Quantitative precursor studies on di- and trihydroxybenzene formation during coffee roasting using "in bean" model experiments and stable isotope dilution analysis

The objective of this study was to investigate the potential of various raw bean components as precursors of pyrogallol (1), hydroxyhydroquinone (2), catechol (3), 4-ethylcatechol (4), 4-methylcatechol (5), and 3-methylcatechol (6) under quasi "natural" roasting conditions by using the recently developed "in bean" model roast experiments. Freeze-dried, fully extracted bean shells were loaded with aqueous solutions of either single coffee compounds or fractions isolated from the raw bean solubles. After freeze-drying, these reconstituted beans were roasted, aqueous coffee brews were prepared, and the target phenols were quantified by means of a stable isotope dilution assay with LC-MS/MS detection. On the basis of the quantitative data, it can be concluded that upon coffee bean roasting, catechol (3) is primarily formed by degradation of caffeoylquinic acids from both the caffeic acid and the quinic acid moiety of the molecule, as well as from Maillard-type reactions from carbohydrates and amino acids. In contrast, pyrogallol (1) and hydroxyhydroquinone (2) are efficiently generated from carbohydrates and amino acids and, in addition, from free or chlorogenic acid bound quinic acid moieties. 4-Ethylcatechol (4) is exclusively generated upon thermal breakdown of caffeic acid moieties. 3-Methylcatechol (6) is formed primarily from the Maillard reactions and, to a minor extent, also from various phenolic precursors, whereas 4-methylcatechol (5) is produced in trace amounts only from all of the different precursors investigated. On the basis of this precursor study, reaction routes explaining the formation of the target phenols are proposed.     

Screening of raw coffee for thiol binding site precursors using "in bean" model roasting experiments

The purpose of the following study was to investigate the influence of coffee roasting on the thiol-binding activity of coffee beverages, and to investigate the potential of various green bean compounds as precursors of thiol-binding sites by using promising "in bean" model roast experiments. Headspace gas chromatographic analysis on coffee brews incubated in the presence of the roasty-sulfury smelling 2-furfurylthiol for 20 min at 30 degrees C in septum-closed vessels revealed that the amounts of "free" thiol decreased drastically with increasing the roasting degree of the beans used for preparation of the brews. A half-maximal binding capacity (BC(50)) of 183 mg of 2-furfurylthiol per liter of standard coffee beverage was determined for a roasted coffee (CTN value of 67), thus demonstrating that enormous amounts of the odor-active thiol are "bound" by the coffee. Furthermore, biomimetic "in bean" precursor experiments have been performed in order to elucidate the precursor for the thiol-binding sites in the raw coffee bean. These experiments opened the possibility of studying coffee model reactions under quasi-natural roasting conditions and undoubtedly identified chlorogenic acids as well as thermal degradation products caffeic acid and quinic acid as important precursors for low-molecular-weight thiol-binding sites. In particular, when roasted in the presence of transition metal ions, chlorogenic acids and even more caffeic acid showed thiol-binding activity which was comparable to the activity measured for the authentic coffee brew.     

Roasting effects on formation mechanisms of coffee brew melanoidins

The effect of the roasting degree on coffee brew melanoidin properties and formation mechanisms was studied. Coffee brew fractions differing in molecular weight (Mw) were isolated from green and light-, medium-, and dark-roasted coffee beans. Isolated fractions were characterized for their melanoidin, nitrogen, protein, phenolic groups, chlorogenic acid, quinic acid, caffeic acid, and sugar content. It was found that the melanoidin level in all fractions correlated with both the nitrogen and the protein content. The melanoidin level also correlated with the phenolic groups' level and ester-linked quinic acid level. It was concluded that proteins and chlorogenic acids should be primarily involved in melanoidin formation. Initial roasting, from green to light-roasted beans, especially led to the formation of intermediate Mw (IMw) melanoidins when compared to high Mw (HMw) melanoidins. Indications were found that this IMw melanoidin formation is mainly due to Maillard reactions and chlorogenic acid incorporation reactions between chlorogenic acids, sucrose, and amino acids/protein fragments. Additionally, it was found that prolonged roasting predominantly led to formation melanoidins with a high Mw. Furthermore, arabinogalactans seem to be relatively more involved in melanoidin formation than galactomannans. It was hypothesized that chromophores may be formed or attached through the arabinose moiety of arabinogalactan proteins (AGP). Finally, it could be concluded that galactomannans are continuously incorporated in AGP-melanoidins upon roasting.     

