Determination of apparent binding constants for aroma compounds with beta-lactoglobulin by dynamic coupled column liquid chromatography

Apparent binding constants of aroma compounds limonene, alpha- and beta-ionone, and terpenyl acetate, with beta-lactoglobulin (BLG), were determined, using dynamic coupled column liquid chromatography, for pH values varying from 3 to 11. K(a) values varied from 2.61 to 3.21 x 10(3) M(-1) for limonene, indicating a strong interaction with BLG. Similarly, significant and close apparent binding constants were obtained for alpha- and beta-ionone, 1.7 x 10(2) and 4.5 to 5.4 x 10(2) M(-1), respectively. These data indicated that a similar mechanism is involved for the binding of these two molecules. The weaker values obtained at low pH, for alpha-ionone relative to beta-ionone, can be explained by the existence of steric hindrance. An increase of the apparent binding constant was observed, for all the compounds studied, when the pH was increased from 3 to 9. At this pH, an apparent binding constant was obtained for terpenyl acetate (1.04 x 10(2) M(-1)), whereas this determination was not possible at pH 3 and 6. The apparent binding constant increase was in agreement with the decrease of aroma compound relative activity coefficient in the presence of BLG, previously observed at this pH. It indicated a best accessibility to the same binding site. The binding constants of all the aroma compounds studied decreased at pH 11 as a result of the important release of the BLG structure previously reported.     

Investigation of binding behavior of alpha- and beta-ionones to beta-lactoglobulin at different pH values using a diffusion-based NOE pumping technique

Diffusion-based NMR techniques were employed to study effects of pH on beta-lactoglobulin (BLG) conformation and binding affinity to alpha- and beta-ionone. In the first part of the study, the influence of pH on the diffusion coefficient of BLG in D(2)O solution was investigated using a stimulated-echo NMR experiment. The diffusion coefficient of BLG decreased with increasing pH values. A significant decrease in the diffusion coefficient observed at pH 11 may be due to total unfolding (denaturation) of the protein, resulting in hydrophobically driven self-aggregation. A diffusion-based NOE pumping technique was then applied to determine the relative binding affinities between alpha- and beta-ionones and BLG at pH values varying from 3 to 11. An increase in signal intensities for beta-ionone with increasing molar concentration ratios between beta-ionone and BLG was observed at all pH ranges studied. The increased signal intensities reflect increased relative binding affinity. The greatest binding affinity occurred at pH 9 and the lowest at pH 11. alpha-Ionone showed binding evidence only at pH 9, and the binding was significantly weaker than that obtained for beta-ionone at the same pH. The high affinity observed for both aroma compounds at pH 9 may be due to a flexible conformation of BLG at this pH so that the flavor ligand accessibility increases. Conversely, alkaline denaturation occurring at pH 11 gives rise to relatively lower binding affinity compared to that observed at the other pH values.     

Effect of xanthan on flavor release from thickened viscous food model systems

The influence of xanthan concentration (0, 0.02, 0.1, 0.4, and 0.8% w/w) and bulk viscosity on the release of 20 aroma compounds of different chemical classes (5 aldehydes, 4 esters, 5 ketones, 3 alcohols, and 3 terpenes) was evaluated in xanthan-thickened food model systems having different viscosities. Interactions between flavor compounds and xanthan were assessed by measuring air-liquid partition coefficients, K, of aroma compounds in pure water and in the xanthan solutions by static headspace gas chromatography. Mass transfer of aroma compounds was estimated by dynamic headspace gas chromatography. Notably, limonene and some of the esters and aldehydes exhibited decreased K values in the presence of xanthan, indicating that the release of these volatile aroma compounds was reduced due to interaction with the xanthan matrix. The degree of interaction depended on the physicochemical characteristics of the aroma compounds. A similar tendency was observed at nonequilibrium with the decreases in release rates being most pronounced for limonene, followed by the esters and aldehydes, with no effect for ketones and an apparent "salting out" effect for alcohols. The reduction in flavor release by xanthan was thus dependent on the physicochemical properties of the aroma compounds and was apparently a result of the aroma-xanthan interactions and not influenced by the viscosity of the system itself.     

