Characterization and fermentability of an ethanol soluble high molecular weight coffee fraction

Brews from differently roasted Arabica coffees were shown to contain 8-12% ethanol soluble substances with molecular masses greater than 2 kDa, possibly contributing to their dietary fiber contents. About 13% of these substances were nondigestible carbohydrates, mainly arabinogalactans. The nondigestible high molecular weight ethanol soluble fraction (HESF) of the medium roasted coffee brew was further characterized and subjected to in vitro fermentation with human fecal bacteria. In addition to carbohydrates, HESF contained proteins/peptides (approximately 20%), but the main fraction was composed of structurally unknown Maillard reaction products. From NMR spectroscopy, we conclude that intact caffeic and ferulic acid derivatives were not incorporated into the melanoidins to a significant extent. Stepwise ultrafiltration and gel filtration indicated a large variation in the molecular weights of HESF constituents. Coffee HESF was shown to be less fermentable by fecal bacteria than soluble coffee fiber isolated by the enzymatic-gravimetric methodology, and because of its lower carbohydrate content, less short-chain fatty acids were produced during the fermentation. Total cell counts, destructive chemical analysis, and NMR spectroscopy indicated that coffee carbohydrates are the preferred substrates for colonic microbiota. However, NMR spectra, absorbances at 405 nm, and nonprotein nitrogen contents showed that noncarbohydrate and nonprotein compounds were also utilized to some extent but the bacterial species involved in this degradation remain to be identified.     

Investigations on the high molecular weight foaming fractions of espresso coffee

The target of the present work was the chemical, technological, and sensorial characterization of the brown polymers (foaming fractions) of freshly prepared espresso coffee. The total foaming fraction (TFF) was precipitated with ammonium sulfate from the defatted freshly prepared beverage and then subfractionated by adding 2-propanol/water to give an insoluble fraction (foaming fraction A, FFA) and a soluble fraction (foaming fraction B, FFB). The former is almost colorless, has a higher molecular weight and a lower nitrogen content, and contains mostly polysaccharides, whereas the latter has a lower molecular weight and a higher protein/melanoidin content, which results in a darker color. FFB showed greater foaming capability, but FFA contributed to the stability of the foam. FFB was further fractionated with solid-phase extraction and characterized by different analytical methods (size exclusion chromatography, UV, HPLC-DAD, 1H NMR). All of the melanoidin-rich fractions showed antioxidant properties with the 2,2-diphenyl-1-picrylhydrazyl hydrate method.     

Low molecular weight melanoidins in coffee brew

Analysis of low molecular weight (LMw) coffee brew melanoidins is challenging due to the presence of many non-melanoidin components that complicate analysis. This study focused on the isolation of LMw coffee brew melanoidins by separation of melanoidins from non-melanoidin components that are present in LMw coffee brew material. LMw coffee fractions differing in polarity were obtained by reversed-phase solid phase extraction and their melanoidin, sugar, nitrogen, caffeine, trigonelline, 5-caffeoylquinic acid, quinic acid, caffeic acid, and phenolic groups contents were determined. The sugar composition, the charge properties, and the absorbance at various wavelengths were investigated as well. The majority of the LMw melanoidins were found to have an apolar character, whereas most non-melanoidins have a polar character. The three isolated melanoidin-rich fractions represented 56% of the LMw coffee melanoidins and were free from non-melanoidin components. Spectroscopic analysis revealed that the melanoidins isolated showed similar features as high molecular weight coffee melanoidins. All three melanoidin fractions contained approximately 3% nitrogen, indicating the presence of incorporated amino acids or proteins. Surprisingly, glucose was the main sugar present in these melanoidins, and it was reasoned that sucrose is the most likely source for this glucose within the melanoidin structure. It was also found that LMw melanoidins exposed a negative charge, and this negative charge was inversely proportional to the apolar character of the melanoidins. Phenolic group levels as high as 47% were found, which could be explained by the incorporation of chlorogenic acids in these melanoidins.     