Insight into the Mechanism of Coffee Melanoidin Formation Using Modified "in Bean" Models

To study the mechanism of coffee melanoidin formation, green coffee beans were prepared by (1) removal of the hot water extractable components (WECoffee); (2) direct incorporation of sucrose (SucCoffee); and (3) direct incorporation of type II arabinogalactan-proteins (AGPCoffee). As a control of sucrose and AGP incorporation, lyophilized green coffee beans were also immersed in water (control). The original coffee and the four modified "in bean" coffee models were roasted and their chemical characteristics compared. The formation of material not identified as carbohydrates or protein, usually referred to as "unknown material" and related to melanoidins, and the development of the brown color during coffee roasting have distinct origins. Therefore, a new parameter for coffee melanoidin evaluation, named the "melanoidin browning index" (MBI), was introduced to handle simultaneously the two concepts. Sucrose is important for the formation of colored structures but not to the formation of "unknown material". Type II AGPs also increase the brown color of the melanoidins, but did not increase the amount of "unknown material". The green coffee hot water extractable components are essential for coffee melanoidin formation during roasting. The cell wall material was able to generate a large amount of "unknown material". The galactomannans modified by the roasting and the melanoidin populations enriched in galactomannans accounted for 47% of the high molecular weight brown color material, showing that these polysaccharides are very relevant for coffee melanoidin formation.     

Two-dimensional 1H-13C nuclear magnetic resonance (NMR)-based comprehensive analysis of roasted coffee bean extract

Coffee was characterized by proton and carbon nuclear magnetic resonance (NMR) spectroscopy. To identify the coffee components, a detailed and approximately 90% signal assignment was carried out using various two-dimensional NMR spectra and a spiking method, in which authentic compounds were added to the roasted coffee bean extract (RCBE) sample. A total of 24 coffee components, including 5 polysaccharide units, 3 stereoisomers of chlorogenic acids, and 2 stereoisomers of quinic acids, were identified with the NMR spectra of RCBE. On the basis of the signal assignment, state analyses were further launched for the metal ion-citrate complexes and caffeine-chlorogenate complexes. On the basis of the signal integration, the coffee components were successfully quantified. This NMR methodology yielded detailed information on RCBE using only a single observation and provides a systemic approach for the analysis of other complex mixtures.     

Hierarchical scheme for LC-MSn identification of chlorogenic acids

The fragmentation behavior of 18 chlorogenic acids that are not substituted at position 1 has been investigated using LC-MS(4) applied to a methanolic coffee bean extract and commercial cider (hard cider). Using LC-MS(3), it is possible to discriminate between each of the three isomers of p-coumaroylquinic acid, caffeoylquinic acid, feruloylquinic acid, and dicaffeoylquinic acid, and a hierarchical key has been prepared to facilitate this process when standards are not available. MS(4) fragmentations further support these assignments, but were not essential in reaching them. The distinctive behavior of 4-acyl and 3-acyl chlorogenic acids compared with the 5-acyl chlorogenic acids is a key factor permitting these assignments. The fragmentation patterns are dependent upon the particular stereochemical relationships between the individual substituents on the quinic acid moiety. Fragmentation is facilitated by 1,2-acyl participation and proceeds through quinic acid conformers in which the relevant substituents transiently adopt a 1,3-syn-diaxial relationship. Selected ion monitoring at m/z 529 clearly indicated the presence in coffee of six caffeoylferuloylquinic acid isomers, whereas previously only two or three had been demonstrated. The hierarchical key permitted specific structures to be assigned to each of the six isomers. These assignments are internally consistent and consistent with the limited data previously available.     

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.     

