Impact of fermentation on nitrogenous compounds of cocoa beans (Theobroma cacao L.) from various origins.

Tangential filtration technique was used to separate and quantify three different fractions of nitrogenous compounds depending on their molecular size, during cocoa fermentation. On every phenotype and origin analyzed, protein profile of non-fermented samples was similar. During fermentation course, proteins get degraded with a concomitant increase in amino acids content. Peptides between 3 and 10 kDa were observed at low levels. A strong correlation between amino acids and ammonia nitrogen, a fermentation marker was found. Attention was drawn on each fraction, and enabled to point out other phenomenon occurring during fermentation. The migration of some nitrogenous compounds towards the bean shell during fermentation was demonstrated. Acetone treatment of cocoa powder prior to SDS-PAGE led to losses of nitrogenous compounds. This result gives clues on the tanning phenomenon carried out by polyphenols on nitrogenous compounds, phenomenon which increases during fermentation.

Near infrared spectroscopy as a new tool to determine cocoa fermentation levels through ammonia nitrogen quantification.

Fermentation is a key step in obtaining fine cocoa through the formation of potent aroma precursors. The fermentation level of cocoa beans is traditionally assessed by measuring the amount of ammonia nitrogen (NH3) using the time-consuming Conway technique. Near infrared spectroscopy (NIRS), a rapid and efficient tool, was used to analyze NH3 levels in several hundred cocoa samples at different fermentation levels from six geographical origins. Fermentation levels were expressed as the number of fermentation days and sum of temperatures. The correlation between Conway results and NIRS spectra enabled the development of a reliable and accurate NIRS calibration to determine NH3 content. We confirm that NH3 is produced during fermentation and its amount depends on the fermentation time, sum of temperatures and geographical origin. NIRS could be used by chocolate manufacturers as a routine method to sort cocoa samples according to their level of fermentation.

Analysis of neutral lipids from microalgae by HPLC-ELSD and APCI-MS/MS.

A method was developed to analyze neutral lipids through the use of three triglycerides, four free fatty acids, six di- and four mono-glycerides standards by high performance liquid chromatography (HPLC) normal phase coupled with either with evaporative light scattering detector (ELSD) or with mass spectrometry (MS) operating in atmospheric pressure chemical ionization (APCI) mode. The method was applied to the determination of the neutral lipid fraction from a Botryococcus braunii race A (B. braunii) culture. This method led us to identify neutral lipids synthesized by B. braunii in a single analysis within 45min through HPLC-APCI-MS/MS technique.

Volatile compounds released by enzymatic hydrolysis of glycoconjugates of leaves and grape berries from Vitis vinifera Muscat of Alexandria and Shiraz cultivars.

Glycoconjugates from Muscat of Alexandria and Shiraz leaves and grape berries were isolated by adsorption on Amberlite XAD-2 resin, and enzymatically released aglycons were analyzed by GC-FID and GC-MS. About 120 aglycons were fully or tentatively identified. Compositional differences were observed between leaves and berries of the two varieties in five aglycon chemical groups: C6 alcohols, aliphatic alcohols, monoterpenes, shikimates, and C(13)-norisoprenoids, which were much more abundant in the leaves than in the berries. The differences observed for C(13)-norisoprenoids were in agreement with their hypothetical independent biosynthesis in leaves and berries. Thus, 3-hydroxy-beta-damascone, an important norisoprenoid aglycon of grape berries, was not detected in leaves, whereas its oxidized derivative, 3-oxo-alpha-damascone, was absent in berries. Compositional differences were also observed between Muscat and Shiraz leaves. 3-Oxo-alpha-ionol was not detected in Shiraz leaves, and its retro derivatives were less abundant than in Muscat of Alexandria leaves. Conversely, in Shiraz leaves the levels of 7,8-dihydroionone derivatives, such as megastigman-3,9-diol and 3-oxo-7,8-dihydro-alpha-ionol, were higher than in Muscat of Alexandria leaves.

