Application of ultra high hydrostatic pressure for investigating the binding of flavor compounds to ß-lactoglobulin via headspace solid phase microextraction-gas chromatography
equilibrium, small variations in the extraction time do not affect the amount of the
analyte extracted by the fiber. However, at the steep part of the curve, even small
variations in the extraction time may result in significant variations in the amount
Gas chromatography (GC) with inactive glass capillary columns is the most
suitable method for the separation of flavor compounds. Gas chromatography is used
extensively to analyze fatty acids, sterols, aldehydes, ketones, carbonyls, alcohols, and
other volatiles and derivatized flavor compounds. Some of the purposes of using GC are
to separate mixtures of compounds; determine the quantity of individual compounds in a
mixture; and analyze aroma components, solvents, pesticides, or additives.
GC is a technique for separating chemical compounds in which a compound is
carried by a mobile phase streaming through a column where separation is being affected
by interaction of individual compounds with the stationary phase of the column. The
mobile phase may be a gas and the stationary phase may be a liquid film on the surface of
an inert support material or a solid surface. Affinity of the solute for the stationary phase
results in retardation of its movement through the chromatography system. The
distribution ratio (K) defines the distribution of compounds between the mobile phase and
the stationary phase at equilibrium, and is described as
where CBSB is the concentration of a compound in the stationary phase per unit volume, and
the concentration of a compound in the mobile phase per unit volume. Each
compound separated will ideally have a different K, reflecting relative affinities for the
The factors that influence the distribution and retention of volatile compounds are
composition and properties of the mobile phase and stationary phase; the intermolecular
forces among component(s), stationary and mobile phases; and temperature. The
intermolecular forces that influence retardation of components are based on Coulomb’s
where F is the force between charged particles, qBaB and qBbB. The separation between the
9 2 2
particles is r, and k is a constant (8.99 x 10P
NmP P/cP P). Coulomb’s Law is the interaction
between charged molecules, such that when F is negative the force between the
molecules is attractive (Pratt and Pumplin; Braithwaite and Smith 1999). The two main
types of electrostatic interactions are polar van der Waal’s retention forces from
interaction between molecules with surface charge and nonpolar dispersion forces
between neutral molecules or functional groups.
An ideal chromatogram is judged by the resolution between peaks where adjacent
peaks are resolved sufficiently so that accurate determination of the peak areas can be
obtained (Braithwaite and Smith 1999). Therefore, there should be a baseline separation
between the peaks and no overlap of the tail of one peak with the leading edge of the next
peak. Resolution is a function of retention characteristics of the components (k), column
efficiency (N) and the selectivity or separating capabilities (α) of the column:
the retention time, wBhB
the width at half height of the peak, and N is column
efficiency. For a given column length, optimum column efficiency is obtained when the
equilibrium step or plate height is at a minimum, described by the van Deemter equation:
where the A term describes the eddy diffusion of component molecules and the variable
pathways the mobile phase may follow through the stationary phase packing in the
capillary column; the B term describes the random motion of molecules dispersed in the
mobile phase along the longitudinal axis of the column, or longitudinal diffusion; and the
C term describes molecules’ constant movement between the mobile and stationary
phases to establish equilibrium defined by the distribution ratio, K.
Many studies on the binding between flavor compounds and proteins focused on
methods to study interactions between protein and flavor compounds (Jung and others
2002; Jung and Ebeler 2003), modeling flavor release (Castellano and Snow 2001),
reduction of off-flavors of soy products (Zhou and Cadwallader 2006), developing new
soy products (Zhou and others 2006), adding flavor to emulsions with proteins as
emulsifiers (Pelletier and others 1998), and adding sulfur-containing compounds for
roasted and savory flavors to meat products (Adams and others 2001). Considine and
others (2005) reported that nonflavor ligands added prior to high pressure treatment
inhibited the formation of intermediate, non-native protein species. Therefore, further
investigation on the binding of flavor compounds associated with high-fat foods with
proteins for use in reduced-fat foods, and the addition of flavor ligands to protein prior to
UHP treatment is warranted.
