Honoré Gnenequidou Ouattara

Acetic acid produced during cocoa fermentation impact strongly the quality of fermented and dried cocoa beans and chocolate. The objective of this study was to analyze acid production in Acetic acid bacteria (AAB) and their response to fermentative conditions. AAB strains were isolated from cocoa fermentation and screened for acid production on both solid and liquid media. From 444 isolates, 15 strains yielded 2 to 38.1 g/L of acid in liquid medium. The best acid producers were identified by biochemical typing as Acetobactersp, Acetobacteraceti, A. peroxydans, A. pasteurianus and Gluconobacteroxydans. Most AAB strains showed strong tolerance to alcohol with round 45% of survival growth rate (SGR) under 12 % ethanol. AAB strains growing well at 2 % acetic acid (SGRround 47 %), failed to grow at 3 % of this acid. Lactic acid has the most hindering effect on AAB growth, provoking a drop of SGR from 100 % to less than 25 % at only 1 % of lactic acid. Maximum growth of acidifying AAB strains occurred at 35 °C. This study evidences the diversity of technological performances of AAB involved in Ivorian cocoa fermentation and allows targeting the most valuable strains as starters.

Microbial preparation containing pectinolytic strains as starter culture should help to standardize cocoa fermentation and reduce the lost due to the variability of cocoa bean. In this study, carbon metabolism, fermentative capacity and effect of environmental conditions on pectinase synthesis were analyzed in four yeast strains previously characterized as highly pectinolytic and stress resistant. The strains showed a restricted carbon metabolism profile with capacity to ferment only glucose and fructose and grown maximally at 5% of these carbon sources. Furthermore, yeasts strains were able to keep round 80% of their relative growth at up to 15% of sugar concentrations and proved to be osmotolerant at 25% glucose. Theses strains expressed their highest fermentative capacity at 35 °C producing up to 10.78 cm3of CO2 and lost more than 50% of this capacity at 40 °C. The isolates studied produced polygacturonase as sole pectinase, optimal production of this enzyme is reached at pH (5-6), at incubation temperature of 30 ºC for strains YS 128, YS 202 and 35 ºC for strains YS 165 and YS 201. However, stress conditions such as 0.5 % acetic acid, 2% lactic acid, 6% citric acid and 6% ethanol repress strongly polygalacturonase production in the strains analyzed. Yeast strains studied present some physiological properties potentially useful for cocoa fermentation but the use of these strains as starter should take into account, the stress conditions susceptible to hinder pectinase production.

Pectinolytic enzymes play an important role in cocoa fermentation. In this study, we characterized three extracellular pectate lyases (Pels) produced by bacilli isolated from fermenting cocoa beans. These enzymes, named Pel-22, Pel-66, and Pel-90, were synthesized by Bacillus pumilus BS22, Bacillus subtilis BS66, and Bacillus fusiformis BS90, respectively. The three Pels were produced under their natural conditions and purified from the supernatants using a one-step chromatography method. The purified enzymes exhibited optimum activity at 60°C, and the half-time of thermoinactivation at this temperature was approximately 30 min. Pel-22 had a low specific activity compared with the other two enzymes. However, it displayed high affinity for the substrate, about 2.5-fold higher than those of Pel-66 and Pel-90. The optimum pHs were 7.5 for Pel-22 and 8.0 for Pel-66 and Pel-90. The three enzymes trans -eliminated polygalacturonate in a random manner to generate two long oligogalacturonides, as well as trimers and dimers. A synergistic effect was observed between Pel-22 and Pel-66 and between Pel-22 and Pel-90, but not between Pel-90 and Pel-66. The Pels were also strongly active on highly methylated pectins (up to 60% for Pel-66 and Pel-90 and up to 75% for Pel-22). Fe ²⁺ was found to be a better cofactor than Ca ²⁺ for Pel-22 activity, while Ca ²⁺ was the best cofactor for Pel-66 and Pel-90. The amino acid sequences deduced from the cloned genes showed the characteristics of Pels belonging to Family 1. The pel - 66 and pel - 90 genes appear to be very similar, but they are different from the pe l- 22 gene. The characterized enzymes form two groups, Pel-66/Pel-90 and Pel-22; members of the different groups might cooperate to depolymerize pectin during the fermentation of cocoa beans.