Molecular affinity and permeability of different molecular weight chitosan membranes

Membranes were prepared from chitosan with different molecular weights by a casting method. The molecular affinity and permeability of the membranes for sodium chloride, glucose, tyrosine, and bovine serum protein were measured at 4 degrees C and pH 7. The molecular permeability of the chitosan membranes was inversely related to molecular weight. All prepared membranes showed a molecular affinity to bovine serum protein. The higher the molecular weight of chitosan membrane, the higher the affinity and the lower permeability of the membrane to bovine serum protein.     

Fractionation, characterization, and study of the emulsifying properties of corn fiber gum

Corn fiber gum (CFG) has been fractionated by hydrophobic interaction chromatography on Amberlite XAD-1180 resin using ionic, acidic, basic, and hydrophobic solvents of different polarities. Characterization, including determination of total carbohydrate, acidic sugar, and protein content, has been done for each fraction together with measurements of molar mass, polydispersity, radius of gyration, Mark-Houwink exponent, and intrinsic viscosity using multiangle laser light scattering and online viscosity measurements. Emulsification properties of all fractions in an oil-in-water emulsion system with 20:1 oil to gum ratio were studied by measuring turbidity over 14 days. The results indicate that CFG consists of different components differing in their molecular weights and carbohydrate and protein contents. The main fraction eluted with NaCl, although low in protein content, has the highest average molecular weight and was determined to be a better emulsifier than the other fractions. The unfractionated CFG, which contains different molecular species, is the best emulsifier.     

Studies on the effect of Amadoriase from Aspergillus fumigatus on peptide and protein glycation in vitro

Amadoriase I is a fructosyl amine oxidase from Aspergillus fumigatus that catalyzes the oxidation of Amadori products (APs) producing glucosone, H2O2, and the corresponding free amine. All the enzymes of this family discovered so far only deglycate small molecular weight products and are inactive toward large molecular weight substrates, such as glycated BSA or ribonuclease A. Therefore, they cannot be used to reverse protein glycation occurring in diabetes or in foods. In this paper, the effect of Amadoriase I added during the in vitro reaction between glucose and peptides having different polarities or proteins with molecular weights ranging from to 5 to 66 kDa was tested. The formation of APs was monitored by ESI-MS of intact glycated protein or peptides and by measuring the Nepsilon-(1-deoxy-d-fructos-1-yl)-L-lysine and furosine concentrations. Results showed that the formation of APs is reduced up to 80% when peptides and glucose are incubated in the presence of Amadoriase. The effect is more evident for hydrophobic peptides. In protein-glucose systems, the effect was dependent on the molecular weight and steric hindrance being negligible for BSA and at a maximum for insulin, where the formation of APs was reduced up to 60%. These findings indicate new potential applications of Amadoriase I as an efficient tool for inhibiting protein glycation in real food systems.     

Impact of ultrafiltration membrane material on Peptide separation from a snow crab byproduct hydrolysate by electrodialysis with ultrafiltration membranes

Electrodialysis with ultrafiltration membrane (EDUF) is a technology based on the separation of molecules according to their charge and molecular mass. Some works have already successfully demonstrated the recovery of bioactive peptide fractions. However, the impact of ultrafiltration membrane (UFM) material, used in the EDUF system, on the peptide migration has never been studied. Consequently, the objectives of this work were (1) to evaluate the effect of two different UFM materials on the selective separation of peptides from a snow crab byproduct hydrolysate by electrodialysis with ultrafiltration membranes and (2) to determine the effect of UFM material on their potential fouling by peptides. It appeared that, after 6 h of EDUF separation using polyether sulfone (PES) and cellulose acetate (CA) UFM, peptides with low molecular weights ranging from 300 to 700 Da represented the most abundant population in the KCl1 (compartment located near the anode for the recovery of anionic/acid peptide fractions) and KCl2 (compartment located near the cathode for the recovery of cationic/basic peptide fractions) permeates. Peptides with molecular weights ranging from 700 to 900 Da did not migrate during the EDUF treatment. Moreover, only CA UFM allowed the recovery of high molecular weight molecules (900-20000 Da) in both KCl compartments. Peptides desorbed from PES and CA UFM after 6 h of EDUF separation had low molecular weights and belonged mainly to the 600-700 Da molecular weight range. These peptides represented a low proportion of the peptides initially present in the snow crab byproduct hydrolysate with individual molecular weight range proportions from 1.52 ± 0.31 to 10.2 ± 2.32%.     