High molecular weight melanoidins from coffee brew

The composition of high molecular weight (HMw) coffee melanoidin populations, obtained after ethanol precipitation, was studied. The specific extinction coefficient (K(mix)) at 280, 325, 405 nm, sugar composition, phenolic group content, nitrogen content, amino acid composition, and non-protein nitrogen (NPN) content were investigated. Results show that most HMw coffee melanoidins are soluble at high ethanol concentrations. The amino acid composition of the HMw fractions was similar, while 17% (w/w) of the nitrogen was NPN, probably originating from degraded amino acids/proteins and now part of melanoidins. A strong correlation between the melanoidin content, the NPN, and protein content was found. It was concluded that proteins are incorporated into the melanoidins and that the degree of chemical modification, for example, by phenolic groups, determines the solubility of melanoidins in ethanol. Although the existence of covalent interaction between melanoidins and polysaccharides were not proven in this study, the findings suggest that especially arabinogalactan is likely involved in melanoidin formation. Finally, phenolic groups were present in the HMw fraction of coffee, and a correlation was found with the melanoidin concentration.     

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.     

High molecular weight coffee melanoidins are inhibitors for matrix metalloproteases

High molecular (above 10 kDa) melanoidins isolated from coffee beans of varying roasting degree were found to be efficient inhibitors for the zinc-containing matrix metalloproteases MMP-1, MMP-2, and MMP-9 with IC(50) values ranging between 0.2 and 1.1 mg/mL in vitro. The inhibitory potential increased with roasting degree. No or only slight inhibition of other zinc-containing peptidases closely related to MMPs, namely, Clostridium histolyticum collagenase and angiotensin converting enzyme, was found, indicating specific structural features of melanoidins to be responsible for the interaction with MMPs. A continuous increase on the apparent molecular weight of melanoidins as well as incorporation of phenolic substances into the melanoidin structure with progress of roasting was observed, concomitant with a significant increase in the carbon/nitrogen of the melanoidins. This suggests that the melanoidins are mainly formed by incorporation of carbohydrates and phenolic compounds onto a proteinaceous backbone. As MMP-1, MMP-2, and MMP-9 play a pivotal role in pathogenesis of colorectal cancer, studies on possible physiological effects of melanoidins are mandatory.     

Antibacterial activity of coffee extracts and selected coffee chemical compounds against enterobacteria

The in vitro antimicrobial activity of commercial coffee extracts and chemical compounds was investigated on nine strains of enterobacteria. The antimicrobial activity investigated by the disc diffusion method was observed in both the extracts and tested chemical compounds. Even though pH, color, and the contents of trigonelline, caffeine, and chlorogenic acids differed significantly among the coffee extracts, no significant differences were observed in their antimicrobial activity. Caffeic acid and trigonelline showed similar inhibitory effect against the growth of the microorganisms. Caffeine, chlorogenic acid, and protocatechuic acid showed particularly strong effect against Serratia marcescens and Enterobacter cloacae. The IC(50) and IC(90) for the compounds determined by the microtiter plate method indicated that trigonelline, caffeine, and protocatechuic acids are potential natural antimicrobial agents against Salmonella enterica. The concentrations of caffeine found in coffee extracts are enough to warrant 50% of the antimicrobial effect against S. enterica, which is relevant to human safety.     