There is an increasing demand for healthier low-fat foods. In reduced-fat foods,
carbohydrates and proteins are dominant components that interact differently with flavor
compounds compared with fat, and thus change the perceived flavor. Therefore, a better
understanding of the science behind protein-flavor interactions is required for the
development of improved food flavor, particularly that of low-fat foods.
Investigation of UHP on the flavor binding capabilities of BLG will assist in the
design of proteins as a flavor carrier system in reduced-fat foods.
The objectives of this research are to:
1) evaluate the combined effects of UHP and holding time at selected pH
values on the hydrophobic properties of BLG using intrinsic
tryptophan and an extrinsic fluorescence probe;
2) develop a method to extract and quantitate selected flavor compounds
using headspace-solid phase microextraction gas chromatography from
a buffer solution; and
3) investigate the binding properties of selected flavor compounds with
both untreated and UHP treated BLG through the use of intrinsic
fluorescence quenching and static headspace analysis.
(Moller and others 1998)
(Considine and others 2005)
Adams RL, Mottram DS, Parker JK, Brown HM. 2001. Flavor-protein binding: disulfide
interchange reactions between ovalbumin and volatile disulfides. J. Agric. Food Chem.
Ai J. 1997. Headspace solid phase microextraction. Dynamics and quantitative analysis
before reaching a partition equilibrium. Anal. Chem. 69: 3260-3266.
Alizadeh-Pazdar N, Li-Chan ECY. 2000. Comparison of protein surface hydrophobicity
measured at various pH values using three different fluorescent probes. J. Agric. Food
Chem. 48: 328-334.
Andriot I, Harrison M, Fournier N, Guichard E. 2000. Interaction between methyl
ketones and β-lactoglobulin: sensory analysis, headspace analysis, and mathematical
modeling. J. Agric. Food Chem. 48: 4246-4251.
Arakawa T, Timasheff SN. 1984. Mechanism of protein salting in and salting out by
divalent cation salts; balance between hydration and salt binding. Biochem. 23: 5912-
Arthur CL, Pawliszyn J. 1990. Solid phase microextraction with thermal desorption using
fused silica optical fibers. Anal. Chem. 62: 2145-2148.
Baldini G, Beretta S, Chirica G, Franz H, Maccioni E, Mariani P, Spinozzi F. 1999. Saltinduced
association of b-lactoglobulin by light and X-ray scattering. Macromolecules 32:
Botelho MM, Valente-Mesquita VL, Oliveira KMG, Polikarpov I, Ferreira ST. 2000.
Pressure denaturation of β-lactoglobulin. Eur. J. Biochem 267: 2235-2241.
Braithwaite A, Smith FJ. 1999. Chromatographic methods. Dordrecht: Kluwer Academic
Bray GA, Popkin BM. 1999. Dietary fat intake does affect obesity. Amer. J. Clin. Nutr.
Brignon G, Dumas BR. 1973. Localisation dans la chaine peptidique de la b
lactoglobuline bovine de la substitution Glu/Gln differenciant les variants genetiques B et
D. FEBS Lett. 33: 73-76.
Brownlow S, Cabral JHM, Cooper R, Flower DR, Yewdall SJ, Polikarpov I, North ACT,
Sawyer L. 1997. Bovine β-lactoglobulin at 1.8Α resolution - still an enigmatic lipocalin.
Structure 5: 481-495.
Burova TV, Grinberg NV, Grinberg VY, Tolstoguzov VB. 2003. Binding of odorants to
individual proteins and their mixtures. Effects of protein denaturation and association. A
plasticized globule state. Colloids Surfaces. A. Physico. Eng. Aspects 213: 235-244.
Castellano J, Snow NH. 2001. Modeling flavor release using inverse gas
chromatography-mass spectrometry. J. Agric. Food Chem. 49: 4296-4299.
