Charge state distribution and hydrogen/deuterium exchange of alpha-lactalbumin and beta-lactoglobulin preparations by electrospray ionization mass spectrometry

Charge state distribution (CSD) and hydrogen/deuterium (H/D) exchange of preparations of alpha-lactalbumin (alpha-Lac) and beta-lactoglobulin (beta-Lg) were investigated using electrospray ionization mass spectrometry (ESI-MS). Storage of alpha-Lac at pH 3 resulted in substantial changes in its CSD, with the emergence of new ion species and shifts toward higher charge state, indicating less stable conformation. ESI spectra of alpha-Lac kept at pH 5.5 for 4 days showed stable conformation; however, extending the storage period resulted in substantial changes in CSD and a decrease in the stability of holo-alpha-Lac (Ca(2+)-bound form). In comparison to apo-alpha-Lac, the relative intensity of holo-alpha-Lac was higher at pH 6.8 but lower at pH 8 during the storage period. beta-Lg showed stable CSD at pH 3, substantial changes at pH 5.5, and minor changes at pH 6.8 and 8 during storage. The H/D exchange results demonstrate that the conformation of holo-alpha-Lac was more stable than that of apo-alpha-Lac and that the conformation of beta-Lg variant B was more stable than that of the beta-Lg variant A. Kinetics of H/D exchange indicated that alpha-Lac and beta-Lg fractions obtained from whey protein preparations have the same or improved conformational stabilities compared to those of alpha-Lac and beta-Lg standards. The presence of four or more hexose residues in alpha-Lac enhanced its conformational stability; the presence of two hexose residues in beta-Lg resulted in a less stable conformation.     

Analysis of zein by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS) was used to analyze the protein composition of corn prolamine (zein). Mass spectra were obtained from commercial zein and zein extracted with aqueous 2-propanol and aqueous ethanol from consumer corn meal. For the commercial zein, three major zein fractions with m/z 26.8k, 24.1k, and 23.4k were clearly seen with two minor fractions (m/z 14.5k and 20.4k) also present. As compared with the results from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), these three fractions were identified as alpha-zeins (24.1k and 23.4k combined as Z19; 26.8k as Z22). When extracted with 55% aqueous 2-propanol, three alpha-zein fractions with m/z 26.8k, 24.1k, and 23.4k were predominant. When extracted with ethanol, extraction temperature had an effect on the final products. When extracted with 75% aqueous ethanol at room temperature, alpha-zein and some 17-18k species were observed, whereas at 60 degrees C, a small amount of delta-zein was also present. Comparison of the MALDI/MS results with SDS-PAGE and gene sequence analysis shows that the MALDI/MS method is superior to SDS-PAGE in having higher resolution and mass accuracy.     

Interactions between bovine beta-lactoglobulin A and various bioactive peptides as studied by front-face fluorescence spectroscopy

Front-face fluorescence spectroscopy was used for the first time to study the interactions between bovine beta-lactoglobulin variant A (beta-Lg A) and various beta-Lg-derived bioactive peptides. Fluorescence spectra were recorded for beta-Lg A-peptide mixtures at 25 degrees C and pH 6.8 with an excitation wavelength of 290 nm to characterize the molecular environment of tryptophan (Trp) residues present in the protein but absent in the peptides. Spectra remained unchanged following addition of peptides beta-Lg f92-100 and beta-Lg f125-135, while Phe-Phe interaction between beta-Lg f69-83 molecules interfered with analysis. Addition of beta-Lg f102-105 produced a blue shift (3 nm) and a significant increase in fluorescence intensity, while addition of beta-Lg f142-148 also caused a significant increase in fluorescence intensity but accompanied by a red shift (3 nm). These results indicate that the polarity of the Trp environment in the beta-Lg A structure may be modified differently depending on the peptide added.     