Interaction of flavonoids with bovine serum albumin: a fluorescence quenching study

The interaction between four flavonoids (catechin, epicatechin, rutin, and quercetin) and bovine serum albumin (BSA) was investigated using tryptophan fluorescence quenching. Quenching constants were determined using the Stern-Volmer equation to provide a measure of the binding affinity between the flavonoids and BSA. The binding affinity was strongest for quercetin and ranked in the order quercetin > rutin > epicatechin = catechin. The pH in the range of 5-7.4 does not affect significantly (p < 0.05) the association of rutin, epicatechin, and catechin with BSA, but quercetin exhibited a stronger affinity at pH 7.4 than at lower pH (p < 0.05). Quercetin has a total quenching effect on BSA tryptophan fluorescence at a molar ratio of 10:1 and rutin at approximately 25:1. However, epicatechin and catechin did not fully quench tryptophan fluorescence over the concentration range studied. Furthermore, the data suggested that the association between flavonoids and BSA did not change molecular conformation of BSA and that hydrogen bonding, ionic, and hydrophobic interaction are equally important driving forces for protein-flavonoid association.     

The speciation of metals in sewage and activated sludge effluent

A gel filtration chromatography technique was used to separate soluble species of Cd, Cu, Co, Mn, Ni, and TI in the influent and effluent of a laboratory scale activated sludge simulation operated at a range of sludge ages from 3 to 18 days. It was found that, whilst there was no general trend for all six metals, Cd and Mn associated mainly with low molecular weight fractions; Co, Cu, and Ni with a range of predominantly high molecular weight fractions; and TI tended towards association with high molecular weight fractions which influenced metal solubility and appeared to be significant in determining metal removal. It appeared that all metals exhibited high affinity for mixed liquor suspended solids (MLSS) and that this affinity was strongest for Cu and Cd. Nickel, Co, Cu, and TI appeared to show a high affinity for polymeric microbial products produced at longer sludge ages. The gel filtration technique was found to be useful in the separation of metal species in conjunction with a sufficiently sensitive detector provided their concentrations in the original sample were relatively high.     

Immunochemical and Electrophoretic Analysis of the Modification of Wheat Proteins in Extruded Flour Products

Antibodies specific for wheat proteins were used to identify protein fractions modified during extrusion of Hard Red Spring wheat flour (14% protein) under four different combinations of extrusion conditions (18 and 24% feed moisture and 145 and 175°C die temperature). Antibody binding was assessed on immunoblots of proteins extracted from flour and extrudates separated by SDS‐PAGE. Antibodies to high molecular weight glutenin subunits (HMW‐GS) and to B‐group low molecular weight glutenin subunits (LMW‐GS) recognized intact subunits from both flour and extrudates. Antibodies to C‐group LMW‐GS had diminished binding to extruded proteins. Glutenin‐specific antibodies also recognized protein in the extrudates migrating as a smear at molecular weights higher than intact subunits, indicating cross‐linked proteins. Antibodies recognized albumins or globulins in flour but not in extrudates, evidence that these fractions undergo significant modification during extrusion. Acid‐PAGE and antibody reaction of gliadins extracted in 1M urea and in 70% ethanol revealed total loss of cysteine‐containing α, β, γ‐gliadins but no obvious effects on sulfur‐poor ω‐gliadins, suggesting gliadin modification involves replacing intramolecular disulfides with intermolecular disulfide cross‐links. Identifying protein fractions modified during different extrusion conditions may provide new options for tailoring extrusion to achieve specific textural characteristics.     

Evolution of Humic Acid Molecular Weight As an Index of Compost Stability

This paper proposes an index for the evaluation of compost maturity based on the evolution of molecular weights of humic acids (HA) during composting. The evolution of HA molecular weight was followed during the composting of both olive mill wastewater (OMW) and olive mill pomace (OMP). The wastes were composted in forced aeration static piles. Samples of the compost were collected at different times during composting. The elution profiles of HA obtained by gel filtration (Sephadex G-150) showed the disappearance of fractions with molecular weights ≤ 50 KDa and the contemporary increase of fraction with molecular weights ≥ 10² KDa. In this range, two fractions can be separated: the first one (A1) with molecular weights greater than 10² KDa and below 2 10² KDa, and the second one (A2) with molecular weight greater than 2 10² KDa. During composting, the ratio A2/A1 tends to reach a constant value which indicates the evolution toward the polymerization of HA. The ratio A2/A1 was named HAEI (Humic Acid Evolution Index). It varies with the material composted and the composting process and represents the maximum possible degree of HA polymerization. A comparison between HAEI and the usually used maturity indexes is also presented.     