Productivity and some properties of immunoglobulin specific against Streptococcus mutans serotype c in chicken egg yolk (IgY)

Hens were immunized on thighs by using whole cells of Streptococcus mutans MT8148 serotype c strain as antigen through intramuscular (im) and subcutaneous (sc) routes to investigate the difference of immunization reactions and the changes in yolk antibody activities against antigen after initial immunization. Several properties of crude IgY were examined to evaluate the stability during food processing. Results showed that the specificity of IgY of im treated hens was nearly 10 times as high as those of sc treated antibody. IgY from the hens immunized with the serotype c strain showed significant cross-reactions against serotypes e and f, while minor reactions against serotypes a, b, d, and g were observed. In thermal stability tests, IgY activity in both yolk and crude IgY decreased with the increasing temperature, from 70 to 80 degrees C, but the thermal denaturation rates between those two samples were not significantly different. The addition of high levels sucrose, maltose, glycerol, or 2% glycine displayed effective protection against thermal denaturation of IgY. Lyophilized yolk-5% gum arabic powder showed better stability against proteases.     

Interactions between volatile and nonvolatile coffee components. 1. Screening of nonvolatile components

This study is the first of two publications that investigate the phenomena of coffee nonvolatiles interacting with coffee volatile compounds. The purpose was to identify which coffee nonvolatile(s) are responsible for the interactions observed between nonvolatile coffee brew constituents and thiols, sulfides, pyrroles, and diketones. The overall interaction of these compounds with coffee brews prepared with green coffee beans roasted at three different roasting levels (light, medium, and dark), purified nonvolatiles, and medium roasted coffee brew fractions (1% solids after 1 or 24 h) was measured using a headspace solid-phase microextraction technique. The dark roasted coffee brew was slightly more reactive toward the selected compounds than the light roasted coffee brew. Selected pure coffee constituents, such as caffeine, trigonelline, arabinogalactans, chlorogenic acid, and caffeic acid, showed few interactions with the coffee volatiles. Upon fractionation of medium roasted coffee brew by solid-phase extraction, dialysis, size exclusion chromatography, or anion exchange chromatography, characterization of each fraction, evaluation of the interactions with the aromas, and correlation between the chemical composition of the fractions and the magnitude of the interactions, the following general conclusions were drawn. (1) Low molecular weight and positively charged melanoidins present significant interactions. (2) Strong correlations were shown between the melanoidin and protein/peptide content, on one hand, and the extent of interactions, on the other hand (R = 0.83-0.98, depending on the volatile compound). (3) Chlorogenic acids and carbohydrates play a secondary role, because only weak correlations with the interactions were found in complex matrixes.     

Antioxidative activities of fractions obtained from brewed coffee

The antioxidative activity of column chromatographic fractions obtained from brewed coffee was investigated to find antioxidants and to assess the benefit of coffee drinking. The dichloromethane extract inhibited hexanal oxidation by 100 and 50% for 15 days and 30 days, respectively, at the level of 5 microg/mL. A GC/MS analysis of fractions, which exhibited oxidative activity, revealed the presence of antioxidative heterocyclic compounds including furans, pyrroles, and maltol. The residual aqueous solution exhibited slight antioxidative activity. The inhibitory activity (%) of the seven fractions from an aqueous solution toward malonaldehde formation from lipid oxidation ranged from 10 to 90 at a level of 300 microg/mL. The results indicate that brewed coffee contains many antioxidants and consumption of antioxidant-rich brewed coffee may inhibit diseases caused by oxidative damages.     

红壤主要土壤组分对低分子量有机酸吸附的研究

通过一次平衡法研究了双氧水去除有机质、添加1%腐殖酸和DCB法(连二亚硫酸钠-柠檬酸钠-碳酸氢钠)去除游离氧化铁、铝对红黏土发育的红壤吸附草酸、柠檬酸、酒石酸和苹果酸的影响。结果表明,去除有机质后,由于受溶液pH、表面吸附点位变化、土壤结构变化、表面基团活性变化、草酸根生成等多种因素的影响,红壤对低分子量有机酸的吸附量虽略有增加,但增加并不明显。添加腐殖酸培养一个月后,由于受土壤表面电荷变化、吸附点位覆盖、氧化铁活性改变、基团质子化等因素的影响,红壤对低分子量有机酸的吸附量虽有轻微的减少,但减少也不明显。去除占土壤总量3.79%的游离氧化铁、铝后,土壤表面正电荷将会显著减少,而导致红壤对低分子量有机酸的吸附量显著减少,其减少程度因有机酸种类而异,与原土对有机酸的最大吸附量(Sm)大小顺序相反。     