Effects of heat treatment and pectin addition on beta-lactoglobulin allergenicity

The specific effects of heat treatment and/or addition of low/high-methylated pectin (LMP/HMP) on the allergenicity of beta-lactoglobulin (beta-Lg) and its hydrolysis products were investigated through a two-step in vitro digestion approach. beta-Lg was first hydrolyzed by pepsin and then by a trypsin/chymotrypsin (T/C) mixture done in a dialysis bag with a molecular weight cutoff of 1000. The protein digestion was followed by SDS-PAGE electrophoresis performed on each digestion product, and their in vitro allergenicity was analyzed by immunoblotting. Such procedure was applied on beta-Lg samples mixed with the two kinds of pectin before or after heating (80 degrees C, 25 min) to determine the respective impact of heat treatment and pectin addition. Heat denaturation improved significantly the susceptibility of beta-Lg against the pepsin and the T/C. This effect, which was coupled to a reduction in immunoreactivity of the digested beta-Lg, appeared to be distinctively modulated by LMP and HMP. Through nonspecific interaction with the beta-Lg, pectin could reduce the accessibility of cleavage sites and/or epitope sequences. This mechanism of action is discussed in relation to the intra- and intermolecular interactions between beta-Lg and pectin initiated under the experimental conditions.     

Hop as an interesting source of resveratrol for brewers: optimization of the extraction and quantitative study by liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

Nowadays, hop is used almost exclusively by brewers for bitterness and flavor. Although hop polyphenols have been widely studied in the past decade for their antioxidant activity in the boiling kettle, very little is known about their real impact on health. The discovery of resveratrol in hop pellets highlights the potential health-promoting effect of moderate beer consumption. Here, we have optimized a quantitative extraction procedure for resveratrol in hop pellets. Preliminary removal of hydrophobic bitter compounds with toluene and cyclohexane at room temperature allows 99% trans-resveratrol recovery by ethanol:water (75:25, v/v) solid/liquid extraction at 60 degrees C. Reverse phase liquid chromatography proves an excellent means of separating isomers. In addition, we have compared two mass spectrometry ionization methods-atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI)-in both the positive and the negative modes. On the basis of standard additions applied with the optimized extraction procedure and reverse phase high-performance liquid chromatography-APCI(+)-tandem mass spectrometry, it appears that Tomahawk hop pellets (T90, harvest 2002) contain 0.5 ppm trans-resveratrol, 2 ppm trans-piceid, no cis-resveratrol, and 0.9 ppm cis-piceid.     

Characterization of vegetable oils: detailed compositional fingerprints derived from electrospray ionization fourier transform ion cyclotron resonance mass spectrometry

Adulteration of vegetable oil is of concern for both commercial and health reasons. Compositional based fingerprints can potentially reveal both the oil source and its possible adulteration. Here, electrospray ionization (ESI) Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) resolves and identifies literally thousands of distinct chemical components of commercial canola, olive, and soybean oils, without extraction or other wet chemical separation pretreatment. In negative-ion ESI FT-ICR MS, the acidic components of soybean oil are easily distinguished from those of canola and olive oil based on relative abundances of C(18) fatty acids, whereas olive oil differs from canola and soybean oil based on relative abundances of tocopherols. In positive-ion ESI FT-ICR MS, the three oils are readily distinguished according to the relative abundances of di- and triacylglycerols with various numbers of double bonds in the fatty acid chains. We demonstrate the detection of soybean oil as an adulterant of olive oil, based on relative abundances of members of each of several chemical families. We suggest that the detailed chemical compositions of vegetable oils can be used to characterize them and to detect and identify adulterants.     

Real-time monitoring of thermal flavor generation in skim milk powder using atmospheric pressure chemical ionization mass spectrometry

The feasibility of monitoring volatile flavor compounds formed by thermal treatment of skimmed milk powder in real time by atmospheric pressure chemical ionization mass spectrometry (APCIMS) was established. Skim milk powder samples were heated isothermally (70 to 120 degrees C) at different moisture contents (2.2 and 12.7 g water/100 g dry solids). Headspace was sampled and analyzed continuously in full scan mode (30-180 amu) by APCIMS. The identity of the volatile compounds monitored by APCIMS was confirmed by coupled GC-EI-APCIMS. The concentration measured by the APCIMS was the net effect of three processes, namely formation of the compound, partition from the skim milk powder into the gas phase, and dilution due to the headspace sampling method used. Preliminary experiments established that the technique could follow the effects of heating temperature and moisture content on the formation of selected compounds from skim milk powder.     