Influence of glycosidic linkages and molecular weight on the fermentation of maltose-based oligosaccharides by human gut bacteria

A structure-function study was carried out to increase knowledge of how glycosidic linkages and molecular weights of carbohydrates contribute toward the selectivity of fermentation by gut bacteria. Oligosaccharides with maltose as the common carbohydrate source were used. Potentially prebiotic alternansucrase and dextransucrase maltose acceptor products were synthesized and separated into different molecular weights using a Bio-gel P2 column. These fractions were characterized by matrix-assisted laser desorption/ionization time-of-flight. Nonprebiotic maltooligosaccharides with degrees of polymerization (DP) from three to seven were commercially obtained for comparison. Growth selectivity of fecal bacteria on these oligosaccharides was studied using an anaerobic in vitro fermentation method. In general, carbohydrates of DP3 showed the highest selectivity towards bifidobacteria; however, oligosaccharides with a higher molecular weight (DP6-DP7) also resulted in a selective fermentation. Oligosaccharides with DPs above seven did not promote the growth of "beneficial" bacteria. The knowledge of how specific structures modify the gut microflora could help to find new prebiotic oligosaccharides.     

Effects of urease and nitrification inhibitors on the efficient use of urea for pastoral systems

Abstract

The humic substances contained in an animal organic waste were extracted and the total extract separated into three humic fractions with different molecular weights (low, F₁ <10³; medium, F₂, with molecular weights ranging from 10³ to 10⁴; and high, F₃ >10⁴). The C content was highest in F₂, the same fraction also showing the lowest N content. The molecular weight of the humic fractions influenced the electrical conductivity, the highest molecular weight resulting in the lowest degree of electrical conductivity. Membrane-controlled ultrafiltra-tion (the method used to separate the various fractions from the whole extract) was also suitable for purifying such enzymes as phosphatase and β-glucosidase: the total activity obtained from the three fractions was considerably greater than that determined in the whole extract, Pyrolysis-gas chromatography (Py-GC) applied to the whole extract and humic fractions showed that in the F₃ fraction (highest molecular weight) benzene was the major fragment while furfural was the major fragment of F₁ (lowest molecular weight). For this reason, the humification index benzene/toluene indicates that the fraction with the highest molecular weight was the most humified while the furfural/pyrrole ratio indicates that the fraction with the lowest molecular weight was the most degradable. The whole extract and the fraction F₁ had a negative effect on seed germination when the concentration was equivalent to 100 mg kg^?1 of C, while the germination index was higher than that of the control when only 10 mg kg^?1 were used. The F₂ fraction had a positive effect on germination regardless of the concentration used. When 10 mg kg^?1 of C of the humic substances studied were added to the nutrient solution for growth experiments with maize plants, F₃ led to increases in root weight and F₂ led to increases in shoot weight. An inhibitor effect was observed for fraction F₁.     

Weitere Untersuchungen über den Einbau von radioaktivem Schwefel in Blattproteine von Ölrettichpflanzen

Further investigations upon the ³⁵S-incorporation into some leaf proteins of oil radish plants . In order to supplement earlier results, concerning the ³⁵S-incorporation into leaf proteins of oil radish plants, a new experiment with increasing amounts of ³⁵S-labelled sulphate-sulphur was started under controlled climatic conditions. The ³⁵S-incorporation into some protein fractions was determined by methods of gel chromatography with various types of Sephadex, by the isoelectric focusing and the liquid scintillation counting. Fresh weights and content of pure protein of the plants were scarcely influenced by increasing amounts of sulphur supplied. In the S-variants 2 and 3 an increased relative incorporation of sulphur from the sulphur fertilization was observed in nitrogen- and sulphurcontaining compounds with lower molecular weights (m.w. < 4000) and into some protein fractions, especially with molecular weights, higher than 4–8 X10⁵ (Sephadex G-200). Determination of total ³⁵S-activity in the leaf material has shown, that the plants of all S-variants have taken up sulphur in nearly equal proportions. Therefore, a pronounced S-incorporation into some protein fractions must have taken place. It is assumed, that the ³⁵S-marked compounds with lower molecular weights are final products, and the ³⁵S-marked protein fractions with higher molecular weights may be enzymes of the secondary sulphur metabolism, the activity of which has probably been intensified in plants, supplied with higher amounts of sulphur. Comparing the earlier and the present investigations, it must be pointed out, that there are some differences in the results of protein fractionation and ³⁵S-patterns, probably caused by a varied performance of these experiments. Nevertheless, similar tendencies may be pointed out in the high rate of S-incorporation into protein fractions.     