Interactions between methyl ketones and beta-lactoglobulin: sensory analysis, headspace analysis, and mathematical modeling

Interaction of flavor compounds with proteins is known to have an influence on the release of flavor from food. Hydrophobic interactions were found between beta-lactoglobulin and methyl ketones; the affinity constant increases by increasing the hydrophobic chain. Addition of beta-lactoglobulin (0.5 and 1%) to aroma solutions (12.5, 50, and 100 microL L(-)(1)) of three methyl ketones induces a significant decrease in odor intensity. The chosen methyl ketones were 2-heptanone (K(b) = 330), 2-octanone (K(b) = 950), and 2-nonanone (K(b) = 2440). The release of these flavor compounds (50 microL L(-)(1)) was studied by static headspace in water solution (50 mM NaCl, pH 3) with different concentrations of beta-lactoglobulin (0, 0.5, 1, 2, 3, and 4%). Increasing the concentration of protein increases the retention of volatiles, and this effect is greatest for 2-nonanone, the compound with the highest affinity constant, and lowest for 2-heptanone. A mathematical model previously developed to describe flavor release from aqueous solutions containing flavor-binding polymers (Harrison, M.; Hills, B. P. J. Agric. Food Chem. 1997, 45, 1883-1890) was used to interpret the data. The model assumes that the polymer-flavor interaction is reversible and the rate-limiting step for release is the transfer of volatiles across the macroscopic gas-liquid interface. This model was used to predict the equilibrium partitioning properties and the rate of release of the three methyl ketones. Increasing the affinity constant leads to decreased release rates and a lower final headspace aroma concentration.     

Air-liquid partition coefficients of aroma volatiles in frozen sugar solutions

Static headspace gas chromatography was used to measure air-solution partition coefficients of homologous series of methyl ketones and ethyl esters in aqueous sugar solutions. The measurements were performed in a temperature range from 25 to -25 degrees C. A rather unexpected temperature dependence of the partition coefficients was observed at subzero temperatures; that is, partition coefficients were found to increase in the temperature interval from 0 to -10 degrees C. A simple model, based on the freeze concentration effect, is proposed to explain the observed temperature dependence.     

Characteristic odor components of kumquat (Fortunella japonica Swingle) peel oil

This study was conducted to determine the composition of kumquat (Fortunella japonica Swingle) cold-pressed peel oil and to determine which volatile components are primarily responsible for the aroma of this oil. Eighty-two compounds were identified in the oil by GC and GC-MS. The major compounds were limonene (93.73%), myrcene (1.84%), and ethyl acetate (1.13%). Flavor dilution (FD) factors and relative flavor activities (RFA) of volatile constituents were evaluated by aroma extract dilution analysis with gas chromatography-olfactometry (GC-O). Camphene, terpinen-4-ol, citronellyl formate, and citronellyl acetate showed high FD factors (>/=5) and RFA (>20). Citronellyl formate and citronellyl acetate were regarded as the characteristic odor components of the kumquat peel oil from the results of FD factor, RFA, and GC-sniffing. Citronellyl acetate is considered to be the odor component most similar to kumquat by organoleptic evaluation with GC-O.     

Volatile release from an emulsion: headspace and in-mouth studies

The headspace concentrations of three esters above solutions containing emulsified lipids were more resistant to dilution by a stream of gas than those above water alone. The effect was greatest for ethyl octanoate, and least for ethyl butyrate, with ethyl hexanoate showing intermediate behavior. This correlated with their solubility in the lipid fraction of the emulsion. Headspace analysis (comparing the emulsion with water) underestimated the release of the esters during consumption. The ratios observed between water and emulsion systems were different for the maximum breath concentration compared with headspace analysis. The emulsion appears to have acted as a reservoir for volatile release, counteracting the effects of sample dilution by saliva.     

Retention of aroma compounds in starch matrices: competitions between aroma compounds toward amylose and amylopectin

The retention of three aroma compounds-isoamyl acetate, ethyl hexanoate, and linalool--from starch-containing model food matrices was measured by headspace analysis, under equilibrium conditions. We studied systems containing standard or waxy corn starch with one or two aroma compounds. The three studied aroma compounds interact differently: ethyl hexanoate and linalool form complexes with amylose, and isoamyl acetate cannot. However, in systems containing one aroma compound, we observed with both starches a significant retention of the three molecules. These results indicate that amylopectin could play a role in the retention of aroma. In systems containing two aroma compounds in a blend, the retentions measured for isoamyl acetate and for linalool were either equal to or less than those in systems where they were added alone. This phenomenon was attributed to competition between aroma compounds to bind with starch. The retention of aroma compounds blended in starch-based systems gave us additional information which confirmed that interactions occur not only with amylose but also with amylopectin.     