Purification and some properties of an Aspergillus niger beta-apiosidase from an enzyme preparation hydrolyzing aroma precursors.

A beta-apiosidase was isolated and purified to electrophoretic homogeneity from an enzyme preparation, Klerzyme 200, through ammonium sulfate precipitation, gel filtration chromatography, ion-exchange chromatography, and HPLC on ion-exchange and size exclusion columns. The purification of the enzyme was aided by the synthesis of 4-methylumbelliferyl beta-D-apiofuranoside for the specific detection of activity on electrophoresis gels. The molecular mass estimated by SDS-PAGE was 120 kDa. The optimum activity of the beta-apiosidase was found at pH 5 and 40 degrees C. The K(m) and V(max) for p-nitrophenyl beta-D-apiofuranoside were 4.2 mM and 2460 nkat/mg of protein, respectively. The enzyme was not inhibited by glucose and ethanol. This enzyme hydrolyzed the intersugar linkages of apiofuranosylglucosides, aroma precursors from grape.

Purification, characterization, and substrate specificity of a novel highly glucose-tolerant beta-glucosidase from Aspergillus oryzae.

Aspergillus oryzae was found to secrete two distinct beta-glucosidases when it was grown in liquid culture on various substrates. The major form had a molecular mass of 130 kDa and was highly inhibited by glucose. The minor form, which was induced most effectively on quercetin (3,3',4',5,7-pentahydroxyflavone)-rich medium, represented no more than 18% of total beta-glucosidase activity but exhibited a high tolerance to glucose inhibition. This highly glucose-tolerant beta-glucosidase (designated HGT-BG) was purified to homogeneity by ammonium sulfate precipitation, gel filtration, and anion-exchange chromatography. HGT-BG is a monomeric protein with an apparent molecular mass of 43 kDa and a pI of 4.2 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing polyacrylamide gel electrophoresis, respectively. Using p-nitrophenyl-beta-D-glucoside as the substrate, we found that the enzyme was optimally active at 50 degreesC and pH 5.0 and had a specific activity of 1,066 micromol min-1 mg of protein-1 and a Km of 0.55 mM under these conditions. The enzyme is particularly resistant to inhibition by glucose (Ki, 1. 36 M) or glucono-delta-lactone (Ki, 12.5 mM), another powerful beta-glucosidase inhibitor present in wine. A comparison of the enzyme activities on various glycosidic substrates indicated that HGT-BG is a broad-specificity type of fungal beta-glucosidase. It exhibits exoglucanase activity and hydrolyzes (1-->3)- and (1-->6)-beta-glucosidic linkages most effectively. This enzyme was able to release flavor compounds, such as geraniol, nerol, and linalol, from the corresponding monoterpenyl-beta-D-glucosides in a grape must (pH 2.9, 90 g of glucose liter-1). Other flavor precursors (benzyl- and 2-phenylethyl-beta-D-glucosides) and prunin (4',5,7-trihydroxyflavanone-7-glucoside), which contribute to the bitterness of citrus juices, are also substrates of the enzyme. Thus, this novel beta-glucosidase is of great potential interest in wine and fruit juice processing because it releases aromatic compounds from flavorless glucosidic precursors.

Analytical methods for monoterpene glycosides in grape and wine. I. XAD-2 extraction and gas chromatographic-mass spectrometric determination of synthetic glycosides.

Synthetic monoterpene and aromatic beta-D-glucopyranosides, beta-rutinosides and 6-O-(alpha-L-arabinofuranosyl)-beta-D-glucopyranosides and their corresponding alcohols, diluted in a synthetic solution imitating wine, were isolated and separated by selective retention on Amberlite XAD-2. The corresponding recoveries and the conditions for the direct determination of these glycosides by gas chromatography and gas chromatography-mass spectrometry after derivatization were determined. Trifluoroacetylation gave the best results but trimethylsilylation provided complementary results. The separation of some diastereoisomeric monoterpene glycosides was also examined.