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Application of ultra high hydrostatic pressure for investigating the binding of flavor compounds to ß-lactoglobulin via headspace solid phase microextraction-gas chromatography

by 1977- Hoang, Tinyee Arden, PhD

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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
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


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compound separated will ideally have a different K, reflecting relative affinities for the
stationary phase.
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:


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where tBRB

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.


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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)


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