Solubility of purified lutein diesters obtained from Tagetes erecta in supercritical CO(2) and the effect of solvent modifiers

Purified lutein diesters deposited on commercial nonporous glass beads were solubilized in supercritical CO(2) in a computerized batch extractor, and their solubilities were compared to their solubilities in hexane. Densities of 0.7, 0.8, and 0.9 g/mL were evaluated without modifiers. Both pressure and temperature increased solubility, although temperatures >50 degrees C promoted carotenoid loss as determined by mass balance. Solubility was enhanced by the use of modifiers and was related to their log P. Chloroform (log P = 2) increased 2.8 times the amount of solubilized lutein diesters compared to pure CO(2) at the same extraction conditions (0.9 g/mL and 40 degrees C) to yield 65% of the amount extracted with hexane. Supercritical CO(2) extraction of lutein diesters could represent a cleaner technology as compared to the current industrial use of hexane with important ecological and health-related implications.     

UHPLC-MS analysis of juvenile hormone II in Mediterranean corn borer (Sesamia nonagrioides) hemolymph using various ionization techniques

The juvenile hormones (JHs) have been considered the most versatile hormones in the animal kingdom. JH-II is the most abundant JH in Sesamia nonagrioides, important maize pests in the Mediterranean basin. This study compared the sensitivities and matrix effects of four ionization modes on analyzing JH-II in S. nonagrioides hemolymph using ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS) in single ion monitoring (SIM) mode. The ionization techniques tested were electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), atmospheric pressure photoionization (APPI), and APPI with the lamp turned off, which corresponds to atmospheric pressure thermospray ionization (APTSI). ESI was discarded because of the high matrix effect. APPI was discarded because the correlation responses between solvent and matrix on the instrumental quality parameters were worse than those for APTSI and APCI. In our analytical conditions, APCI has shown the best validation parameter values. APCI ionization is widely available in instrumental laboratories.     

Effect of heat treatment on the circular dichroism spectra of bovine beta-lactoglobulin A, B, and C.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated in phosphate buffer at temperatures between 40 and 94 degrees C for 10 min, cooled, and analyzed using near-UV and far-UV circular dichroism (CD). The decrease in near-UV CD intensity at 293 nm (Deltaepsilon(293)) could be analyzed in terms of a two-state model, and the stability was beta-Lg C > beta-Lg A > beta-Lg B on the basis of the midpoint temperatures for samples heated at pH 6.7 and 7.4. However, the slopes of the curves at the midpoint temperature for variant A were generally less than those for beta-Lg B and beta-Lg C, indicating that the substitution of Val (beta-Lg A) for Ala (beta-Lg B or beta-Lg C) at position 118 had altered the entropic contribution to unfolding of the protein. The changes in CD at 270 nm (Deltaepsilon(270)), an index of significant alteration to disulfide bond dihedral angles, occurred at higher temperatures than those for the Deltaepsilon(293) results. The far-UV CD showed some small changes as a consequence of heat treatment, and the shifts at 205 nm ([theta](205)) fitted a two-state model. Plotting the changes in both Deltaepsilon(293) and [theta](205) against the loss of nativelike and sodium dodecyl sulfate-monomeric protein (assessed by polyacrylamide gel electrophoresis) showed a strong 1:1 relationship between Deltaepsilon(293) or [theta](205) and the loss of nativelike beta-Lg. These results indicated that the initial irreversible stage in the heat-induced aggregation of beta-Lg (nativelike monomer to unfolded monomer) altered the chirality of the environment of Trp(19) and modified the secondary structure of beta-Lg slightly. The differences in the behavior of variants A-C were explicable on the basis of generalized electrostatic and hydrophobicity effects as well as specific amino acid effects.     

