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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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LIST OF FIGURES
Chapter 1: Introduction

1. Ribbon diagram of a single subunit of β-lactoglobulin .......................................28
Chapter 2: Surface hydrophobicity of ultra high pressure treated β-lactoglobulin –
PRODAN fluorescent probe

1. Intrinsic tryptophan emission spectra of native BLG solutions at pH 3.0,

5.0, 7.0, or 9.0 .........................................................................................................49
2. Intrinsic tryptophan emission spectra of BLG solutions at pH 3 treated

with UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................50

3. Intrinsic tryptophan emission spectra of BLG solutions at pH 5 treated with
UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................51

4. Intrinsic tryptophan emission spectra of BLG solutions at pH 7 treated with
UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................52

5. Intrinsic tryptophan emission spectra of BLG solutions at pH 9 treated with
UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................53

6. Extrinsic PRODAN emission spectra of BLG solutions at pH 3 treated with
UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................54

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7. Extrinsic PRODAN emission spectra of BLG solutions at pH 5 treated with
UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................55
8. Extrinsic PRODAN emission spectra of BLG solutions at pH 7 treated with

UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................56
9. Extrinsic PRODAN emission spectra of BLG solutions at pH 9 treated with

UHP at 600 MPa for selected holding times from 0 to 32 min (UHP0-
UHP32) ...................................................................................................................57
10. Extrinsic PRODAN emission spectra of native BLG solutions at pH 3.0-9.0 .......58
11. Surface hydrophobicity of BLG determined at pH 3.0-9.0 after ultra high

pressure (UHP) treatment times 0, 4, 8, 16, or 32 min with PRODAN..................59
12. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for native BLG at pH 3.0...............................................................................60
13. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 0 min holding time at pH 3.0...........61
14. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 4 min holding time at pH 3.0...........62
15. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 8 min holding time at pH 3.0...........63
16. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 16 min holding time at pH 3.0.........64

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17. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 32 min holding time at pH 3.0.........65
18. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for native BLG at pH 3.0...............................................................................66
19. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 0 min holding time at pH 5.0...........67
20. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 4 min holding time at pH 5.0...........68
21. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 8 min holding time at pH 5.0...........69
22. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 16 min holding time at pH 5.0.........70
23. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 32 min holding time at pH 5.0.........71
24. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for native BLG at pH 7.0...............................................................................72
25. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 0 min holding time at pH 7.0...........73
26. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 4 min holding time at pH 7.0...........74
27. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 8 min holding time at pH 7.0...........75

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RP

28. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 16 min holding time at pH 7.0.........76
29. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 32 min holding time at pH 7.0.........77
30. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for native BLG at pH 9.0...............................................................................78
31. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 0 min holding time at pH 9.0...........79
32. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 4 min holding time at pH 9.0...........80
33. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 8 min holding time at pH 9.0...........81
34. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB

and n for UHP treated (600 MPa) BLG with 16 min holding time at pH 9.0.........82

35. PRODAN binding to BLG plotted by Cogan method (1976) to calculate KBdB
and n for UHP treated (600 MPa) BLG with 32 min holding time at pH 9.0.........83
Chapter 3: Application of solid-phase microextraction with headspace gas
chromatography to the analysis of diacetyl, 2-methylbutyraldehyde, ethyl lactate,
and δ-decalactone in model buffer solution

1. Exposure time of selected compounds adsorbed by a PDMS/DVB fiber ..............99
2. Exposure time of selected compounds adsorbed by a CAR/PDMS fiber.............100
3. Figure 3. Linearity data of ethyl lactate (▲, RP P=0.995), diacetyl (♦,

2 2

P=0.997), d-decalactone (■, RP P=0.951), and 2-methylbutyraldehyde (●,

2

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RP

2

P=0.954) in sodium phosphate buffer (0.01 M, pH 9.0) extracted by HS-
SPME. ..................................................................................................................101
Chapter 4: Application of solid-phase microextraction with headspace gas
chromatography to the analysis of diacetyl, 2-methylbutyraldehyde, ethyl lactate,
and d-decalactone in ultra high pressure-treated β-lactoglobulin solutions

1. Stern-Volmer plots of the fluorescence quenching of native and UHP

treated BLG after 0, 8, or 32 min by diacetyl .......................................................120
2. Stern-Volmer plots of the fluorescence quenching of native and UHP treated

BLG after 0, 8, or 32 min by ethyl lactate ............................................................121
3. Stern-Volmer plots of the fluorescence quenching of native and UHP treated

BLG after 0, 8, or 32 min by 2-methylbutyraldehyde ..........................................122
4. Stern-Volmer plots of the fluorescence quenching of native and UHP treated