Electron spin resonance (ESR) studies on the formation of roasting-induced antioxidative structures in coffee brews at different degrees of roast

The antioxidative properties of coffee brew fractions were studied using electron spin resonance spectroscopy using 2,2,6,6-tetramethyl-1-piperidin-1-oxyl (TEMPO) and Fremy's salt (nitrosodisulfonate) as stabilized radicals. TEMPO was scavenged by antioxidants formed during roasting and not by chlorogenic acid, whereas Fremy's salt was scavenged by all antioxidants tested including chlorogenic acid. The stabilized radical TEMPO allowed the exclusive measurement of roasting-induced antioxidants. The roasting-induced antioxidant activity of coffee brews increased with increasing degree of roast, and most of these antioxidants were formed during the initial roasting stage. The majority of these roasting-induced antioxidants were present in the high molecular weight fractions, indicating that the formation of these antioxidants preferably occurs at specific high molecular weight structures, likely being arabinogalactan and/or protein moieties which might be part of the melanoidin complex. It was found that chlorogenic acids most probably do not lose their antioxidant activity and phenolic characteristics upon incorporation in coffee melanoidins. The parameter fast reacting antioxidants (FRA) was introduced as an alternative for the antioxidative potential. FRA levels showed that coffee fractions rich in roasting-induced antioxidants exposed their antioxidant activity relatively slowly, which must be a consequence of its complex structure. Finally, the melanoidin content and the roasting-induced antioxidant activity showed a positive and linear correlation for the coffee brew fractions, showing that roasting-induced antioxidants are present within melanoidins. This is the first time that the formation of roasting-induced antioxidants could be directly correlated with the extent of Maillard reaction and melanoidin formation in a complex product such as coffee.     

Effect of Mechanical Dough Development on the Extractability of Wheat Storage Proteins from Bread Dough

Six wheat cultivars covering a range of quality parameters were mixed to various proportions of their optimum work input using mechanical dough development (MDD) mixers. Mixing and baking characteristics were determined and each dough was subsampled. The proteins were extracted for analysis by reversed-phase HPLC. Considerable protein mobilization appeared to occur during the MDD process, but the changes appeared to be cultivar-specific and did not indicate how mixing or baking behavior could be predicted. Protein content in extracted fractions was lowest for the weakest, poorest quality wheat but failed to consistently rank the stronger samples. Acetic acid insoluble protein level decreased with mixing as did extractable high molecular weight glutenin subunits. Gliadin protein level initially decreased with mixing before rising sharply with overmixing, while low molecular weight glutenin subunits displayed the reverse pattern. The rate of change of the extractability of the protein fractions with work input was greatest for the weakest samples and least for the stronger samples. However, when the protein quantity in the extractable fractions was plotted against relative work input, the rate of change of protein extractability did not appear to vary significantly between cultivars of different strengths.     

Characterization of white wine mannoproteins

Mannoproteins, Saccharomyces cerevisiae yeast polysaccharides, play a major role in wine processing and characteristics. A systematic characterization of these polymers in terms of chemical composition and molecular structure is addressed in this study. Mannoproteins were isolated from white wine through a sequence of operations that consisted of nanofiltration for concentration of macromolecules, polysaccharide precipitation with ethanol, and affinity chromatography on concanavalin A. The whole wine mannoproteins present a very broad molecular mass distribution with several populations. Two major populations with very different compositions were separated by size exclusion chromatography. The mannoproteins with higher molecular mass are a mannose homopolymer containing 10.3% protein. The mannoproteins with lower molecular mass consisted of 87.5% of mannose and some other residues and a protein content of 2.5%. The highest molecular weight mannoprotein structure was examined by (1)H and (13)C NMR spectroscopic techniques such as 1-D TOCSY, 2-D COSY, and 2-D HMQC.     