Atmospheric pressure ionization LC-MS-MS determination of urushiol congeners

This paper describes atmospheric pressure ionization (API) LC-MS-MS determination of urushiols, 3-n-alkenyl- and -alkyl-substituted catechols responsible for poison oak dermatitis. Urushiol was isolated from Western poison oak according to the method of Elsohly et al. (1) (J. Nat. Prod. 1982, 45, 532-538)-the purified preparation contained C(17)- and C(15)-substituted urushiols with zero, one, two, and three double bonds as determined from GC-MS analysis of trimethylsilyl derivatives. Urushiol mixtures were separated on a C(18) reversed phase HPLC column with a methanol-water gradient with urushiols eluting in 100% methanol. Atmospheric pressure chemical ionization (APCI) produced primarily [M - H](-) and MH(+) molecule ions. Electrospray ionization (ESI) yielded [M - H](-) and adduct ions including [M + Cl](-). Daughter ions of [M - H](-) included quinoid radical anions ([M - H - H(2)](-) and m/z 122(-)) and a benzofuran phenate (m/z 135(-)). A suite of hydrocarbon fragments were produced by collision-induced dissociation of MH(+) directly or via an intermediate [MH - H(2)O](+) daughter ion. Six urushiol congeners, one not previously reported in poison oak, were determined by negative ion API-LC-MS-MS with detection limits of approximately 8 pg/microL (ESI) and approximately 800 pg/microL (APCI). API-LC-MS-MS was used to determine urushiol in surface wipes, air samples, and plant materials.     

Steric effects governing disulfide bond interchange during thermal aggregation in solutions of beta-lactoglobulin B and alpha-lactalbumin

Intermolecular disulfide bond formation in pure beta-lactoglobulin (beta-Lg) B and in its 1:1 mixture with alpha-lactalbumin (alpha-La), heated at 85 degrees C for 10 min in solutions of low and high (100 mM NaCl) ionic strength and pH 6.0, was studied by reverse-phase HPLC and MALDI-TOF mass spectrometry. Disulfide bonding between beta-Lg monomers was more extensive than reported in the literature for a temperature of 68.5 degrees C, including formation of trimers connected by two of the three adjacent cysteines, C106/C119/C121. The participation of the different thiol groups in disulfide bonds appeared to depend on their location in the native structure, with surface-located cysteines more involved than internally located ones. This also applied to alpha-La-beta-Lg interactions, where the predominant participants were the surface-located alphaC111, alphaC120, alphaC61, and alphaC6. The least active participant was alphaC28, suggesting that it becomes sterically inaccessible during unfolding of the protein. High ionic strength apparently promoted disulfide bonding. The order of cysteine participation at the high ionic strength was similar to that at low ionic strength, with fewer native-location bonds observed and a lower activity of some groups, such as beta-C106/C119/C121 and alphaC61.     

Electrospray ionization Fourier transform mass spectrometric analysis of wine.

Positive- and negative-ion electrospray ionization Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry has been used for direct analysis of five wines (California Red, Corbiere, Zinfandel, Beaujolais, and Sauvignon Blanc), without any prior separation or purification steps. The high mass resolving power (typically m/Delta m(50%) > or = 80,000, in which Delta m(50%) is mass spectral peak full width at half-maximum peak height) and mass accuracy (< or =1 ppm) of FT-ICR mass spectrometry make it ideal for the study of complex mixtures such as wine, because the components are simultaneously resolved and identified as to elemental composition. Moreover, the high dynamic range of the instrument is advantageous for identifying trace components. The positive-ion mass spectra obtained from the wines were somewhat similar and were dominated by sucrose and (for red wines) anthocyanins. More than 30 compounds (phenolics and carbohydrates) were identified. The negative-ion mass spectra exhibited much greater variation among different wines, with several compounds peculiar to each wine. Elemental compositions could be assigned with high confidence to 76-94% of negative ions of >10% relative abundance. The present results suggest that it may be possible to fingerprint a wine on the basis of its negative-ion ESI FT-ICR mass spectrum.     