Isolation and determination of alpha-dicarbonyl compounds by RP-HPLC-DAD in green and roasted coffee

Glyoxal, methylglyoxal, and diacetyl formed as Maillard reaction products in heat-treated food were determined in coffee extracts (coffee brews) obtained from green beans and beans with different degrees of roast. The compounds have been reported to be mutagenic in vitro and genotoxic in experimental animals in a number of papers. More recently, alpha-dicarbonyl compounds have been implicated in the glycation process. Our data show that small amounts of glyoxal and methylglyoxal occur naturally in green coffee beans. Their concentrations increase in the early phases of the roasting process and then decline. Conversely, diacetyl is not found in green beans and forms later in the roasting process. Therefore, light and medium roasted coffees had the highest glyoxal and methylglyoxal content, whereas dark roasted coffee contained smaller amounts of glyoxal, methylglyoxal, and diacetyl. For the determination of coffee alpha-dicarbonyl compounds, a reversed-phase high performance liquid chromatography with a diode array detector (RP-HPLC-DAD) method was devised that involved the elimination of interfering compounds, such as chlorogenic acids, by solid phase extraction (SPE) and their derivatization with 1,2-diaminobenzene to give quinoxaline derivatives. Checks of SPE and derivatization conditions to verify recovery and yield, respectively, resulted in rates of 100%. The results of the validation procedure showed that the proposed method is selective, precise, accurate, and sensitive.     

Evolution of green coffee protein profiles with maturation and relationship to coffee cup quality

Coffee flavor is the product of a complex chain of chemical transformations. The green bean has only a faint odor that is not at all reminiscent of coffee aroma. It contains, however, all of the necessary precursors to generate the unmistakable coffee flavor during roasting. The levels and biochemical status of these precursors may vary in relation to genetic traits, environmental factors, maturation level, postharvest treatment, and storage. To improve our understanding of coffee flavor generation, the sensory and biochemical impact of maturation was assessed. Maturation clearly favored the development of high-quality flavor in the coffee brew. A specific subclass of green coffee beans, however, generated high-quality coffee flavor irrespective of maturation. Biochemical aspects were examined using a dynamic system: immature and mature green coffee suspensions were incubated under air or argon. On the analytical side, a specific pool of flavor precursors was monitored: chlorogenic acids, green coffee proteins, and free amino acids. A link between maturation, the redox behavior of green coffee suspensions, and their sensory scores was identified. Compared to ripe beans, unripe beans were found to be more sensitive to oxidation of chlorogenic acids. Aerobic incubation also triggered the fragmentation or digestion of the 11S seed storage protein and the release of free amino acids.     

Effect of roasting on the antioxidant activity of coffee brews

Colombian Arabica coffee beans were roasted to give light, medium, and dark samples. Their aqueous extracts were analyzed by gel filtration chromatography, UV-visible spectrophotometry, capillary electrophoresis, and the ABTS(*)(+) assay. A progressive decrease in antioxidant activity (associated mainly with chlorogenic acids in the green beans) with degree of roasting was observed with the simultaneous generation of high (HMM) and low molecular mass (LMM) compounds possessing antioxidant activity. Maximum antioxidant activity was observed for the medium-roasted coffee; the dark coffee had a lower antioxidant activity despite the increase in color. Analysis of the gel filtration chromatography fractions showed that the LMM fraction made a greater contribution to total antioxidant activity than the HMM components.     

Isolation, identification, and quantification of roasted coffee antibacterial compounds