BLG after 0, 8, or 32 min by δ-decalactone .........................................................123
5. Static headspace analysis of 2-methylbutyraldehyde (5 ppm) in BLG or

UHP treated BLG (600 MPa) with 0, 8, or 32 min holding time (UHP0,
UHP8, and UHP32) ..............................................................................................125

6. Static headspace analysis of δ-decalactone (5 ppm) in BLG or UHP treated
BLG (600 MPa) with 0, 8, or 32 min holding time (UHP0, UHP8, and
UHP32) .................................................................................................................126

7. Static headspace analysis of diacetyl (5 ppm) in BLG or UHP treated BLG

(600 MPa) with 0, 8, or 32 min holding time (UHP0, UHP8, and UHP32).........127
8. Static headspace analysis of ethyl lactate (5 ppm) in BLG or UHP treated
BLG (600 MPa) with 0, 8, or 32 min holding time (UHP0, UHP8, and UHP32)......128
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CHAPTER ONE
INTRODUCTION
In the United States, health is a powerful driver for the food industry, especially
when nearly 60% of grocery shoppers are overweight and 12.5% of children are
diagnosed with two or more risk factors for heart disease (Sloan 2006). In Western
societies, excessive consumption of dietary fat is linked to obesity (Bray and Popkin
1999) and cardiovascular diseases (Law 2000). High intake of saturated fats, trans fats,
and cholesterol increase the risk of coronary heart disease (Law 2000). Currently, dietary
fat comprises nearly 36% of the energy content of the American diet. . The recommended
total fat intake is between 20 and 35 percent of calories for adults, and for Americans, it
is important to decrease their intake of saturated fats to less than 10 percent based on
population studies of American diets (Thompson and Veneman 2005).
One way to decrease energy from fat in the diet is to substitute low-fat foods for
high-fat foods. However, it may be difficult for some people to limit their dietary choices
to the low-fat foods. The National Dairy Council reports that foods high in energy density
are more palatable because of a high fat content, which may lead to overeating and
consequent weight gain (McBean 2000). Fat modifies the overall perception of flavor of
many foods through their effect on mouthfeel (e.g. creaminess of ice cream, richness of
whole milk) and on the volatility and threshold value of flavor compounds present
(Nawar 1996). Most flavor compounds are nonpolar, and fat is a flavor carrier for these
hydrophobic compounds (Guyot and others 1996). Therefore, when a considerable
amount of fat is removed, the flavor will be lost resulting in food that is often bland and
monotonous.
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Advancements in food technology may offer ways of reducing fat and energy
consumption while satisfying consumer preference for high fat foods. High-fat foods
appeal to consumers because the foods are often flavorful and rich. Fat replacers or fat
substitutes are used to describe ingredients that replace fat or provide the physical and
sensory properties of fat to reduce fat in food applications, and are derived from several
chemical compounds that include carbohydrates, proteins, lipids, and synthetic
compounds (Lindsay 1996). Among the sensory qualities of reduced-fat foods, flavor is
one of the most important attributes consumers use as an index of acceptability, and
within the last 30 years, there is continued interest in improving protein functionality
through the binding of flavor compounds to proteins to improve flavor release and
perception of foods (Gremli 1974; Damodaran and Kinsella 1980; Dumont and Land
1986; Lubbers and others 1998; Guichard and Langourieux 2000; Burova and others
2003).
Basic properties of β-lactoglobulin
The Food and Drug Administration defines whey as “the liquid substance
obtained by separating the coagulum from milk, cream, or skim milk in cheese making”
(FDA 2005). For every 1 pound of cheese produced, approximately 9 pounds of liquid
whey result, and according to USDA statistics in 1999, total cheese production in the US
was 7.94 billion pounds, which equates to almost 72 billion pounds of liquid whey
(USDA 1999). Although not every dairy facility has the capability to process whey into a
usable product and often rely on disposing the whey by-product, whey can be recovered
and processed into animal feed, human food products, and pharmaceuticals.

