Fernando M. Nunes

Modification of wheat straw lignin by solid state fermentation with white-rot fungi

The potential of crude enzyme extracts, obtained from solid state cultivation of four white-rot fungi (Trametes versicolor, Bjerkandera adusta, Ganoderma applanatum and Phlebia rufa), was exploited to modify wheat straw cell wall. At different fermentation times, manganese-dependent peroxidase (MnP), lignin peroxidase (LiP), laccase, carboxymethylcellulase (CMCase), avicelase, xylanase and feruloyl esterase activities were screened and the content of lignin as well as hydroxycinnamic acids in fermented straw were determined. All fungi secreted feruloyl esterase while LiP was only detected in crude extracts from B. adusta. Since no significant differences (P>0.05) were observed in remaining lignin content of fermented straw, LiP activity was not a limiting factor of enzymatic lignin removal process. The levels of esterified hydroxycinnamic acids degradation were considerably higher than previous reports with lignocellulosic biomass. The data show that P. rufa, may be considered for more specific studies as higher ferulic and p-coumaric acids degradation was observed for earlier incubation times.

Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains as structural features of coffee arabinogalactans.

The hot water soluble green coffee arabinogalactans, representing nearly 7% of total coffee bean arabinogalactans, were characterized by (1)H and (13)C NMR and, after partial acid hydrolysis, by ESI-MS/MS. Data obtained showed that these are highly branched type II arabinogalactans covalently linked to proteins (AGP), with a protein moiety containing 10% of 4-hydroxyproline residues. They possess a beta-(1-->3)-Galp/beta-(1-->3,6)-Galp ratio of 0.80, with a sugars composition of Rha:Ara:Gal of 0.25:1.0:1.5, and containing 2mol% of glucuronic acid residues. Beyond the occurrence of single alpha-L-Araf residues and [alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] disaccharide residues as side chains, these AGPs contain unusual side chains at O-3 position of the beta-(1-->6)-linked galactopyranosyl residues composed by [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->] and [alpha-L-Rhap-(1-->5)-alpha-L-Araf-(1-->5)-alpha-L-Araf-(1-->] oligosaccharides. Rhamnoarabinosyl and rhamnoarabinoarabinosyl side chains are reported for the first time as structural features of plant arabinogalactan-proteins.

Characterization of Galactomannan Derivatives in Roasted Coffee Beverages

In this work, the galactomannans from roasted coffee infusions were purified by 50% ethanol precipitation, anion exchange chromatography, and phenylboronic acid-immobilized Sepharose chromatography. Specific enzymatic hydrolysis of the beta-(1-->4)-D-mannan backbone allowed us to conclude that the galactomannans of roasted coffee infusions are high molecular weight supports of low molecular weight brown compounds. Also, the molecular weight of the brown compounds linked to the galactomannan increases with the increase of the coffee degree of roast. The reaction pathways of galactomannans during the coffee roasting process were inferred from the detection of specific chemical markers by gas chromatography-electron impact mass spectrometry and/or electrospray ionization tandem mass spectrometry. Maillard reaction, caramelization, isomerization, oxidation, and decarboxylation pathways were identified by detection of Amadori compounds, 1,6-beta-anhydromannose, fructose, glucose, mannonic acid, 2-ketogluconic acid, and arabinonic acid in the reducing end of the obtained oligosaccharides. The implication of the several competitive reaction pathways is discussed and related to the structural changes of the galactomannans present in the roasted coffee infusions.

Chemical composition and functional properties of native chestnut starch (Castanea sativa Mill).

