Elucidation of protein-lipid interactions in a lysozyme-corn oil system by Fourier transform Raman spectroscopy

Lysozyme (25% in D2O, corn oil, and their emulsions (10% w/w oil/D2O solution) were examined by Fourier transform Raman spectroscopy. Emulsions showed three layers, namely, top oil, middle cream, and bottom aqueous layers. Raman spectral analysis revealed hydrophobic interactions involving both protein and lipid components. Compared to lysozyme in D2O, the difference spectrum obtained after subtraction of oil from the cream layer spectrum showed reduced intensity of tryptophan bands at 760, 1013, 1340, and 1360 cm(-)(1), reduced intensity ratio of the tyrosine doublet at 850 and 830 cm(-)(1), and increased intensity of the C-H bending band at 1455 cm(-)(1). Compared to corn oil, the difference spectrum after subtraction of lysozyme from the cream layer spectrum indicated decreased intensity at 2855 cm(-)(1) (lipid CH(2) symmetric stretch) and 3011 cm(-)(1) (unsaturated fatty acid hydrocarbon chain =C-H stretch) and a higher intensity ratio of the C-H stretching band at 2900 cm(-)(1) to bands at 2885 and 2933 cm(-)(1). Spectra of the top and bottom layers resembled corn oil and lysozyme, respectively, except for changes in the D2O band. Raman spectroscopy can be used to detect structural changes in proteins, lipids, and D2O due to protein-lipid interactions.     

Structural evaluation of myofibrillar proteins during processing of Cantonese sausage by Raman spectroscopy

Structural changes of myofibrillar proteins from raw pork muscle and Cantonese sausage at different processing periods were elucidated using Raman spectroscopy. Fourier deconvolution combined with iterative curve fitting were used to analyze the amide I Raman band. Results from amide I, amide III, and C-C stretching vibrations in 890-1060 cm(-1) showed that α-helix decreased accompanied by an increase in β-sheet structure during the first 18 h, and a rebuilding process of secondary structures was observed at the rest stage due to proteolysis. The hierarchical cluster analysis results of amide I and amide III confirmed this rebuilding process. Changes in a doublet near 850 and 830 cm(-1) suggested that some tyrosine residues became buried in a more hydrophobic environment due to intermolecular interactions. Raman spectra in the 2855-2940 cm(-1) region suggested that the environment of aliphatic side chains might have been changed during the final stage and further confirmed above rebuilding process.     

Comparison of changes in the secondary structure of unheated, heated, and high-pressure-treated beta-lactoglobulin and ovalbumin proteins using fourier transform raman spectroscopy and self-deconvolution

Changes in protein secondary structure and conformation of ovalbumin and beta-lactoglobulin (15% protein w/w) were investigated by Fourier transform Raman spectroscopy and self-deconvolution. The amounts of alpha-helix, beta-sheets, random coil, and beta-turns in native beta-lactoglobulin were 15, 54, 6, and 25%, respectively, and those for ovalbumin (41, 34, 13, and 12%) compared well with published values obtained by X-ray crystallography. The proteins were heated at 90 degrees C for 30 min and high-pressure-treated at 600 MPa for 20 min. Heating increased beta-sheet structures in both proteins at the expense of alpha-helix; for beta-lactoglobulin beta-sheet structures increased from 54 to 70% and for ovalbumin, from 34 to 54%. Random coil increased from 6% in the native protein to 30% in high-pressure-treated beta-lactoglobulin. However, for ovalbumin, the contribution from beta-turns doubled in high-pressure-treated samples, with little change in random coil. Further examination of the deconvoluted amide I band in heated samples revealed several component bands. Bands at 1626 and 1682 cm(-1) for ovalbumin and at 1625 and 1680 cm(-1) for beta-lactoglobulin were observed and are associated with aggregated, intermolecular beta-sheet (beta-aggregation), indicative of heat denaturation. The band seen at 1632-1640 cm(-1) corresponded to intramolecular beta-sheet structures, whereas the band at 1625 cm(-1) is associated with exposed beta-sheets (for example, beta-strands with strong hydrogen bonding that are not part of the core of beta-sheets). In high-pressure-treated samples bands were also observed at 1628 and 1680 cm(-1) for ovalbumin and at 1626 and 1684 cm(-1) for beta-lactoglobulin, suggesting involvement of beta-sheet structures in protein aggregation. Raman bands were observed at 1665-1670 cm(-1) for ovalbumin and at 1663-1675 cm(-1) for beta-lactoglobulin due to random coil structures. The bands at 1650-1660 cm(-1) due to alpha-helices were observed in both heated and high-pressure-treated samples. In addition, in heated samples of both ovalbumin and beta-lactoglobulin, peak intensity increased for beta-sheet in the amide III region, 980-990 cm(-1), and decreased for helix structures (900-960 cm(-1)). In contrast, there was no peak at 1240 cm(-1) (amide III beta-sheet structures) in either high-pressure-treated ovalbumin or beta-lactoglobulin, suggesting that high-pressure denaturation at 600 MPa for 20 min is less extensive than heat denaturation at 90 degrees C for 30 min.     