Effect of heat treatment on bovine beta-lactoglobulin A, B, and C explored using thiol availability and fluorescence.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.     

Characterization of the temperature activation of pectin methylesterase in green beans and tomatoes

Low-temperature blanching of vegetables activates the enzyme pectin methylesterase (PME), which demethylates cell wall pectins and improves tissue firmness. This temperature activation of PME has been investigated by measuring the formation of methanol in intact tissue of green beans and tomatoes. Rates of methanol formation at temperatures of 35-65 degrees C were obtained by measuring the release of methanol from thin slices of tomato pericarp or green bean pod material. Activation energies of 112 and 97 kJ mol(-1) were calculated for PME activity in green beans and tomatoes, respectively. These activation energies indicate that the rate of pectin demethylation at 65 degrees C will be nearly 100 times that at 25 degrees C. PME activity was also determined titrimetrically using a solubilized form of the enzyme and purified pectin at temperatures from 30 to 60 degrees C. Under these conditions, much lower activation energies of 37 and 35 kJ mol(-1) were obtained for green beans and tomatoes, respectively. Methanol accumulation during heating of whole intact green beans was also determined and yielded an activation energy similar to that obtained with sliced beans. Whole green beans held at room temperature did not accumulate any methanol, but sliced or homogenized beans did. If whole beans were first heated to 45 degrees C and then cooled, methanol accumulation was observed at room temperature. These results indicate that two factors contribute to the observed high rate of pectin de-esterification during low-temperature blanching: (1) An irreversible change, causing PME to become active, occurs by heating to > or = 45 degrees C. (2) The high activation energy for pectin de-esterification means that the rate of de-esterification increases substantially with increasing temperature.     

Inhibition against heat coagulation of ovotransferrin by ovalbumin dry-heated at 120 degrees C

Ovalbumin (OVA) was aggregated stepwise by dry heating at 120 degrees C with a gradual increase in its heating times (10 min-6 h). The inhibiting effects of DHOVAs (OVAs dry-heated for various times up to 6 h) on the heat coagulation of ovotransferrin (OT) were studied. DHOVAs and OT were solubilized at 5% (w/w) concentration with 10 mM sodium phosphate buffer, pH 7.4. Their solutions were mixed at the volume ratio of 1:1 and reheated at 60 degrees C for 3. 5 min. Some remarkable differences according to dry-heating time were observed: coagulum formations were greatly inhibited in the solutions mixed with DHOVAs treated for more than around 2.0 h, with decreasing turbidity as dry-heating time increased. In addition, the effects of reheating time and temperature, as well as those of pH and ionic strength, were also examined on coagulum formation and turbidity development in connection with dry-heating time. Thus, the inhibiting effects of dry-heated egg white on the heat coagulation of fresh egg white previously described were confirmed on the molecular level of OVA and OT.     

Interaction of fusarium mycotoxins, fusaproliferin and fumonisin B1, with DNA studied by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) in negative ion mode was used to monitor the possible noncovalent adduct formations between DNA analogue oligonucleotides and two Fusarium mycotoxins, fumonisin B1 and fusaproliferin. Using mild experimental ESI conditions specific noncovalent interactions were detected between both single- and double-stranded model oligonucleotides and fusaproliferin with 1:1 stoichiometry. Similar association complexes were observed for the deacetyl derivative of fusaproliferin. There were no peaks due to adduct formation present in the mass spectra of fumonisin B1, incubated with oligonucleotides in a wide concentration range, suggesting no specific interaction for this molecule. In a competitive complexation reaction, another mycotoxin, the beauvericin, forms more stable association complex with DNA than fusaproliferin. These findings can be of use in the understanding of molecular mechanisms of action during apoptosis and can be correlated with the teratogenic effect of fusaproliferin.     

