Investigation into the roles of yeasts, lactic acid bacteria and acetic acid bacteria in cocoa bean fermentation

摘要

Cocoa beans (Theobroma cacao L.) are the main raw material for chocolate production. Fermentation of cocoa beans by microorganisms is essential for development of the precursors of chocolate flavour. Despite more than 100 years of research into the microbial ecology of cocoa fermentation, the beans are still fermented by an uncontrolled, traditional process conducted by a consortium of indigenous species of yeasts, lactic acid bacteria (LAB) and acetic acid bacteria (AAB). The roles of the different microbial groups and associated species in contributing to the fermentation and the quality of the resultant beans and chocolate derived from them remain unclear and speculative. An understanding of these roles is needed to develop starter culture technology for conducting a better controlled process. The objective of this thesis is to understand how individual microbial groups and species contribute to cocoa bean fermentation and chocolate character.Cocoa bean fermentations were conducted under conditions where the growth of yeasts was inhibited by the addition of natamycin and the growth of LAB was restricted by the use of nisin and lysozyme. Fermentations were also conducted with aseptically extracted beans inoculated with four different combinations of yeasts and bacteria (yeasts only, yeasts + LAB, yeasts + AAB and yeasts + LAB + AAB). Traditional, indigenous fermentations were conducted as controls. The growth of individual species of yeasts, LAB and AAB was determined throughout fermentation and correlated with changes in cocoa bean pH, sugars, organic acids, flavour volatiles (higher alcohols, esters, aldehydes, ketone and pyrazines) and, in some cases, amino acids. The microbiological and chemical data were correlated with sensory evaluation of chocolates prepared from the beans.Beans fermented without the growth of yeasts showed normal growth of LAB and AAB. However, they did not undergo characteristic alcoholic fermentation and there was insignificant production of ethanol, higher alcohol and ester volatiles. Dried beans from these fermentations did not meet standard criteria for shell weight and fermentation score, and chocolate prepared from them lacked characteristic flavour. It was concluded that yeast growth and activity were essential for cocoa bean fermentation and the development of chocolate characteristics.Normal growth of yeasts and AAB bacteria occurred in fermentations where the growth of LAB was prevented or restricted. These beans underwent characteristic alcoholic fermentation where the utilization of sugars and the production of ethanol, organic acids and volatile compounds in the bean pulp and nibs were similar for beans fermented in the presence of LAB. Lactic acid was produced during both fermentations but more so when LAB grew. Beans fermented in the presence or absence of LAB were fully fermented, had similar shell weights and gave acceptable chocolates with no differences in sensory rankings. It was concluded that LAB were not necessary for successful cocoa fermentation.Beans aseptically extracted and fermented with inoculated species gave the growth of these species only, with no interference from indigenous microflora. Despite some differences in the concentrations of lactic acid and acetic acid, fermentations with inoculated yeasts only, or yeasts + LAB, or yeasts + AAB, or yeasts + LAB + AAB had similar chemical profiles and chocolates prepared from these beans showed no significant differences in sensory rankings for chocolate flavour and liking. It was concluded that fermentations with inoculated yeasts only give acceptable quality chocolate and that the growth of LAB and AAB was not essential for a successful cocoa fermentation. Only yeasts would be necessary in developing starter culture technology for cocoa bean fermentation.