Elucidation of the emulsification properties of sugar beet pectin

A protocol has been developed to fractionate sugar beet pectin using hydrophobic affinity chromatography. Three samples eluted from the column using 4 M NaCl as solvent (fractions 1A, 1B, and 1C), two fractions eluted using 2 M NaCl (fractions 2A and 2B), and one fraction eluted using water (fraction 3). The fractions were shown to be very polydisperse, and differences between the GPC refractive index and UV absorbance (214 nm) elution profiles demonstrated chemical heterogeneity. They were found to contain significantly different proportions of protein (1A, 2.79%; 1B, 0.97%; 1C, 0.77%; 2A, 1.41%; 2B, 5.09%; and 3, 5.89%) and ferulic acid (approximately 1A, 0.5%; 1B, 0.5%; 1C, 0.9%; 2B, 1.5%; and 3, 2%). The weight-average molecular mass, M(w), of the fractions also varied (1A, 153 kDa; 1B, 155 kDa; 1C, 306 kDa; 2A, 562 kDa; 2B, 470 kDa; 3, 282 kDa). Three fractions, that is, 1A, 1B, and 3, produced orange oil emulsions with a relatively small droplet size that were stable over a period of weeks. The other three fractions (1C, 2A, and 2B with higher M(w) values) produced emulsions with an initially larger droplet size, and the droplet size increased considerably over time. The increased droplet size may be influenced by the viscosity of the aqueous continuous phase. There was no simple relationship between protein or ferulic acid content and emulsification ability. For example, fraction 1B, which contained the lowest proportion of both protein and ferulic acid, produced stable emulsions of similar droplet size to fraction 3, which contained the largest proportion of protein and ferulic acid. The role of protein in the emulsification process was investigated by measuring the amount of protein in the aqueous phase before and after emulsification. It was clearly demonstrated that proteinaceous material adsorbed at the oil-water interface. It is evident that the emulsification properties of sugar beet pectin are influenced by the accessibility of the protein and ferulic acid groups to the surface of the oil droplets, the proportion of ester groups, and the molecular mass distribution of the fractions.     

Effect of ascorbic acid in dough: reaction of oxidized glutathione with reactive thiol groups of wheat glutelin

The reactions of oxidized glutathione generated from endogenous glutathione by the addition of ascorbic acid (AA) prior to dough mixing on free thiol groups of gluten proteins have been investigated. A small amount of (35)S-labeled glutathione was added as a tracer to identify the reaction products of GSSG and free protein thiols by radioactivity measurement. First, gluten was isolated from the dough, then the gliadins were extracted, and residual glutenin was partially hydrolyzed with thermolysin. After preseparation by gel permeation chromatography, the fractions with the highest radioactivity were separated by high-performance liquid chromatography. Radioactive peptides were identified, isolated, sequenced, and assigned to amino acid sequences of gluten protein components. The isolated peptides contained exclusively the cysteine residues C(b) and C(x) of low molecular weight subunits of glutenin, which are supposed to be highly reactive in forming intermolecular disulfide bonds. From these results it can be assumed that the cysteine residues C(b) and C(x) of the low molecular weight subunits of glutenin are at least partly present in the thiol form in flour. During dough mixing they are converted to protein-protein disulfides or glutathione-protein mixed disulfides by thiol/disulfide interchange reactions. Oxidized glutathione necessary for this reaction is generated from glutathione by the action of AA. These results are in accordance with the major hypothesis about the mechanism of action of AA.     

Evaluation of mushroom dietary fiber (nonstarch polysaccharides) from sclerotia of Pleurotus tuber-regium (Fries) singer as a potential antitumor agent

Mushroom dietary fiber or nonstarch polysaccharides (NSPs) that were soluble in hot alkali and belonged to the beta-glucan type were isolated from the sclerotia of an edible mushroom, Pleurotus tuber-regium. The mushroom NSPs were further separated into a number of fractions [hot alkali extracts (HAEs)] with weight-average molecular weights (M(w)) ranging from 1 x 10(4) to 42.2 x 10(4). The HAE fractions [with M(w) of (5.8-17.1) x 10(4)] administered intraperitoneally at a dose of 20 mg/kg of body weight to BALB/c mice implanted with solid tumor Sarcoma 180 were found to be effective in inhibiting tumor proliferation with an inhibition ratio of > or =50%. In vitro experiments using human tumor cell lines HL-60 and HepG2 had shown that HAE fractions with M(w) of (5.8-42.2) x 10(4) also had antiproliferative activity at three different concentrations (50, 100, and 200 microg/mL) toward the tumor cell lines tested. All HAE fractions did not inhibit the growth of a normal kidney cell line (Vero) from monkey. It is therefore postulated that the antitumoral effect of NSPs from the sclerotia of P. tuber-regium is probably host-mediated and cytocidal.     