Structural characteristics of purified beta-conglycinin from soybeans stored under four conditions

Four storage conditions including adverse conditions [84% relative humidity (RH), 30 degrees C], mild conditions (57% RH, 20 degrees C), cold conditions (4 degrees C), and uncontrolled ambient conditions were used for storing soybeans. The storage time was 9 months for the adverse conditions and 18 months for the other three conditions. Beta-conglycinin was purified and characterized with respect to its molecular properties. After storage under the adverse conditions, beta-conglycinin showed no significant changes in total sugar content, surface hydrophobicity, free SH and SS bonds, and amino acid composition within 6 months; however, it showed a significant decrease in surface hydrophobicity and a significant increase in total free SH and total SH including SS content after 6 months. Analysis of the secondary structure showed a significant increase in alpha-helix content, but a significant decrease in beta-sheet content after 3 months. For the other three conditions, no significant changes occurred to the structures of beta-conglycinin when compared to the control. The molecular mass of beta-conglycinin remained in the range of 199-212 kDa for all conditions during the entire storage periods.     

Specificity of disulfide bond formation during thermal aggregation in solutions of beta-lactoglobulin B and kappa-casein A

Heat-induced (90 degrees C, 10 min, pH 6.7) intermolecular disulfide bond formation in 1:1 mixtures of beta-lactoglobulin B (beta-Lg) and kappa-casein A (kappa-CN) was studied by enzymatic digestion with trypsin or glu-C, reverse-phase HPLC, and MALDI-TOF-MS. Observed masses were compared to theoretically calculated masses of disulfide-bonded peptide dimers and trimers, and the number of different masses matching peptide combinations involving each bond was used as a measure of confidence of identification. The beta-Lg cysteine residues 121 or 119 were involved in bonds with both cysteines of kappa-CN and all cysteines of beta-Lg. This agrees with the supposed initiatory role of beta-C121 in heat-induced SH/SS interchange. The largest numbers of matches corresponded to bonds linking beta-C119/C121 with kappa-C11 or with beta-C66. Multiple matches were recorded for beta-C119/C121 bonding with beta-C119/C121, with beta-C160, or with kappa-C88. However, beta-C106 was observed only in bonds with beta-C119/C121 and did not appear to bond to kappa-CN, suggesting it remains buried in the core of the protein.     

Anthocyanins in wild blueberries of Quebec: extraction and identification

Anthocyanins were extracted from a mixture of berries of Vaccinium angustifolium and Vaccinium myrtillo?des at 7.7 degrees C, 26 degrees C, and 79 degrees C using ethanol alone or ethanol acidified with hydrochloric, citric, tartaric, lactic, or phosphoric acids at a solvent to solid ratio of 10. The effect of these parameters on extracted anthocyanins stability was investigated. The pH-differential and HPLC-DAD methods were used to determine anthocyanin contents. Extracted anthocyanins were purified on a C-18 solid-phase extraction cartridge and characterized by HPLC/electrospray ionization/mass spectrometry (HPLC-ESI-MS/MS). Anthocyanins were identified according to their HPLC retention times, elution order, and MS fragmentation pattern and by comparison with standards and published data. Anthocyanin extractions gave different yields depending on the type of added acid and the extraction temperature. High yields of monomeric and total anthocyanins (26.3 and 28.9 mg/g of dry matter) were obtained at 79 degrees C using phosphoric acid. Extraction using tartaric acid at 79 degrees C provided the lowest degradation index (1.05). Anthocyanins were stable and browning by polyphenol oxidase was inhibited under these conditions. Of the six common anthocyanindins, five were identified in the extracts, namely, delpinidin, cyanidin, peonidin, petunidin, and malvidin; pelargonidin was not found. In addition to well-known major anthocyanins, new anthocyanins were identified for the first time in extracts of wild blueberries from Quebec.