Starch isolation methods can change their physico-chemical and functional characteristics hindering the establishment of a starch-food functionality relation. A simple high yield and soft isolation method was applied for chestnut (Castanea sativa Mill) starch consisting in steeping and fruit disintegration in a 25 mM sodium bisulfite solution and purification by sedimentation. Starch integrity, physico-chemical composition, morphology and functional properties were determined, being observed significant differences from previous described methods for chestnut starch isolation. The X-ray pattern was of B-type, with a degree of crystallinity ranging from 51% to 9%, dependent on the starch moisture content. The onset, peak, and conclusion gelatinization temperatures were 57.1°C, 61.9°C and 67.9°C, respectively. Total amylose content was 26.6%, and there was not found any evidence for lipid complexed amylose. Swelling power at 90°C was 19 g/g starch, and the amount of leached amylose was 78% of the total amylose content. Native chestnut starch presents a type B pasting profile similar to corn starch but with a lower gelatinization (56.1°C) and peak viscosity (79.5°C) temperatures, making native chestnut starch a potential technological alternative to corn starch, especially in application where lower processing temperatures are needed.

Effect of cooking on total vitamin C contents and antioxidant activity of sweet chestnuts (Castanea sativa Mill.)

In this work the total vitamin C contents (ascorbic acid+dehydroascorbic acid) and antioxidant activity of raw and cooked chestnuts was evaluated. The vitamin C contents of raw chestnuts varied significantly between the different cultivars (cv) studied and it varied from 400mg/kg dry weight (cv Lada) to 693mg/kg dry weight (cv Martaínha). The different cultivars behave differently during the cooking process concerning the loss of vitamin C. A significant decrease in the vitamin C content of the chestnuts was observed, 25-54% for the boiling process and 2-77% for the roasting process. Boiled and roasted chestnuts can be good sources of vitamin C since it may represent 22.4%, 16.2%, 26.8% and 19.4%, respectively, of the recommended dietary intake for an adult man and woman. The cooking process significantly changed the antioxidant activity of the chestnuts. A difference was observed between the cultivars during the cooking processes, concerning the antioxidant activity. For the raw chestnuts the variation in vitamin C content of the chestnuts explains 99% of the antioxidant activity variation but for the roasted and boiled chestnuts this percentage significantly decreases to 51% and 88%, respectively. Although a high antioxidant activity is still present in the cooked chestnuts, the cause for this antioxidant activity is less dependent on the vitamin C content of the chestnuts, probably due to the conversion of ascorbic acid to dehydroascorbic acid. The increase in gallic acid during the cooking process, presumably transferred from the peels to the fruit, also contributes to the high antioxidant activity observed for the cooked chestnuts.

Immunostimulatory properties of coffee mannans.

Coffee infusion mannans are acetylated polysaccharides containing single Galp and Araf residues as side chains of a beta-(1 --> 4)-Manp backbone. These mannans are structurally similar to the bioactive acetylated mannans from Aloe vera (AV). In this study, acetylated mannans were obtained from two coffee infusions prepared from light and dark roasted beans. These samples were tested for their immunostimulatory activity and compared with an extract of AV mannan and with locust bean gum (LBG) galactomannans. The coffee samples, as well as the AV extract, stimulated murine B- and T-lymphocytes, as evaluated by the in vitro expression of the surface lymphocyte activation marker CD69, more marked on B- than on T-lymphocytes. In coffee samples, contrarily to the AV, no proliferative effect was noticed. LBG sample did not show any immunostimulatory activity. Because the material that remains in the residue of the hot water extraction was still very rich in mannans, a sequential extraction was performed and a main fraction was recovered with a 4 M NaOH solution. Because this material was insoluble in water, a partial acetylation was performed. These polysaccharides also showed immunostimulatory activity, opening the possibility of exploitation of coffee infusion and coffee residue as sources of bioactive polysaccharides.

Evaluation of the effect of roasting on the structure of coffee galactomannans using model oligosaccharides.