Coffee brew is a widely consumed beverage with multiple biological activities due both to naturally occurring components and to the hundreds of chemicals that are formed during the roasting process. Roasted coffee extract possesses antibacterial activity against a wide range of microorganisms, including Staphylococcus aureus and Streptococcus mutans, whereas green coffee extract exhibits no such activity. The naturally occurring coffee compounds, such as chlorogenic acids and caffeine, cannot therefore be responsible for the significant antibacterial activity exerted by coffee beverages against both bacteria. The very low minimum inhibitory concentration (MIC) found for standard glyoxal, methylglyoxal, and diacetyl compounds formed during the roasting process points to these alpha-dicarbonyl compounds as the main agents responsible for the antibacterial activity of brewed coffee against Sa. aureus and St. mutans. However, their low concentrations determined in the beverage account for only 50% of its antibacterial activity. The addition of caffeine, which has weak intrinsic antibacterial activity, to a mixture of alpha-dicarbonyl compounds at the concentrations found in coffee demonstrated that caffeine synergistically enhances the antibacterial activity of alpha-dicarbonyl compounds and that glyoxal, methylglyoxal, and diacetyl in the presence of caffeine account for the whole antibacterial activity of roasted coffee.     

Fourier transform infrared and physicochemical analyses of roasted coffee

In this study, Brazilian coffee beans processed to different stages of roast at 210, 220, 230, and 240 °C were analyzed for pH value, titratable acidity, moisture content, and color lightness. Fourier transform infrared (FTIR) spectroscopy, in conjunction with principal component analysis, was conducted to study the effects of process time and temperature on the IR-active components of the acetyl acetate extract of the roasted coffee. The results showed that high-temperature-short-time resulted in higher moisture content, higher pH value, and higher titratable acidity when the beans were roasted beyond the start-of-second-crack stage, as compare to low-temperature-long-time process (LTLT). The LTLT process also resulted in greater IR absorbance for aldehydes, ketones, aliphatic acids, aromatic acids, and caffeine carbonyl bands on the FTIR spectra. Clusters for principal component score plots were well separated, indicating that the changes IR-active components in the coffee extracts, due to the different roasting treatments, can be discriminated by the FTIR technique. On the basis of the loading plots of principal components, changes of IR-active compounds in the coffee extract at various stages of roasting were discussed.     

The antioxidant and chlorogenic acid profiles of whole coffee fruits are influenced by the extraction procedures

Commercial whole coffee fruit extracts and powder samples were analyzed for chlorogenic acids (CGA), caffeine and antioxidant activities. CGA and caffeine were characterized by LC-MS(n) and HPLC accordingly, and quantified by UV absorbance. ORAC, HORAC, NORAC, SORAC and SOAC (antioxidant capacities) were assessed. Three caffeoylquinic acids, three feruloylquinic acids, three dicaffeoylquinic acids, one p-coumaroylquinic acid, two caffeoylferuloylquinic acids and three putative chlorogenic lactones were quantified, along with a methyl ester of 5-caffeoylquinic acid (detected in one sample, the first such report in any coffee material). Multistep whole coffee fruit extracts displayed higher CGA content than single-step extracts, freeze-dried, or air-dried whole raw fruits. Caffeine in multistep extracts was lower than in the single-step extracts and powders. Antioxidant activity in whole coffee fruit extracts was up to 25-fold higher than in powders dependent upon the radical. Total antioxidant activity of samples displayed strong correlation to CGA content.     

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.     

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.     

Incorporation of chlorogenic acids in coffee brew melanoidins

The incorporation of chlorogenic acids (CGAs) and their subunits quinic and caffeic acids (QA and CA) in coffee brew melanoidins was studied. Fractions with different molecular weights, ionic charges, and ethanol solubilities were isolated from coffee brew. Fractions were saponified, and the released QA and CA were quantified. For all melanoidin fractions, it was found that more QA than CA was released. QA levels correlated with melanoidin levels, indicating that QA is incorporated in melanoidins. The QA level was correlated with increasing ionic charge of the melanoidin populations, suggesting that QA may contribute to the negative charge and consequently is, most likely, not linked via its carboxyl group. The QA level correlated with the phenolic acid group level, as determined by Folin-Ciocalteu, indicating that QA was incorporated to a similar extent as the polyphenolic moiety from CGA. The QA and CA released from brew fractions by enzymes confirmed the incorporation of intact CGAs. Intact CGAs are proposed to be incorporated in melanoidins upon roasting via CA through mainly nonester linkages. This complex can be written as Mel=CA-QA, in which Mel represents the melanoidin backbone, =CA represents CA nonester-linked to the melanoidin backbone, and -QA represents QA ester-linked to CA. Additionally, a total of 12% of QA was identified in coffee brew, whereas only 6% was reported in the literature so far. The relevance of the additional QA on coffee brew stability is discussed.     