Identification of new strawberry sulfur volatiles and changes during maturation

Two Florida strawberry cultivars, 'Strawberry Festival' and 'Florida Radiance', were harvested at five fruit developmental stages (white, half red, three-quarter red, full ripe, and overripe) at four harvest dates. A static headspace solid-phase microextraction (SPME) sampling technique coupled with gas chromatography (GC) using pulsed flame photometric detection (PFPD) was employed to measure 16 sulfur volatiles in these strawberries. A total of 7 sulfur volatiles have been previously reported, and 9 are reported for the first time in strawberries. Newly identified sulfur volatiles include methyl thiopropionate, ethyl thiobutanoate, methyl thiohexanoate, methyl (methylthio)acetate, ethyl (methylthio)acetate, methyl 2-(methylthio)butyrate, methyl 3-(methylthio)propionate, ethyl 3-(methylthio)propionate, and methyl thiooctanoate. Identifications were based on matching sulfur peak linear retention indexes (LRIs) of unknowns with authentic standards and gas chromatography-mass spectrometry (GC-MS) data. Concentrations were determined using both internal and external standards. Most sulfur volatiles increased with increasing maturity, with only concentrations of hydrogen sulfide and methanethiol remaining relatively consistent at all five stages. At the white and half red stages, most sulfur volatiles consisted of various alkyl sulfides. At three-quarter red (commercial ripe), full ripe, and overripe stages, the majority of sulfur volatiles consisted of sulfur esters. Most sulfur volatiles increased dramatically between the commercial ripe, full ripe, and overripe stages, increasing as much as 100% between full ripe and overripe. Principal component analysis indicated that sulfur volatiles could be used to distinguish overripe from full ripe and commercial ripe berries.     

Comparison of the most odor-active compounds in fresh and dried hop cones (Humulus lupulus L. variety spalter select) based on GC-olfactometry and odor dilution techniques

Application of aroma extract dilution analysis on the volatiles obtained from dried cones of Spalter Select hops grown in the German hop-growing area of Hallertau revealed 23 odorants in the flavor dilution (FD) factor range of 16-4096, 20 of which could be identified. On the basis of high FD factors, trans-4, 5-epoxy-(E)-2-decenal, linalool, and myrcene were identified as the most potent odorants, followed by ethyl 2-methylpropanoate, methyl 2-methylbutanoate, (Z)-1,5-octadien-3-one, nonanal, (E,Z)-1,3, 5-undecatriene, 1,3(E),5(Z),9-undecatetraene, propyl 2-methylbutanoate, 4-ethenyl-2-methoxyphenol, and 1-octen-3-one. Ten of the high-impact hop aroma compounds had previously not been identified as hop constituents and, in particular, 1,3(E),5(Z), 9-undecatetraene has not yet been reported as a food odorant. In an extract obtained from fresh hops, in addition to the odorants found in dry hops, (Z)-3-hexenal was characterized as a further key odorant rendering an additional green aroma note to the fresh material.     

Effects of hydration,lipids, and temperature on the binding of the volatile aroma terpenes by beta-lactoglobulin powders

The binding properties of dry proteins are relatively poorly known. Many proteins are present in emulsions and suspensions and also in dry forms. This is particularly true of dairy proteins, which are often stored and sold in powdered form. In the present work, the binding of three terpenes (alpha-terpinene, gamma-terpinene, and terpinolene), which belong to the basic aroma components, and of decane by powdered beta-lactoglobulin (BLG) was studied at different hydration levels (0.05-0.40 g of H(2)O/g of protein) and temperatures (298 and 309.5 K), in the presence or absence of lipids and small concentrations of ethanol. Vapor sorption isotherms were determined for these systems by a static method of headspace gas chromatographic analysis. A cooperative effect of hydrophobic hydration was observed for the binding of aroma terpenes and decane by the solid BLG. The temperature increase from 298 to 309.5 K reduced the observed hydration threshold of BLG by 0.05-0.08 g of H(2)O/g of protein. Lipids (1.2% w/w) in hydrated BLG gave at least a 2-fold increase in its binding affinity for the hydrocarbons studied, and synergic effects of the hydration and lipid on this affinity were observed.     