Raman spectroscopic study of structural changes in Hake (Merluccius merluccius L.) muscle proteins during frozen storage

This paper examines changes in the structure and functionality of fish muscle proteins at frozen storage temperatures known to render very different practical storage lives (-10 and -30 degrees C). Apparent viscosity and dimethylamine (DMA) content showed drastic temperature-related differences during storage. Raman spectroscopy revealed the occurrence of some structural changes involving secondary and tertiary protein structures. The changes in secondary structure were quantified, showing an increase of beta-sheet at the expense of alpha-helix structure. The nuC-H stretching band near 2935 cm(-)(1) increased in intensity, indicating denaturation of the muscle proteins through the exposure of aliphatic hydrophobic groups to the solvent. These structural changes were more pronounced at -10 degrees C but occurred at both storage temperatures, whereas changes in apparent viscosity and DMA only occurred in storage at -10 degrees C. The possible utility of these structural changes for quality assessment is discussed.     

THE HUMIC ACIDS EXTRACTED BY VARIOUS REAGENTS FROM A SOIL

The infra-red, visible, and ultra-violet absorption spectra of humic acids extracted from a red-brown earth by various reagents, are described. The variations in the intensity of various absorption bands in the infra-red spectra are related to the method of extraction, yield, and titration data. For example, the humic acids extracted by milder reagents give rise to spectra which show relatively weak aliphatic C-H absorption whilst the intensity of the bands arising from oxygen-containing groups (carboxyl and ketone carbonyl) is relatively strong. The opposite behaviour is shown by the corresponding bands in the spectra of humic acids extracted by stronger reagents. The intensities of other infra-red bands are also considered. The optical density of the C═O band at 1720 cm^?1 in the humic-acid spectra and of the carboxylate ion band at 1380 cm^?1 in the spectra of the K salt is linearly related to the exchange capacity. The presence of carboxyl groups ionizing above pH 7 and extending to as high as pH 11, is demonstrated.     

FTIR‐spectroscopic characterization of humic acids and humin fractions obtained by advanced NaOH,Na4P2O7, and Na2CO3 extraction procedures

Aim of our study was the development of the methodological basis for the characterization of humic fractions of a long‐term field experiment. Humic acids (HAs) were extracted from three layers of a nontilled soil using three different extractants (1 M NaOH, 0.1 M Na₄P₂O₇, 1 M Na₂CO₃), and the humin fraction was enriched. NaOH as extractant for FTIR analysis of humic substances yields higher resolved IR spectra, especially in the important regions of stretching vibrations including aromatic and aliphatic groups and in the fingerprint area including amides, aliphats, and aromats than the other extractants. The NaOH extraction has lower extraction yields as compared to Na₄P₂O₇ and Na₂CO₃ and represents a different part of the soil organic matter (SOM). This is reflected by lower C : N ratios and higher E₄ : E₆ and fulvic acid–to–humic acid ratios as compared to the other extractants. The FTIR band areas of HA fraction obtained by NaOH showed an increase of the aromatic and carbonyl groups and a decrease of amide groups with increasing soil depth. Aliphatic groups showed contradicting results: The bands of the stretching vibrations increased, and the band of the bending vibrations decreased. We assume that band interactions in the bending vibrations were responsible for that phenomenon under the assumption of an increase of aliphatic groups with increasing soil depth. The IR bands of the enriched humin fraction showed a decreasing trend in case of both aliphatic bands deriving from stretching vibrations and an increase of aromatic characteristics with depth. Our study led to the conclusion that HA fractions obtained by 1 M NaOH represent a small and dynamic fraction indicated by the measured yields in combination with values of N_t, C : N, E₄ : E₆ ratios, and ratios of fulvic acids (FA) to HA. The humin fraction has a high contribution to the total organic C and represents a more stabilized fraction of SOM which still shows changes in its aromatic and aliphatic characteristics with soil depth.     