Characterization of methanol-soluble and methanol-insoluble proteins from developing peanut seed

The 85% methanol-soluble proteins are known to specifically contribute to the production of flavor of roasted peanut. To determine the nature of the 85% methanol-soluble proteins, they were isolated from the peanut seed, and the 85% methanol-soluble (MS) and 85% methanol-insoluble (MIS) fractions were characterized using polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis. The results showed that the 85% MS fraction contained lower amounts (9-10%) of protein than the MIS fraction (15-33%). Protein content of the MIS fraction increased more significantly during seed maturation than it did in the MS fraction. Unlike the protein, free amino acids and soluble sugars levels of the MS fraction decreased significantly during seed maturation. The 85% MS fraction contained predominantly low molecular weight (<20 kDa) proteins/polypeptides, whereas the MIS fraction contained a mixture of polypeptides with molecular weight between 14 kDa and 90 kDa. SDS-PAGE showed no major changes in the polypeptide composition of the MS fraction during seed maturation. Capillary electrophoretic analysis revealed major qualitative and quantitative changes in the protein and polypeptide composition of the MS and MIS fractions during seed maturation. Fatty acid analysis of these fractions indicated that the MS fraction is lipoprotein in nature and rich in oleic and linoleic acids.     

鳖甲胶原蛋白酶解物的分离纯化及体外抗氧化性研究

为建立中华鳖背甲中胶原蛋白酶解物(CH)的制备条件,并明确其体外抗氧化活性,本研究采用单因素试验探讨鳖甲胶原蛋白酶解物的分离条件。结果表明,其最佳提取工艺参数:胃蛋白酶添加量 4 000 U·g^-1、pH值2.5、酶解温度35℃。通过透析纯化后获得体外抗氧化活性最好的组分为分子量<50 kDa的样品。该样品经Sephadex G-75凝胶色谱分离纯化得到2个组分,其中GP-2组分对O2-·、OH·和DPPH·3种自由基清除能力最强;之后对GP-2组分进行离子交换层析分离同样获得2个组分,其中P1组分体外抗氧化活性高于GP-2,其对DPPH·、OH·、O2-·清除率分别为81.67%、65.45%和6.78%。经SDS-PAGE电泳确定P1组分的分子量约40 kDa。本研究结果为鳖甲的开发利用及鳖源新型抗氧化物质的研发提供了一定的理论基础。     

Chemical characterization of the high molecular weight material extracted with hot water from green and roasted arabica coffee

The polysaccharides present in coffee infusions are known to contribute to the organoleptic characteristics of the drink, such as the creamy sensation perceived in the mouth known as "body", the release of aroma substances, and the stability of espresso coffee foam. To increase the knowledge about the origin, composition, and structure of the polysaccharide fraction, the high molecular weight material (HMWM) was extracted with hot water from two green and roasted ground arabica coffees: Costa Rica (wet processed) and Brazil (dry processed). The polysaccharides present in the green coffees HMWM were arabinogalactans (62%), galactomannans (24%), and glucans, and those found in roasted coffees were galactomannans (69%) and arabinogalactans (28%). The polysaccharides of the HMWM of the roasted coffees were less branched than those of the green coffees. The major green coffee proteins had molecular weights of 58 and 38 kDa, and the 58 kDa protein had two subunits, of 38 and 20 kDa, possibly linked by disulfide bonds. The protein fraction obtained from roasted coffees had only a defined band with < or =14 kDa and a diffuse band with >200 kDa. The majority of the galactomannans were precipitated with solutions of 50% ethanol, and the size-exclusion chromatography of the roasted fractions showed coelution of polysaccharides, proteins, phenolics, and brown compounds. The use of strong hydrogen and hydrophobic dissociation conditions allowed us to conclude that the phenolics and brown compounds were linked by covalent bonds to the polymeric material.