Characterization and quantitation of low and high molecular weight phenolic compounds in apple seeds

The phenolic constituents of seeds of 12 different apple cultivars were fractionated by sequential extraction with aqueous acetone (30:70, v/v) and ethyl acetate after hexane extraction of the lipids. Low molecular weight phenolic compounds were individually quantitated by RP-HPLC-DAD. The contents of extractable and nonextractable procyanidins were determined by applying RP-HPLC following thiolysis and n-butanol/HCl hydrolysis, respectively. As expected, the results revealed marked differences of the ethyl acetate extracts, aqueous acetone extracts, and insoluble residues with regard to contents and mean degrees of polymerization of procyanidins. Total phenolic contents in the defatted apple seed residues ranged between 18.4 and 99.8 mg/g. Phloridzin was the most abundant phenolic compound, representing 79-92% of monomeric polyphenols. Yields of phenolic compounds significantly differed among the cultivars under study, with seeds of cider apples generally being richer in phloridzin and catechins than seeds of dessert apple cultivars. This is the first study presenting comprehensive data on the contents of phenolic compounds in apple seeds comprising extractable and nonextractable procyanidins. Furthermore, the present work points out a strategy for the sustainable and complete exploitation of apple seeds as valuable agro-industrial byproducts, in particular as a rich source of phloridzin and antioxidant flavanols.     

Polymorphism of high molecular weight glutenin subunits in three species of Aegilops

A collection of 136 accessions of Aegilops umbellulata (39), Ae. comosa (75) and Ae. markgrafii (22) was analysed for high-molecular-weight (HMW) glutenin subunits composition. The homogeneity of the accessions was studied and 55.1% of the collection was homogeneous for HMW glutenin subunits (29 Ae. umbellulata, 33 Ae. comosa and 14 Ae. markgrafii). The HMW glutenin subunits of Ae. umbellulata are encoded by the Glu-U1 locus; in Ae. comosa results showed that this proteins are encoded at the 1M chromosome, and the locus was named Glu-M1. In Ae. markgrafii it was assumed that HMW glutenin subunits were encoded by an homoeologous locus and it was named Glu-C1. All the accessions of Ae. umbellulata and Ae. markgrafii expressed both, x-type and y-type subunits. Among the Ae. comosa accessions, only one expressed an x-type subunit alone. All the accessions of Ae. umbellulata and some of Ae. comosa had x-type glutenins of higher molecular weights than those commonly present in bread wheat. A total of 8 alleles were detected at the Glu-U1 locus, 11 at the Glu-M1 and 4 at the Glu-C1. The new HMW glutenin variation found in this work suggests their possible utilisation in breeding for wheat quality.     

Antioxidative activities of heterocyclic compounds formed in brewed coffee

Typical volatile heterocyclic compounds found in brewed coffee extracts-pyrroles, furans, thiophenes, and thiazoles-were examined for antioxidative activity, which was determined by measuring the oxidative conversion of hexanal to hexanoic acid using gas chromatography. 2-Acetylpyrrole, 1-methylpyrrole, and pyrrole inhibited hexanal oxidation by 98, 87, and 78%, respectively, at a concentration of 500 microgram/mL over a period of 30 days. 2-Methylfuran, which inhibited hexanal oxidation by 90% at all concentrations tested (500, 200, and 100 microgram/mL) for a 30-day period, exhibited the greatest activity among furans tested. Similarly, 2-methylthiophene, which inhibited hexanal oxidation by almost 100% at a concentration of 500 microgram/mL over 30 days, exhibited the greatest activity among the thiophenes tested. In general, thiazoles were ineffective antioxidants at all concentrations tested. However, 4,5-dimethylthiazole was able to inhibit hexanal oxidation by 50% at the highest level tested (500 microgram/mL). 2-Acetylpyrrole, 2-methylfuran, and 2-methylthiophene at concentrations of 500, 200, and 100 microgram/mL and furan at a concentration of 500 microgram/mL exhibited antioxidative activities comparable to that of the synthetic antioxidant butylated hydroxytoluene at a concentration of 50 microgram/mL.     

