Carl Erik Hansen

Enzyme activities in cocoa beans during fermentation

The activities of endoprotease, aminopeptidase, carboxypeptidase, invertase (cotyledon and pulp), polyphenol oxidase and glycosidases were studied during heap fermentation of ICS-95 cocoa beans. These enzymes are of key importance in flavour precursor formation and in pigment degradation during cocoa fermentation. Optimal extraction and assay conditions were established to characterise the enzymes reactions and to quantify and compare enzyme activities during cocoa fermentation. The enzymes exhibited large differences in pH optima and stability during fermentation. Aminopeptidase, invertase (cotyledon and pulp) and polyphenol oxidase were strongly inactivated, carboxypeptidase was partly inactivated, whereas endoprotease and glycosidases remained active throughout the fermentation. Since many enzymes are inactivated during fermentation, it is generally recognised that the actual period of enzyme action is short. Although our results confirmed total inactivation for some enzymes, we show that several key enzymes are not completely inactivated during fermentation. Therefore, some enzyme reactions can continue throughout the whole fermentation process. Only polyphenol oxidase was strongly inactivated during sun and artificial drying of the beans. The other enzymes were stable during the drying process. Enzymes like endoprotease and glycosidases are still active in properly fermented and dried beans.

Comparison of enzyme activities involved in flavour precursor formation in unfermented beans of different cocoa genotypes.

The activities of endoprotease, aminopeptidase, carboxypeptidase and invertase (cotyledon and pulp) were studied in unfermented beans of 10 genotype samples with different flavour characteristics (high and low cocoa flavour). Analysis of variance showed that significant differences in enzyme activities exist between certain genotypes. Aminopeptidase and endoprotease activities in beans of the PA7 genotype were higher than in all others. Principal component analysis (PCA) showed that the PA7 genotype (high cocoa flavour) was very different from the UIT1 genotype (low cocoa flavour). Although significant differences exist, no simple and general relationship is established between the flavour potential of a genotype and the level of key enzyme activities in unfermented beans. Carboxypeptidase is of key importance for peptide and free amino acid formation, but differences in enzyme activity could not be correlated to flavour potential of the genotype. It is suggested that the level of enzyme activities present in unfermented beans is not a limiting factor for optimal formation of flavour precursors during the fermentation process. © 2000 Society of Chemical Industry

Effects of culture conditions on accumulation of arachidonic and eicosapentaenoic acids in cultured cells of Rhytidiadelphus squarrosus and Eurhynchium striatum

Abstract The effects of various culture conditions on the amounts of arachidonic acid (AA) and eicosapentaenoic acid (EPA) were analysed in axenic cultures of the moss species, Rhytidiadelphus squarrosus and Eurhynchium striatum . The mosses contained ca 30% AA and 5% EPA of total fatty acids when cultured at 20°. Low temperature was accompanied by a shift towards higher percentages of EPA, α-linolenic acid and dihomo γ-linolenic acid and a slight decrease in AA and linoleic acid. Stabilization of the culture medium with 0. 1% 2-morpholinoethanesulphonic acid resulted in the induction of buds and leafy gametophytes and a 5–10-fold increase in the yield of biomass (up to 7 g dry wt 1 −1 culture medium). Protonema cells contained the highest lipid concentrations, 15.6% (w/w); bud formation in these cultures was prevented by using a high salt concentration in the medium. The moss cells required light and the addition of glucose, but no plant hormones and vitamins, for proliferation in culture.

Isolation and characterisation of cell wall polysaccharides from cocoa (Theobroma cacao L.) beans

Abstract. Cell wall material (CWM) was prepared from sun-dried cocoa (Theobroma cacao L.) bean cotyledons before and after fermentation. The monosaccharide composition of the CWM was identical for unfermented and fermented beans. Polysaccharides of the CWM were solubilised by sequential extraction with 0.05 M trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA), 0.05 M Na2CO3, and 1 M, 4 M and 8 M KOH. The non-cellulosic sugar composition for each fraction was similar for unfermented and fermented samples, indicating that fermentation caused no significant modification of the structural features of individual cell wall polysaccharides. Pectic polysaccharides accounted for 60% of the cell wall polysaccharides but only small amounts could be solubilised in solutions of CDTA, Na2CO3, and 1 M and 4 M KOH. The bulk of the pectic polysaccharides were solubilised in 8 M KOH and were characterised by a rhamnogalacturonan backbone heavily substituted with side-chains of 5-linked arabinose and 4-linked galactose. Linkage analysis indicated the presence of additional acidic polysaccharides, including a xylogalacturonan and a glucuronoxylan. Cellulose, xyloglucan and a galactoglucomannan accounted for 28%, 8% and 3% of the cell wall polysaccharides, respectively. It is concluded that the types and structural features of cell wall polysaccharides in cocoa beans resemble those found in the parenchymatous tissue of many fruits and vegetables rather than those reported for many seed storage polysaccharides.

Incorporation and metabolism of radiolabelled linoleic acid in cultured cells of the moss Rhytidiadelphus squarrosus

Abstract Exogenously added [ 14 C]linoleic acid was readily taken up by axenically cultured Rhytidiadelphus squarrosus moss protonema cells. The radiolabelled acid was rapidly incorporated into phosphatidylcholine (PC), followed by a transfer of radiolabel into monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and phosphatidylglycerol (PG). The distribution of radiolabelled linoleic acid, linolenic acid n-3(LNA)+n-6(GLA), dihomo γ-linolenic acid (DHGLA) and arachidonic acid (AA) was analysed in the complex lipids. The data suggest major roles of PC and MGDG in the desaturation reactions from linoleic acid to LNA + GLA and DHGLA to AA.