2594 (HR5) and 0.1722 µg pNP/µg protein/min (HR7). GC-MS results shows that the optimum formation of cocoa aroma was obtained in 5-day old fermented samples in which H5 (without treatment) and HR5 (treated with Rhizopus oligosporus) samples showed many compounds detected. Aroma-forming compounds in cocoa beans were caused by exogenous lipase, where compounds such as dodecane, tridecane, tetradecane, 1.3- dimethylbenzene and caffeine have a higher peak in the chromatogram of cacao’s aroma produced due to the fermentation of Rhizopus oligosporus for 5 (HR5) and 7 (HR7 ) days on deactivated beans.