Mechanisms of the antioxidant activity of a high molecular weight fraction of whey

The antioxidant mechanisms of whey proteins in a Tween 20-stabilized salmon oil-in-water emulsion were investigated. The antioxidant activity of the high molecular weight (HMW) fraction of whey from pasteurized milk was found to increase with concentration, as determined by its ability to inhibit TBARS and lipid peroxide formation. The ability of sulfhydryl-blocked whey to inhibit TBARS formation was reduced 60% compared to the HMW fraction alone at 7 days of storage. HMW fraction was able to scavenge peroxyl radicals, with scavenging decreasing approximately 20% when sulfhydryls were blocked. HMW fraction was able to chelate iron away from the surface of negatively charged BSA-stabilized emulsion droplets, indicating that the whey proteins were able to chelate iron. A better understanding of the mechanisms by which whey proteins inhibit lipid oxidation could increase the use of whey proteins as food antioxidants.     

Chemical characterization of red wine grape (Vitis vinifera and Vitis interspecific hybrids) and pomace phenolic extracts and their biological activity against Streptococcus mutans

Grapes are rich sources of potentially bioactive polyphenols. However, the phenolic content is variable depending on grape variety, and may be modified during vinification. In this study, we examined the chemical composition and biological activity of phenolic extracts prepared from several red wine grape varieties and their fermented byproduct of winemaking (pomace) on some of the virulence properties of Streptococcus mutans a well-known dental pathogen. Grape phenolic extracts were obtained from Vitis vinifera varieties Cabernet Franc and Pinot Noir and Vitis interspecific hybrid varieties Baco Noir and Noiret. The anthocyanins and flavan-3-ols content were highly variable depending on grape variety and type of extract (whole fruit vs fermented pomace). Nevertheless, all grape phenolic extracts remarkably inhibited glucosyltransferases B and C (70-85% inhibition) at concentrations as low as 62.5 microg/mL (P < 0.01). Furthermore, the glycolytic pH-drop by S. mutans cells was inhibited by the grape extracts without affecting the bacterial viability; an effect that can be attributed to partial inhibition of F-ATPase activity (30-65% inhibition at 125 microg/mL; P < 0.01). The biological activity of fermented pomace was either as effective as or significantly better than whole fruit grape extracts. The results showed that grape phenolic extracts, especially from pomace, are highly effective against specific virulence traits of S. mutans despite major differences in their phenolic content.     

Evolution of robusta green coffee redox enzymatic activities with maturation

Oxidation reactions in coffee involve redox-sensitive polyphenols and appear to control the fragmentation of coffee storage proteins both in solution and during roasting. Coffee-specific nitrogenous flavor precursors may derive from this process. Accordingly, data converge to suggest that the redox status of the green bean before roasting might control the development of subsequent redox reactions during roasting. Consequently, we decided to identify biological events that may trigger or prevent oxidation during maturation of the coffee cherry and set the final redox status of the green bean. In a previous study, we observed that the sensitivity of green coffee to oxidative processes decreased along maturation. By using the very same samples originating from open-pollinated Robusta clones, we followed the activity of three essential redox enzymes: catalase (CAT), peroxidase (POD) and polyphenoloxidase (PPO). While CAT and POD activities increased with maturation, PPO activities decreased. Thanks to the identification of an atypical immature subclass, it appeared that CAT might be an essential factor in setting the final redox status of the green bean before the roasting event.     