Influence of coffee roasting on the incorporation of phenolic compounds into melanoidins and their relationship with antioxidant activity of the brew

In the present study, the influence of coffee roasting on free and melanoidin-bound phenolic compounds and their relationship with the brews' antioxidant activity (AA), evaluated by TRAP, TEAC, and TRAP, were investigated. Changes in the relative content of free chlorogenic acids (CGA), free lactones, and melanoidin-bound phenolic acids during roasting indicate that phenolic compounds were incorporated into melanoidins mainly at early stages of the process, being thereafter partly oxidized to dihydrocaffeic acid, and degraded. Although less than 1% of CGA in green coffee was incorporated into melanoidins during roasting, the relative content of melanoidin-bound phenolic acids increased significantly during this process, reaching up to 29% of total phenolic compounds in brews from dark roasted coffees. Regardless of the AA assay used and considering all roasting degrees, the overall contribution of CGA to the AA of the whole brews was higher than that of melanoidin-bound phenolic compounds. It was estimated that the latter compounds contributed to 25-47% of the AA, depending on the assay used.     

Effect of roasting on the formation of chlorogenic acid lactones in coffee

Of all plant constituents, coffee has one of the highest concentrations of chlorogenic acids. When roasting coffee, some of these are transformed into chlorogenic acid lactones (CGL). We have studied the formation of CGL during the roasting of coffee beans in Coffea arabica cv. Bourbon; C. arabicacv. Longberry; and C. canephora cv. Robusta. Individual CGL levels were determined by comparison of HPLC peaks with those of synthetic CGL standards. Seven CGL were identified: 3-caffeoylquinic-1,5-lactone (3-CQL), 4- caffeoylquinic-1,5-lactone (4-CQL), 3-coumaroylquinic-1,5-lactone (3-pCoQL), 4-coumaroylquinic-1,5-lactone (4-pCoQL), 3-feruloylquinic-1,5-lactone (3-FQL), 4-feruloylquinic-1,5-lactone (4-FQL), and 3,4-dicaffeoylquinic-1,5-lactone (3,4-diCQL). 3-CQL was the most abundant lactone in C. arabica and C. canephora, reaching peak values of 230 +/- 9 and 254 +/- 4 mg/100 g (dry weight), respectively, at light medium roast ( approximately 14% weight loss). 4-CQL was the second most abundant lactone (116 +/- 3 and 139 +/- 2 mg/100 g, respectively. The maximum amount of CGL represents approximately 30% of the available precursors. The relative levels of 3-CQL and 4-CQL in roasted coffee were reverse to those of their precursors in green coffee. This suggests that roasting causes isomerization of chlorogenic acids prior to the formation of lactones and that the levels of lactones in roasted coffee do not reflect the levels of precursors in green coffee.     

Contribution of chlorogenic acids to the iron-reducing activity of coffee beverages

The iron-reducing activity of coffee beverages was determined by the ferric reducing antioxidant power (FRAP) assay. The influence on FRAP due to the degree of roasting (light, medium, and dark), species (Coffea arabica and Coffea robusta), and caffeine content (regular and decaffeinated) was investigated using ground and soluble coffee samples. The concentration of specific chlorogenic acids and caffeine in the beverages was determined by high-performance liquid chromatography and related to FRAP using Pearson correlation coefficients. All measurements were expressed per unit of soluble solids. Beverages prepared with ground coffee had, on average, 27% higher FRAP values than those prepared with soluble coffee (p < 0.05). In the former beverages, FRAP of C. robusta samples was significantly higher (on average, 50.3%) when compared to that of C. arabica samples, and FRAP values decreased with increasing degree of roasting (p < 0.05). A strong correlation (r > 0.91) was found between FRAP and the total content of chlorogenic acids, particularly that of the caffeoylquinic acid isomers. The iron-reducing activity of coffee beverages was not influenced by caffeine.     

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.