Imaging molecular chemistry of Pioneer corn

Synchrotron Fourier transform infrared (FTIR) microspectroscopy as a rapid, direct, and nondestructive analytical technique can explore molecular chemical features of the microstructure of biological samples. The objective of this study was to use synchrotron FTIR microspectroscopy to image the molecular chemistry of corn (cv. Pioneer 39P78) to reveal spatial intensity and distribution of chemical functional groups in corn tissue. This experiment was performed at the U2B station of the National Synchrotron Light Source in Brookhaven National Laboratory (NSLS-BNL, Upton, NY). The Pioneer corn tissue was imaged from the pericarp, seed coat, aleurone, and endosperm under peaks at 1736 (carbonyl C=O ester), 1510 (aromatic compound), 1650 (amide I), 1550 (amide II), 1246 (cellulosic material), 1160 (CHO), 1150 (CHO), 1080 (CHO), 929 (CHO), 860 (CHO), 3350 (OH and NH stretching), 2929 (CH(2) stretching band), and 2885 cm(-1) (CH(3) stretching band). The results showed that with synchrotron FTIR microspectroscopy, the images of the molecular chemistry of Pioneer corn could be generated. Such information on the microstructural-chemical features of grain corn can also be used for corn breeding programs for selecting superior varieties of corn for targeted food and feed purposes and for prediction of corn quality and nutritive value for humans and animals.     

Principal component similarity analysis of Raman spectra to study the effects of pH,heating, and kappa-carrageenan on whey protein structure

Raman spectroscopy was used to elucidate structural changes of beta-lactoglobulin (BLG), whey protein isolate (WPI), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (5.0, 7.0, and 9.0), heating (80 degrees C, 30 min), and presence of 0.24% kappa-carrageenan. Three data-processing techniques were used to assist in identifying significant changes in Raman spectral data. Analysis of variance showed that of 12 characteristics examined in the Raman spectra, only a few were significantly affected by pH, heating, kappa-carrageenan, and their interactions. These included amide I (1658 cm(-1)) for WPI and BLG, alpha-helix for BLG and BSA, beta-sheet for BSA, CH stretching (2880 cm(-1)) for BLG and BSA, and CH stretching (2930 cm(-1)) for BSA. Principal component analysis reduced dimensionality of the characteristics. Heating and its interaction with kappa-carrageenan were identified as the most influential in overall structure of the whey proteins, using principal component similarity analysis.     

Study of protein-lipid interactions at the bovine serum albumin/oil interface by Raman microspectroscopy

The interface of 10 or 25% (w/v) bovine serum albumin (BSA), pH 7, buffered solution against mineral or corn oil was studied with a Raman microscope. A gradient of distribution of protein and oil at the interface was observed. The difference spectrum obtained by subtracting the spectrum of mineral or corn oil from that of the BSA/oil interface indicated interactions involving different functional groups of the BSA and the oil molecules. Against mineral oil, the BSA spectrum showed reduced intensity of the tryptophan band at 750 cm(-1) and reduced intensity ratio of the tyrosine doublet at 850-830 cm(-1), indicating changes in the microenvironment of these hydrophobic residues. A negative band at 2850 cm(-1) indicated the involvement of the CH groups in the mineral oil. However, the amide regions, normally assigned to protein secondary structure, were not significantly changed. When the spectrum of BSA was subtracted from the BSA/mineral oil interface spectrum, the resultant difference spectrum showed changes of symmetric and antisymmetric CCC stretches at 980 and 1071 cm(-1), respectively. In contrast, the difference spectrum of BSA/corn oil interface - BSA showed a decrease of CH(2) symmetric stretching at 2850 cm(-1) and a decrease of unsaturated fatty acid hydrocarbon chain stretch at 3010 cm(-1). Raman spectroscopy is a useful tool to study the nature of protein-lipid interactions.     