Interactions between wine polyphenols and aroma substances. An insight at the molecular level

Control of the organoleptic quality of wine or grape-derived beverages requires the study of the interactions between flavor volatiles and polyphenols. The influence of catechin and a wine highly condensed tannin fraction on the volatility of aroma substances was investigated using a dynamic headspace technique. In a hydroalcoholic solution, isoamyl acetate, ethyl hexanoate, and benzaldehyde appeared to be more retained than limonene at low catechin concentrations (0-5 g/L). The tannin fraction induced a slight decrease of benzaldehyde volatility and a salting out of limonene and had no effect on the two esters. Furthermore, investigations at the molecular level were conducted using (1)H NMR spectroscopy. Chemical shift changes registered upon addition of a ligand to a substrate kept at constant concentration allowed the determination of the dissociation constant in a 1:1 binding model. Complexation with catechin was evaluated to be similarly weak for benzaldehyde and the two esters. In addition, catechin and epicatechin displayed a higher affinity for benzaldehyde than for 3, 5-dimethoxyphenol, supporting the hypothesis of a hydrophobic driving force.     

Behavior of flavor compounds in model food systems: a thermodynamic study

Physicochemical parameters, such as hydrophobicity, water solubility, and volatility, of four flavor compounds (ethyl acetate, ethyl butyrate, ethyl hexanoate, and 2-pentanone) were determined. The amount of flavor compounds released from different model matrices (mineral water, purified triolein, an oil-in-water emulsion, a carbohydrate matrix, and a complex matrix containing lipids and carbohydrates) into the gaseous phase was determined at thermodynamic equilibrium, at 37 degrees C, by static headspace gas chromatography. The degree of interaction between the flavor compounds and the matrix components was shown by measuring the percentage retention using the water matrix as the reference. The partition of flavor compounds was principally dependent on their hydrophobicity. Physicochemical interactions that occurred in the different media led to different degrees of flavor retention. An impact of fat on flavor retention was demonstrated when a water matrix and an oil-in-water matrix or carbohydrate and complex matrices were compared. A carbohydrate impact on flavor compound retention was also detected, which was evident even in the presence of lipids.     

Aroma constituents and alkylamides of red and green huajiao (Zanthoxylum bungeanum and Zanthoxylum schinifolium)

Huajiao denotes the fruits of various species of Zanthoxylum in the plant family Rutaceare used for cooking. The two most commercially popular species are bungeanum (red huajiao) and schinifolium (green huajiao). Fresh huajiao has a very high content of essential oil, up to 11%, which is described as having fresh, spicy, floral, cooling, and green aroma notes. A comprehensive analysis of the essential oils by GC-MS using advanced peak deconvolution and data processing software, revealed many overlapping components. A total of 120 aroma compounds for each species has been found. In the essential oils, linalyl acetate (15%), linalool (13%), and limonene (12%) are the major components of red huajiao, whereas linalool (29%), limonene (14%), and sabinene (13%) are the major components of green huajiao. For estimation of the aroma contribution of individual components, a new concept, "aroma character impact value" (ACI), is introduced as the percentage of the ratio of the concentration of an aroma component to its odor threshold value. Despite the differences in major components, both species have six common compounds of top aroma character impact: linalool, alpha-terpineol, myrcene, 1,8-cineole, limonene, and geraniol. The tingling sensation of huajiao is caused mainly by the alkylamide hydroxy-alpha-sanshool. The tingling compound decomposes easily under hydrolytic conditions or under UV light.     

Effect of beta-cyclodextrin on aroma release and flavor perception

Binding and release of volatile compounds to and from beta-cyclodextrin were measured in model aqueous systems using static equilibrium headspace and dynamic headspace dilution. Beta-cyclodextrin decreased the static equilibrium headspace for some volatiles (e.g., ethyl octanoate and decanone) due to binding, but dilution studies demonstrated that binding was readily reversible. Dynamic release of hydrophobic volatile compounds was similar to that observed from emulsions. When beta-cyclodextrin was added to fat free yogurt, the release of a commercial lemon flavoring was modified and was similar to release from a regular fat yogurt. Sensory difference testing confirmed the release results. The data demonstrate that beta-cyclodextrin can be used to modify flavor delivery in both model and real systems; the effects in the latter are sensorially significant.     