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β-Lactoglobulin (BLG) is a major whey protein in mammalian species, except for
rodents, lagomorphs and humans. In bovine whey, BLG is the predominant protein, at a
concentration of 2-4 g/L compared to α-lactalbumin (1-1.5 g/L), serum albumin (0.1-0.4
g/L), and immunoglobulin (0.6-1.0 g/L) (Swaisgood 1996). The characterization of BLG
by amino acid sequencing and isoelectric focusing has identified seven genetic
polymorphisms, A, B, C, D, E, F, and G (Phillips and others 1994). The most prevalent
are the A and B forms (Croguennec and others 2004) that differ by two amino acids
(substitutions Asp64 (A) Gly (B); Val118 (A) Ala (B)) (Brignon and Dumas 1973),
and is commercially available in pure form of either variant or as a mixture of both. BLG
is a polypeptide with a molecular weight of 18,350 Da (Brownlow and others 1997),
consisting of 162 amino acid residues. In milk, it exists as a dimer with a molecular
weight of 32,400 Da. BLG is a globular protein containing thiol and disulfide groups.
The orthorhombic crystal structure of BLG at pH 7.6 contains a nine-stranded,
flattened β-barrel or calyx and a flanking three-turn α-helix (Phillips and others 1994).
One curved face of the calyx is formed from a twisted antiparallel β-sheet containing the
N-terminal half of strand A, together with strands B-D (Figure 1) (Brownlow and others
1997). The other curved face is a similarly twisted antiparallel β-sheet containing strands
E-H and the C-terminal half of strand A. Strand A (residues 17-26) has a 90° bend at its
midpoint, Leu22, reflecting its participation in both sheets. A ninth strand (I) extends the
EFGHA sheet. The extended A-B loop (i.e. the loop connecting strand A to strand B) is
involved in hydrogen bonding interactions that form part of the dimer interface along
with strand I. The α-helix lies in the sequence between strands H and I. A disulfide bond
(Cys66-Cys160) connects the C-D loop to the carboxyl-terminal region and is on the
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outside of the molecule, close to the mouth of the hydrophobic pocket (Brownlow and
others 1997). The second disulfide bond (Cys106-Cys119) links strands G and H. Cys121
is buried at the sheet-helix interface (Phillips and others 1994), and does not normally
participate in a disulfide linkage in BLG (Uhrinova and others 2000).
The structure of BLG is pH dependent. At acidic pH less than 3.5, the dimer
reversibly dissociates due to strong electrostatic repulsive interactions, but no
conformation changes occur (Molinari and others 1996). At pH 2.6, an overall positive
charge of BLG may cause a disruption of tertiary as well as quaternary structure
(Uhrinova and others 2000). At pH 2 to 3, at which the protein has a net charge of ~+20,
BLG is essentially monomeric under salt-free conditions (Timasheff and Townend 1961;
Baldini and others 1999; Sakurai and others 2001). In the pH range of 3.7 to 5.2, BLG
reversibly forms a larger oligomer. This self-association process has a maximum around
pH 4.6, just below the isoelectric point, and is more pronounced for BLG-A than for
BLG-B (Townend and Timasheff 1960; Timasheff and Townend 1961; McKenzie and
others 1967). Light scattering studies indicated that intermediate oligomers (tetramers
and hexamers) are present in significant amounts at 8 °C and 15 °C and at BLG
concentrations of less than 1 mM (Kumosinski and Timasheff 1966). At neutral pH and
BLG concentration greater than 50µM, BLG is predominantly dimeric. Six percent of the
monomer surface area is buried at the dimer interface where 12 intermolecular hydrogen
bonds and 2 ion pairs are involved (Brownlow and others 1997; Qin and others 1998). In
the pH range of 6-8, a reversible refolding of BLG polypeptide chains is called the
Tanford transition (Tanford and others 1959). At pH greater than 8, the open end of the
BLG calyx provides an access route to a cavity at the center of BLG, which is a binding
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site for small hydrophobic molecules; this may be considered as an “open” conformation,
as opposed to a “closed” conformation occurring at acidic pH (2.6) (Uhrinova and others
2000).
Most lipocalins bind small hydrophobic molecules within the calyx. It has been
suggested that BLG is involved in the transport of retinol and/or fatty acids (Cogan and
others 1976; Sawyer and others 1998). A proposed retinol binding site, or hydrophobic
pocket, is located in the cavity surrounded by β strands BCDEFG where residue side
chains that line the cavity are all hydrophobic (Sawyer and others 1998). There is a
concentration of hydrophobic residues lining the cleft formed between the α-helix and
the β-sheet with charged or polar groups at one end of the cleft, thus forming a possible
site for fatty acid binding (Figure 1).
Intrinsic Protein Fluorescence
Ultraviolet spectrophotometry is a fast, accurate quantitative and nondestructive
technique used in studies of protein structure and function of membranes, polymers, and
biological macromolecules (Demchenko 1992). Fluorescence is the emission of photons
which results from the transition of paired electrons from a higher-energy orbital to the
lower orbital (Lakowicz 1983). Following light absorption, a fluorophore is excited to a
higher vibrational level. The electronic transition down to the lowest electronic level
results in an excited vibrational state. The absorption spectrum of the molecule reflects
the vibrational levels of the electronically excited states, and the emission spectrum
reflects the vibrational levels of the ground electronic state. As a result, the vibrational
structures seen in the absorption and the emission spectra are similar (Lakowicz 1983).

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