The roasting process induces structural changes in coffee galactomannans. To know more about the reaction pathways that occur during the roasting of coffee, mannosyl and galactomannosyl oligosaccharides, having a degree of polymerization (DP) between 3 and 4, were used as models for galactomannans. These compounds were dry-heated under air atmosphere from room temperature to 200 °C, being maintained at 200 °C for different periods of time. The roasted materials were analyzed by mass spectrometry (ESI-MS, MALDI-MS, and ESI-MSn) and methylation analysis. In the MS spectra were identified several [M+Na]+ ions belonging to a series from a single hexose to 10 hexose residues ([Hex1-10+Na]+). The ions corresponding to their respective mono- and tridehydrated derivatives ([Hex2-10-H2O+Na]+ and [Hex2-10-3H2O+Na]+, respectively) were also identified. ESI-MSn as well as deuterium-labeling and alditol derivatization experiments showed that the tridehydrations occur at the reducing end of the oligosaccharides. The identification of (1→2)- and (1→6)-linked mannose residues and (1→4)-linked glucose residues by methylation analysis allowed the conclusion that transglycosylation and isomerization reactions occur during dry thermal processing.

Role of hydroxycinnamates in coffee melanoidin formation

Melanoidins are the high molecular weight brown end products of the Maillard reaction. They are formed during heat processing of foods like coffee, bread, malt, and beef. A chemical definition of these food polymers is still impossible, despite several efforts to determine their structure. In the last years, the interest in research on melanoidins has increased due to their biological activities. Coffee brew is one of the main sources of melanoidins in human diet. Various melanoidin fractions were obtained by applying chromatographic separation techniques or specific isolation procedures, allowing, a partial view on structural features and diversity of coffee brew melanoidins. Different melanoidin populations can be found with respect to total carbohydrate contents and their structural features. In this paper, the recent advances in research on coffee melanoidin structures and formation mechanisms are reviewed. The participation of hydroxycinnamates in melanoidin formation, especially true for coffee melanoidins, is a hypothesis older than three decades, but only recently more consistent data have been obtained for their presence. Although the role of hydroxycinnamates in melanoidin formation is not yet completely understood, it was demonstrated that the interaction between phenolic compounds and melanoidins can be of non-covalent or of covalent nature. The most likely linkage point is through the protein fragments incorporated in the coffee melanoidin during the roasting process, although carbohydrates, such as arabinose, seem to be possible binding sites for the chlorogenic acid derivatives on these brown structures, too.

The potential of white‐rot fungi to degrade phorbol esters of Jatropha curcas L. seed cake

The potential of solid‐state cultivation, with three white‐rot fungi (Bjerkandera adusta, Ganoderma resinaceum and Phlebia rufa), to decrease phorbol esters concentration of Jatropha curcas L. was evaluated in this study. Incubation was conducted in 250 mL Erlenmeyer flasks without agitation at 28°C for 30 days. Phorbol esters were analyzed by reverse‐phase HPLC after an extraction procedure using dichloromethane. All fungi studied were able to decrease the concentration of phorbol esters, mainly B. adusta and P. rufa which significantly reduced (p<0.05) phorbol esters contents to non‐toxic levels. These results suggest that white‐rot fungi could be potentially used as a possible approach for the biological treatment of the oilseed cake.

Comparison between different types of carboxylmethylcellulose and other oenological additives used for white wine tartaric stabilization

Carboxylmethylcellulose (CMC) is authorised to prevent wine tartaric instability. The effect of CMC structural characteristics on their effectiveness is not well understood. The main purpose of this study was to compare the impact of CMCs with different degrees of substitution and molecular weight, on tartaric stability, tartaric acid, mineral concentration, phenolic compounds, chromatic and sensory characteristics in white wines, and compare its effectiveness with other oenological additives. Mini-contact test showed that all CMCs and metatartaric acid stabilized the wines; however, some arabic gums and mannoproteins do not stabilized the wines. CMCs had no significant effect on tartaric acid, potassium, calcium and sensory attributes. Tartaric stabilization effectiveness depends on CMCs degree of substitution, but also on wine matrix, probably its initial potassium content. Results suggest that CMC is a good alternative to white wine tartaric stabilization; nevertheless deeper structure knowledge is necessary in order to choose the appropriate CMC for a given tartaric instability.