Determination of molecular weight of agars and effect of the molecular weight on the glass transition

A novel procedure to determine the molecular weight (MW) and MW distributions for various agars is described. The MW values of commercial agars, an agarose, an agaropectin, and hydrolyzed agaroses were determined by size exclusion chromatography-low angle laser light scattering, using 4.0 M guanidine hydrochloride as eluent to avoid gelation. The MW for the commercial agars was between 106 400 and 243 500 with polydispersity between 1.283 and 6. 600. The MW of the agarose separated from a commercial agar was lower than that of the agaropectin. To prepare agaroses with different MW values, the obtained agarose was hydrolyzed. The MW of the agarose decreased with hydrolysis time, and the polydispersity, on the contrary, increased. The glass transition temperature (T(g)) of agarose with different MW values and that of agaropectin were measured by differential scanning calorimetry. The T(g) of the agarose was higher than that of the agaropectin with higher MW. The T(g) of agarose increased with MW.     

In vitro antioxidant and ex vivo protective activities of green and roasted coffee

The antioxidant properties of green and roasted coffee, in relation to species (Coffea arabica and Coffea robusta) and degree of roasting (light, medium, dark), were investigated. These properties were evaluated by determining the reducing substances (RS) of coffee and its antioxidant activity (AA) in vitro (model system beta-carotene-linoleic acid) and ex vivo as protective activity (PA) against rat liver cell microsome lipid peroxidation measured as TBA-reacting substances. RS of C. robustasamples were found to be significantly higher when compared to those of C. arabica samples (p < 0.001). AA for green coffee samples were slightly higher than for the corresponding roasted samples while PA was significantly lower in green coffee compared to that of all roasted samples (p < 0.001). Extraction with three different organic solvents (ethyl acetate, ethyl ether, and dichloromethane) showed that the most protective compounds are extracted from acidified dark roasted coffee solutions with ethyl acetate. The analysis of acidic extract by gel filtration chromatography (GFC) gave five fractions. Higher molecular mass fractions were found to possess antioxidant activity while the lower molecular mass fractions showed protective activity. The small amounts of these acidic, low molecular mass protective fractions isolated indicate that they contain very strong protective agents.     

Flavoring components of raw monsooned arabica coffee and their changes during radiation processing

Volatile aroma principles, nonvolatile taste constituents (caffeine and chlorogenic and caffeic acids), and glycosidically bound aroma compounds of monsooned and nonmonsooned raw arabica coffee were analyzed using gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC). Among the most potent odor active constituents known to contribute to the aroma of the green beans, 3-isopropyl-2-methoxypyrazine, 3-isobutyl-2-methoxypyrazine, 4-vinylguaiacol, beta-damascenone, (E)-2-nonenal, trans,trans-2,4-decadienal, phenylacetaldehyde, and 3-methylbutyric acid were detected by GC-MS in both samples. A decrease in content of methoxypyrazines and an increase in 4-vinylguaiacol and isoeugenol resulted in a dominant spicy note of monsooned coffee. These phenolic compounds exist partly as their glycosides, and their release from the bound precursors during monsooning accounted for their higher content in monsooned coffee. A considerable decrease in astringent chlorogenic acid as a consequence of hydrolysis to bitter caffeic acid was noted in monsooned coffee. Radiation processing of nonmonsooned beans at a dose of 5 kGy resulted in an increased rate of monsooning. At this dose a quantitative increase in most of the aroma active components could be observed in all samples studied. Hydrolysis of chlorogenic acid to caffeic acid was noted in radiation-processed monsooned coffee beans irrespective of whether the treatment was carried out before or after monsooning. These changes were, however, not observed in irradiated, nonmonsooned coffee beans, suggesting an enzymatic rather than a radiolytic cleavage of chlorogenic acid. A rationale behind the mechanism of monsooning and radiation-induced enhancement of the monsooning process is discussed.