Chong PL. 1988. Effects of hydrostatic pressure on the location of PRODAN in lipid
bilayers and cellular membranes. Biochem. 27: 399-404.
Chong PL, Weber G. 1983. Pressure dependence of 1,6-diphenyl-1,3,5-hexatrine
fluorescence in single-component phosphatidylcholine liposomes. Biochem. 22: 5544-
Cogan U, Kopelman M, Mokady S, Shinitzky M. 1976. Binding affinities of retinol and
related compounds to retinol bindin proteins. Eur. J. Biochem. 65: 71-78.
Considine T, Singh H, Patel HA, Creamer LK. 2005. Influence of binding of sodium
dodecyl sulfate, all-trans-retinol, and 8-anilino-1-naphthalenesulfonate on the highpressure-induced
unfolding and aggregation of b-lactoglobulin B. J. Agric. Food Chem.
Croguennec T, O'Kennedy BT, Mehra R. 2004. Heat-induced denaturation/aggregation of
β-lactoglobulin A and B: kinetics of the first intermediates formed. Int. Dairy J. 14: 399-
Dagget V, Levitt M. 1992. A model of the molten globule state from molecular dynamics
simulations. Proc. Natl. Acad. Sci. 89: 5142-5146.
Damodaran S, Kinsella JE. 1980. Flavor protein interactions. Binding of carbonyls to
bovine serum albumin: thermodynamic and conformation effects. J. Agric. Food Chem.
Demchenko AP. 1992. Fluorescence and dynamics in proteins. In: Lakowicz JR. Topics
in fluorescence spectroscopy. New York: Plenum Press. p 65-111.
Dumont JP, Land DG. 1986. Binding of diacetyl by pea proteins. J. Agric. Food Chem.
Engelhard M, Evans PA. 1995. Kinetics of interaction of partially folded proteins with a
hydrophobic dye: evidence that molten globule character is maximal in early folding
intermediates. Prot. Sci. 4: 1553-1562.
Food and Drug Administration. 2005. Direct food substances affirmed as generally
recognized as safe. FDA.
Fischer N, Widder S. 1997. How proteins influence food flavor. Food Technol. 51(1): 68-
Futenberger S, Dumay E, Cheftel JC. 1997. High-pressure promotes β-lactoglobulin
aggregation through SH/S-S interchange reactions. J. Agric. Food Chem. 45: 912-921.
Gremli HA. 1974. Interaction of flavor compounds with soy protein. J. Am. Oil Chem.
Soc. 51: A95-A97.
Guichard E. 2006. Flavour retention and release from protein solutions. Biotech. Adv. 24:
Guichard E, Langourieux S. 2000. Interactions between β-lactoglobulin and flavor
compounds. Food Chem. 71: 301-308.
Guyot C, Bonnafont C, Lesschaeve I, Issanchou S, Voilley A, Spinnler HE. 1996. Effect
of fat content on odor intensity of three aroma compounds in model emulsions: D-
decalactone, diacetyl, and butyric acid. J. Agric. Food Chem. 44(8): 2341-2348.
Iametti S, Transidico P, Bonomi F, Vecchio G, Pittia P, Rovere P, Dall'Aglio G. 1997.
Molecular modifications of β-lactoglobulin upon exposure to high pressure. J. Agric.
Food Chem. 45: 23-29.
Izzo HV, Ho CT. 1993. Effect of residual amide content on aroma generation and
browning in heated gluten-glucose model systems. J. Agric. Food Chem. 41: 2364-2367.
Jelen HH, Majcher M, Zawirska-Wojtasiak R, Wiewiorowska M, Wasowicz E. 2003.
Determination of geosmin, 2-methylisoborneol, and a musty-earthy odor in wheat grain
by SPME-GC-MS, profiling volatiles, and sensory analysis. J. Agric. Food Chem. 51:
Johnston DE. 1995. High pressure effects on milk and meat. In: Ledward DA, Johnston
DE, Earnshaw RG and Hasting APM. High pressure processing of foods. Loughborough:
Nottingham University Press. p 99-121.