In situ simultaneous analysis of polyacetylenes, carotenoids and polysaccharides in carrot roots

This paper presents an approach to simultaneously analyze polyacetylenes, carotenoids, and polysaccharides in carrot (Daucus carota L.) roots by means of Raman spectroscopy. The components were measured in situ in the plant tissue without any preliminary sample preparation. The analysis is based on the intensive and characteristic key bands observed in the Raman spectrum of carrot root. The molecular structures of the main carrot polyacetylenes, falcarinol and falcarindiol, are similar, but their Raman spectra exhibit specific differences demonstrated by the shift of their -C[triple bond]C- mode from 2258 to 2252 cm(-)(1), respectively. Carotenoids can be identified by -C=C- stretching vibrations (about 1520 and 1155 cm(-)(1)) of the conjugated system of their polyene chain, whereas the characteristic Raman band at 478 cm(-)(1) indicates the skeletal vibration mode of starch molecule. The other polysaccharide, pectin, can be identified by the characteristic band at 854 cm(-)(1), which is due to the -C-O-C- skeletal mode of alpha-anomer carbohydrates. The Raman mapping technique applied here has revealed detailed information regarding the relative distribution of polyacetylenes, carotenoids, starch, and pectin in the investigated plant tissues. The distribution of these components varies among various carrot cultivars, and especially a significant difference can be seen between cultivated carrot and the wild relative D. carota ssp. maritimus.     

Raman spectroscopic study of oat globulin conformation

Analysis of Raman spectra of oat globulin showed that extreme pH values caused an increase in the amide and C-H stretching band intensity, indicating changes in the secondary structures of the protein due to denaturation. Similar changes were observed when oat globulin was treated with chaotropic salts and several protein perturbants. Sodium dodecyl sulfate, beta-mercaptoethanol, and ethylene glycol also caused a shift in the amide III' band, suggesting a transition from beta-sheet to a random coil conformation. Heating at temperatures near the denaturation temperature of oat globulin led to increases in the amide and C-H band intensity, indicating unfolding of the protein. The data indicate that FT-Raman spectroscopy is suitable for studying the secondary structure of plant proteins such as oat globulin.     

FT-IR investigation of cell wall polysaccharides from cereal grains. Arabinoxylan infrared assignment

The FT-IR fingerprint of wheat endosperm arabinoxylan (AX) was investigated using a set of polysaccharides exhibiting variation of their degree of substitution and xylo-oligosaccharides comprising xylose units mono- or disubstituted by arabinose residues. Substitution of the xylose backbone by arabinose side units was more particularly studied in the 1000-800 cm(-1) spectral region, by taking advantage of second-derivative enhancement. The 920-1020 cm(-1) spectral region revealed two absorption bands at 984 and 958 cm(-1), the intensities of which varied according to the degree of substitution. Whereas the intensity of the band at 958 cm(-1) increased with the degree of substitution, that at 984 cm(-1) decreased. The second-derivative spectral data of xylo-oligosaccharides indicated that these changes could be attributed to substitution of the xylan backbone by arabinose residues, and the band at 958 cm(-1) was ascribed to the presence of disubstituted xylose residues. Principal component analysis of FT-IR spectra of model mixtures of AX, beta-glucans, and arabinogalactans suggested that it is possible to evaluate the relative proportions of the polymers and degree of substitution of AX in complex mixtures such as the cell wall of cereal grains.     

Study of succinylated food proteins by Raman spectroscopy

Three food protein products, soy protein isolates, spray-dried egg white, and whey protein isolates, were chemically modified to varying levels with succinic anhydride, and the extent of modification of these proteins were determined by standard wet chemistry methods. Raman spectra (500-2000 cm(-1)) of the modified proteins were obtained. New C=O stretching vibrations were observed at 1420 and 1737 cm(-1) and were attributed to the carboxylate (COO-) and ester carbonyl (RCOO-) groups, respectively, which were appended to the proteins during succinylation. Two series of calibration curves were obtained by plotting the intensity ratio of the 1420 and 1737 cm(-1) to 1003 cm(-1) phenylalanine stretching band (used as an internal standard) against the extent of substituted epsilon-amino (and alpha-amino) groups and aliphatic hydroxyl groups, respectively. Linear fits were obtained with correlation coefficient r > 0.988. The Raman spectral data were also analyzed to study the effect of succinylation on the conformation of the three proteins. Some conformational changes were observed, including a transition from ordered to disordered structures, an exposure of tryptophan residues from a buried, hydrophobic microenvironment, and probably conformational shift of cystine residues.     