Influence of carrot psyllid (Trioza apicalis) feeding or exogenous limonene or methyl jasmonate treatment on composition of carrot (Daucus carota) leaf essential oil and headspace volatiles

The effect of carrot psyllid (Trioza apicalis F?rster) feeding and limonene and methyl jasmonate (MeJA) treatments on the essential oil composition and headspace volatiles of carrot (Daucus carota ssp. sativus), cvs. Parano and Splendid, leaves was studied. Carrot psyllid feeding significantly increased the concentrations of sabinene, beta-pinene, and limonene, whereas limonene treatment increased the concentration of (Z)-beta-ocimene in the leaves of both cultivars. The limonene treatment significantly increased the concentration of total phenolics in the leaves of both cultivars, and MeJA treatment increased phenolic concentration in the leaves of Parano. Exogenous limonene spray did not decrease the number of carrot psyllid eggs laid either 2 or 24 h after treatment. The results suggest that carrot psyllid feeding induces changes in the endogenous monoterpene pool in the carrot leaves. Limonene and MeJA treatments affect some induced defenses of the carrot, but the exogenous limonene spray is not an effective oviposition deterrent against carrot psyllid.     

Evolution of chemical and sensory properties during aging of top-fermented beer

The aging and consequent changes in flavor molecules of a top-fermented beer were studied. Different aging conditions were imposed on freshly bottled beer. After 6 months of aging, the concentration changes were recorded for acetate esters, ethyl esters, carbonyls, Maillard compounds, dioxolanes, and furanic ethers. For some flavor compounds, the changes with time of storage were monitored at different temperatures, either with CO(2) or with air in the headspace of the bottles. For some molecules a relationship was determined between concentration changes and sensory evaluation results. A decrease in volatile esters was responsible for a reduced fruity flavor during aging. On the contrary, various carbonyl compounds, some ethyl esters, Maillard compounds, dioxolanes, and furanic ethers showed a marked increase, due to oxidative and nonoxidative reactions. A very high increase was found for furfural, 2-furanmethanol, and especially the furanic ether, 2-furfuryl ethyl ether (FEE). For FEE a flavor threshold in beer of 6 mug/L was determined. In the aged top-fermented beer, FEE concentrations multiple times the flavor threshold were observed. This was associated with the appearance of a typical solvent-like flavor. As the FEE concentration increased with time at an almost constant rate, with or without air in the headspace, FEE (and probably other furanic ethers) is proposed as a good candidate to evaluate the thermal stress imposed on beer.     

Effect of gelatinization and starch-emulsifier interactions on aroma release from starch-rich model systems

Release of selected volatile aldehyde compounds from starch-rich matrices was studied by headspace extraction, using solid-phase microextraction, and gas chromatography. Changes in the rheological properties of the starch-based matrices, due to starch concentration, gelatinization, and interactions with emulsifiers, were studied by steady shear and dynamic methods. The degree of volatile retention was found to depend on the compound properties, starch concentration, native structure of the granules, and presence of emulsifiers. The nongelatinized starch granules were more effective in lowering the volatile headspace quantities. Loss of the native structural integrity of the granules decreases the retention ability. For the nongelatinized starch dispersions, the more hydrophilic emulsifier showed a more pronounced effect on the matrix rheology and also on the aroma retention. A different behavior was observed for the gelatinized systems. Interpretation of the volatile release profiles was made on the basis of the matrix physical properties and interactions among components.     

Analysis of volatile compounds of taperebá (Spondias mombin L.) and cajá (Spondias mombin L.) by simultaneous distillation and extraction (SDE) and solid phase microextraction (SPME)

Volatile compounds of cajá and taperebá fruits, both classified as Spondias mombin, but from different geographic origins, were extracted (and analyzed) using solid phase microextraction (SPME) and simultaneous distillation and extraction (SDE). Forty-eight compounds were identified in taperebá and 47 in cajá by SPME using a DVB/CAR/PMDS fiber. (E)-Caryophyllene (18.7%), ethyl butyrate (10.0%), and ethyl hexanoate (7.0%) were the most abundant components in taperebá volatiles extracted by SPME, whereas myrcene (41.1%) and beta-phellandrene (8.5%) were the major compounds in cajá. In the taperebá SDE extract, 46 substances were identified, and (Z)-caryophyllene (13.2%) and limonene (9.5%) were predominant. From the 42 substances found in the SDE extract of cajá, the major components were myrcene (38.0%) and p-cymene (6.2%). The two fruits showed similar chromatograms upon the use of SDE and SPME. These methods made it possible to determine 30 identical components in both fruits by using SDE and 32 by using SPME.