Jung D, Ebeler SE. 2003. Headspace solid-phase microextraction method for the study of
the volatility of selected flavor compounds. J. Agric. Food Chem. 51: 200-205.
Jung D, Ebeler SE. 2003. Investigation of binding behavior of α- and β-ionones to β-
lactoglobulin at different pH values using a diffusion-based NOE pumping technique. J.
Agric. Food Chem. 51: 1988-1993.
Jung D-M, de Ropp JS, Ebeler SE. 2002. Application of pulsed field gradient NMR
techniques for investigating binding of flavor compounds to macromolecules. J. Agric.
Food Chem. 50: 4262-4269.
Kataoka H, Lord HL, Pawliszyn J. 2000. Applications of solid-phase microextraction in
food analysis. J. Chrom. A 880: 35-62.
Kuhn J, Considine T, Singh H. 2006. Interactions of milk proteins and volatile flavor
compounds: Implications in the development of protein foods. J. Food Sci. 71: R72-R82.
Kumar Y, Tayyab S, Muzammil S. 2004. Molten-globule like partially folded states of
human serum albumin induced by fluoro and alkyl alcohols at low pH. Arch. Biochem.
Biophys. 426: 3-10.
Kumosinski TF, Timasheff SN. 1966. Molecular interactions in b-lactoglobulin, X: The
stoichiometry of the β-lactoglobulin mixed tertramerization. J. Am. Chem. Soc.
Lakowicz JR. 1983. Principles of fluorescence spectroscopy. New York: Plenum Press.
Lasalle MW, Li H, Yamada H, Akasaka H, Redfield C. 2003. Pressure-induced unfolding
of the molten globule of all-Ala α-lactalbumin. Prot. Sci. 12: 66-72.
Law M. 2000. Dietary fat and adult diseases and the implications for childhood nutrition:
an epidemic approach. Am. J. Clin. Nutr. 72: 1291S-1296S.
Ledward DA. 2000. Effects of pressure on protein structure. High Pressure Res. 19: 1-10.
Lindsay RC. 1996. Food additives. In: Fennema OR. Food Chemistry. Third Edition.
New York: Marcel Dekker, Inc. p 767-821.
Liu X, Powers JR, Swanson BG, Hill HH, Clark S. 2005. Modification of whey protein
concentrate hydrophobicity by high hydrostatic pressure. Innov. Food Sci. Emerg.
Technol. 6(3): 310-317.
Lubbers S, Landy P, Voilley A. 1998. Retention and release of aroma compounds in
foods containing proteins. Food Technol. 52: 68-74, 208-214.
Masson P, Clery C. 1996. Pressure-induced molten globule states of proteins. In: Hayashi
R. High pressure bioscience and biotechnology. London: Elsevier Applied Science
Publishers. p 117-126.
Mazon H, Marcillat O, Forest E, Smith DL, Vial C. 2004. Conformational dynamics of
the GdmHCl-induced molten globule state of creatine kinase monitored by hydrogen
exchange and mass spectrometry. Biochem. 43: 5045-5054.
McBean LD. 2000. Insights into weight management. Dietary determinants of obesity.
McGregor RB, Weber G. 1986. Estimation of the polarity of the protein interior by
optical spectroscopy. Nature 319: 70-73.
McKenzie HA, Sawyer WH, Smith MB. 1967. Optical rotatory dispersion and
sedimentation in the study of association-dissociation: bovine β-lactglobulins near pH 5.
Biochim. Biophys. Acta 147: 73-92.
Molinari H, Ragona L, Varani L, Musco G, Consonni R, Zetta L, Monaco HL. 1996.
Partially folded structure of monomeric bovine β-lactoglobulin. FEBS Letters 381: 237-
Moller RE, Stapelfeldt H, Skibsted LH. 1998. Thiol reactivity in pressure-unfolded β-
lactoglobulin. Antioxidative properties and thermal refolding. J. Agric. Food Chem. 46:
Moreno F, Cortijo M, Gonzalez-Jimenez J. 1999. The fluorescent probe Prodan
characterizes the warfarin binding site on human serum albumin. Photochem. and
Photobiol. 69(1): 8-15.