Differentiation of natural and synthetic phenylalanine and tyrosine through natural abundance 2H nuclear magnetic resonance

The natural abundance deuterium NMR characterization of samples of the amino acids tyrosine (1) and phenylalanine (2), examined as the acetylated methyl esters 4 and 6, has been performed with the aim to identify by these means the contribution in animals of the hydroxylation of the diet l-phenylalanine (2) to the formation of l-tyrosine (1), a feature previously revealed on the same samples through the determination of the phenolic delta(18)O values. The study, which includes also the NMR examination of benzoic acid (5) from 2 and of tyrosol (7) from 1, substantially fails in providing the required information because the mode of deuterium labeling of tyrosine samples of different origins is quite similar but indicates a dramatic difference in the deuterium labeling pattern of the two amino acids 1 and 2. The most relevant variation is with regard to the deuterium enrichments at the CH(2) and CH positions, which are inverted in the two amino acids of natural derivation. Moreover, whereas the diastereotopic benzylic hydrogen atoms of l-tyrosine (1) appear to be equally deuterium enriched, in l-phenylalanine (2) the (D/H)(3)(R)() > (D/H)(3)(S)(). Similarly, benzoic acid (5) shows separate signals for the aromatic deuterium nuclei, which are quite indicative of the natural or synthetic derivation. The mode of deuterium labeling of the side chain of 1 and 2 is tentatively correlated to the different origins of the two amino acids, natural from animal sources for l-tyrosine and biotechnological probably from genetically modified microorganisms for l-phenylalanine.     

Diffuse-Reflectance Mid-infrared Spectral Properties of Soils under Alternative Crop Rotations in a Semi-arid Climate

We carried out mid-infrared (mid-IR) spectral interpretation of soils 0–5 and 5–15 cm deep in selected alternative crop rotations (ACR) treatments and an adjacent native prairie soil. Ashing and spectral subtraction shows that absorbance at 3700–2850 and 1700–1550 cm^?1 indicates organic absorbance. Prairie soils, with their greater carbon (C) content, have different spectral properties from the cropped soils. Prairie soils have greater absorbance at the 2950–2870 cm^?1 and the 1230 cm^?1 CH bands. The soils from the different depths had different spectral properties, with the soils 0–5 cm deep having stronger absorbance at the 1055 cm^?1 carbohydrate band, at 1270–1460 cm^?1, and at the 1730 cm^?1 ester band. The soils 5–15 cm deep are characterized by greater absorbance at the clay band. Soil C and nitrogen (N) correlated negatively with the 3700 cm^?1 clay band and the 1830 cm^?1 quartz band and correlated positively with the 2920 cm^?1 because of aliphatic CH absorbance.     

ATR-FT/IR study on the interactions between gliadins and dextrin and their effects on protein secondary structure

The effects of heat treatment and dextrin addition on the secondary structure of gliadins were investigated by means of attenuated total reflection Fourier transform infrared spectroscopy (ATR-FT/IR). Gliadins and gliadin/dextrin mixtures (before and after thermal treatment) were prepared as a dried protein film on the ATR-FT/IR zinc selenide cell plate and equilibrated at a water activity (a(w)) of 0.06. The results show that gliadins undergo conformational changes upon thermal treatment both in the absence and in the presence of dextrin. In particular, in the thermally treated gliadins, the decrease of the band at around 1651 cm(-)(1) and the increase of the bands at around 1628 and 1690 cm(-)(1) suggest a loss of alpha-helix structure and a higher content of protein aggregates. The same trend was observed in the presence of dextrin. Concerning the interactions between gliadins and dextrin, gliadin/dextrin mixtures show variations in the amide I region compared to native gliadins (e.g., an increase of the band at 1645 cm(-)(1) and the absence of the band at around 1668 cm(-)(1)) that might be due to hydrogen bond formation between gliadins and dextrin. It was also found that the spectrum of gliadin/dextrin mixtures was less affected by the hydration state than that of native gliadins, as observed from the differential spectra obtained by subtraction of the spectrum obtained at a(w) = 0.06 (driest condition tested) from the spectrum of the sample equilibrated at a(w) = 0.84. This could be due to the fact that C=O and N-H groups of gliadins are engaged to form hydrogen bonds with the hydroxyl groups of dextrin, and so they are not perturbed by the presence of water molecules. Finally, water activity effects on the secondary structure of gliadins are also discussed.     