Nawar WW. 1996. Lipids. In: Fennema OR. Food chemistry. Third Edition. New York:
Marcel Dekker, Inc. p 225-319.
Paci E, Greene LH, Jones RM, Smith LJ. 2005. Characterization of the molten globule
state of retinol-binding protein using a molecular dynamics simulation approach. FEBS J.
Page B, Lacroix G. 1993. Application of solidphase microextraction to the headspace gas
chromatographic analysis of halogenated volatiles in selected foods. J. Chrom. 48: 199-
Pawliszyn J. 1997. Solid phase microextraction: theory and practice. New York: Wiley-
VCH, Inc. 247p.
Pawliszyn J. 1999. Applications of solid phase microextraction. Cambridge: The Royal
Society of Chemistry. 655.
Pelletier E, Sostmann K, Guichard E. 1998. Measurement of interactions between b-
lactoglobulin and flavor compounds (esters, acids, and pyrazines) by affinity and
exclusion size chromatography. J. Agric. Food Chem. 46(4): 1506-1509.
Phillips LG, Whitehead DM, Kinsella JE. 1994. Structural and chemical properties of β-
lactoglobulin. In: Structure-function properties of food proteins. New York: Academic
Press. p 75-106.
Pratt S, Pumplin J. Coulomb's law.
HTThttp://www.pa.msu.edu/courses/1997spring/PHY232/lectures/coulombslaw/TTH. July 15,
Ptitsyn OB. 1987. Protein folding: hypotheses and experiments. J. Prot. Chem. 6: 273-
Ptitsyn OB. 1995. Molten globule and protein folding. Adv. Prot. Chem. 47: 83-229.
Qin BY, Bewley MC, Creamer LK, Baker HM, Baker EN, Jameson GB. 1998. Structural
basis of the tanford transition of bovine β-lactoglobulin. Biochem. 37: 14014-14023.
Rega B, Fournier N, Guichard E. 2003. Solid phase microextraction (SPME) of oragne
juice flavor: odor representativeness by direct gas chromatography olfactometry (D-GC-
O). J. Agric. Food Chem. 51: 7092-7099.
Rocha S, Ramalheira V, Barros A, Delgadillo I, Coimbra MA. 2001. Headspace solid
phase microextraction (SPME) analysis of flavor compounds in wine. Effect of the
matrix volatile composition in the relative response factors in a wine model. J. Agric.
Food Chem. 49: 5142-5151.
Sakurai K, Oobatake M, Goto Y. 2001. Salt-dependent monomer-dimer equilibrium of
bovine β-lactoglobulin at pH 3. Prot. Sci. 10: 2325-2335.
Sawyer L, Brownlow S, Polikarpov I, Wu SY. 1998. β-Lactoglobulin: structural studies,
biological clues. Int. Dairy J. 8: 65-72.
Sloan AE. 2006. Top 10 functional food trends. Food Technol. 60(4): 22-39.
Steffen A, Pawliszyn J. 1996. Analysis of flavor volatiles using headspace solid-phase
microextraction. J. Agric. Food Chem. 44: 2187-2193.
Swaisgood HE. 1996. Characteristics of milk. In: Fennema OR. Food chemistry. New
York: Marcel Dekker, Inc. p 841-878.
Tanford C, Bunville LG, Nozaki Y. 1959. The reversible transformation of β-
lactoglobulin at pH 7.5. J. Am. Chem. Soc. 81: 4032-4035.
Tedford L-A, Smith D, Schaschke CJ. 1999. High pressure processing effect on the
molecular structure of ovalbumin, lysozyme and β-lactoglobulin. Food Res. Int. 32: 101-
Thompson TG, Veneman AM. 2005. Dietary guidelines for Americans 2005.