Site-specific formation of Maillard, oxidation, and condensation products from whey proteins during reaction with lactose

Heat treatment of dairy products leads to structural changes of proteins, which can severely decrease the nutritional value [Mauron, J. J. Nutr. Sci. Vitaminol. (Tokyo) 1990, 36 (Suppl. 1), S57-69]. In this study, model solutions of the two main whey proteins, alpha-lactalbumin and beta-lactoglobulin, respectively, were incubated with lactose, and modifications were monitored by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Lactulosyl residues were the most abundant modifications of alpha-lactalbumin and beta-lactoglobulin. Up to four of these adducts were identified on the proteins. Enzymatical digest with endoproteinase AspN prior to mass spectrometric analysis allowed the detection of further modifications and their localization in the amino acid sequence. Most prominent modifications were lactulosyllysine, Nepsilon-carboxymethyllysine, oxidation of lysine to aminoadipic semialdehyde, oxidation of methionine to methionine sulfoxide, cyclization of N-terminal glutamic acid to a pyrrolidone, and oxidation of cysteine or tryptophan. The presence of methionine oxidation was deduced from a control protein that had been oxidized by hydrogen peroxide. These studies establish MALDI-TOF-MS as a reliable tool to monitor chemical modifications of nutritional proteins during food processing.     

Amino acid and protein scavenging of radicals generated by iron/hydroperoxide system: an electron spin resonance spin trapping study

Reduction of free radicals generated by Fe(II)/cumene-hydroperoxide (CumOOH) by amino acids (Gly, Cys, Met, His, and Trp) and proteins (bovine serum albumin (BSA), beta-lactoglobulin, and lactoferrin) was followed by electron spin resonance spectroscopy using alpha-phenyl-N-tert-butylnitrone (PBN), 2-methyl-2-nitrosopropane (MNP), and 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) as spin traps. The radical species detected were mostly carbon-centered radicals from CumOOH fragmentation (methyl/*H3 and ethyl/*H2CH3), although carbon-centered radicals originated from amino acids could be formed in the presence of Cys, Met, His, or Trp. All proteins and amino acids, except Cys, were effective at inhibiting generation of radicals from the Fe(II)/CumOOH system. Trp was the amino acid with the highest antiradical activity, followed by His > Gly approximately Met. Lactoferrin was the protein showing the most efficient inhibition of radical formation from the Fe(II)/CumOOH system, and BSA and beta-lactoglobulin were not significantly different in their antiradical activities. These results suggest that proteins with higher inhibitory activity on lipid oxidation promoted by transition metal catalytic decomposition of hydroperoxides should be those with elevated metal-chelating and radical-scavenging properties as well as low concentration and accessibility of reducing groups from amino acids capable of activating metals, such as sulfhydryl groups.     

Ultrastructural changes and structure and mobility of myowater in frozen-stored hake (Merluccius merluccius L.) muscle: relationship with functionality and texture

The ultrastructural changes and the main Raman spectral features of water (3100-3500 and 50-600 cm(-)(1) ranges) in frozen-stored hake were studied with the aim of connecting these changes with loss of some functional properties such as water holding capacity, and with modifications of muscle texture. The following results were obtained: (a) The changes in the spaces between myofibrils can be related to modifications of shear resistance. (b) The behavior of the strong 160 cm(-)(1) band can be related to conformational transitions of muscle proteins, to changes in the structure of muscle water, and/or to alterations in protein-water interactions. (c) There were intensity changes in the nu(s)(OH) band that may be attributable to transfer of water to larger spatial domains during frozen storage.     

Formation of soluble and micelle-bound protein aggregates in heated milk

The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.