Dissertation For Graduate School - Ting Wang 2nd Edition

The Authentication of Whey Protein Powder Ingredients and Understanding
Factors Regulating Astringency in Acidic Whey Protein Beverages to Estimate

Astringency by Infrared Spectroscopy ― An Instrumental Approach
Dissertation
Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
in the Graduate School of The Ohio State University
By
Ting Wang
Graduate Program in Food Science and Technology
The Ohio State University
2014
Dissertation Committee:
Dr. Luis E. Rodriguez-Saona, Advisor
Dr. Monica Giusti
Dr. Michael E Mangino
Dr. Lynn Knipe

Copyrighted by
Ting Wang
2014
Abstract
Whey protein, recognized as a healthy ingredient nowadays, has been widely used
in beverages, bakery products, meat products, and infant formula with attractive
functionalities such as water binding, gelation, emulsification, and foaming. The
processing of whey protein from cheese manufacture has become highly automated and
the production amount has increased largely, which indicated that whey protein has
become to an essential ingredient in food industry. The advanced techniques involving
membrane filtration and ion exchange have made it possible to manufacture specific
product to fulfill the demand of customers. While whey protein has been successfully
transformed from waste to a value-added ingredient, it still confronts problems: The
adulteration of raw material and astringency induced when whey protein beverage was
formulated under acidic pH.
The overall objectives of this dissertation were to provide an accurate and
convenient instrumental method, Infrared Spectroscopy (IR), to authenticate the whey
protein powder and predict the astringency of acidic whey protein beverage, and
understand the factors regulating the whey protein astringency, hence benefit the
application of whey protein as a key ingredient in food products.
Whey protein powders including whey protein isolate (WPI), whey protein
concentrate (WPC), and whey protein hydrolysate (WPH) were obtained from different
ii
manufactures, with different sample characteristics and processing techniques. Each
powder was formulated to a corresponding beverage. The first batch was controlled under
uniform pH value (pH=3.5) after formulation from different powders, and beverage of the
second batch formulated by each whey protein powder was further adjusted to 5 different
pH values (ranging from 2.2 to 3.9). Quantitative sensory test of astringency on all whey
protein beverage samples was conducted at the Sensory Service Center in North Carolina
State University. Infrared spectra of all samples were collected in the wavenumber ranges
from 4000 to 700 cm-1. Classification model and prediction model were built through
multivariate analysis. Multiple linear regression and Pearson correlation were used to
reveal the factors modulating whey protein astringency.
SIMCA classification models with interclass distances larger than 3 were built to
authenticate raw ingredients, and the unknown samples were successfully predicted using
calibration models. The Partial Least Square Regression (PLSR) analysis showed strong
ability to predict astringency based on infrared spectra of different protein products (WPI,
WPC, and WPH beverages). IR bands associated with protein secondary conformation
and carbonyl side chain of amino acids were identified to be significant in both
authentication and prediction. Whey protein types and pH values were suggested to be
significant factors in the development of astringency in whey protein products.
In conclusion, with the comprehensive information obtained from food samples
spectra, IR combined with multivariable chemometrics was an accurate, easy and high
throughput method in understanding and predicting whey protein astringency, which will
be extremely attractive to both scientific research and food industry practice.
iii
Acknowledgments
I sincerely thank Dr. Luis Rodriguez-Saona for his advising, which is not only on
the knowledge and skills as a Ph. D student, but also on the personality in the future of
my life. He taught me how to become a confident people; he guided me how to choose
the career; he supported me to gain experience from food industry; He encouraged me to
conquer problems and fix mistakes. Whenever I became confused, he was always there to
help and gave me the right direction. He is the best advisor I have ever met and it is the
best decision I have ever made to choose him as my advisor.
I also would like to thank my dissertation committee members, Dr. Monica
Giusti, Dr. Michael E Mangino, Dr. Lynn Knipe and Dr. Celia E. Wills. The blessing,
help and guidance given by them always encouraged me; their feedback helped me with a
wonderful dissertation.
I would like to express my special thanks of gratitude to PepsiCo, Inc. for their
generous financial and samples support, as well as Siow Ying Tan, Mutilangi William
and Connie Cerdena who gave me this excellent opportunity to work with them on this
excited project.
I would like to thank the Sensory Service Center at North Carolina State
University, leading by Dr. MaryAnne Drake, for their great efforts on the sensory
evaluation of my samples.
iv
I would like to thank my husband, the most important one in my life.
v
Vita
September 14, 1982 ....................................... Yang Quan, China
2001 – 2005 ................................................... B.S. Bioengineering, South China
University of Technology
2008 - 2011 .................................................. M.S. Food Science and Technology, The
Ohio State University
2011 - present ............................................... Ph.D. Food Science and Technology, The
Ohio State University
Fields of Study
Major Field: Food Science and Technology
vi
Table of Contents
Abstract .............................................................................................................................. ii
Acknowledgments ............................................................................................................. iv
Vita .................................................................................................................................... vi
List of Tables..................................................................................................................... xi
List of Figures ................................................................................................................. xiii
Chapter 1: Literature Review ............................................................................................ 1
1.1 A Review of whey protein processing and functionality .......................................... 1
1.1.1 Whey and whey protein fractions ...................................................................... 1
1.1.2 Whey protein processing and production ........................................................... 5
1.1.3 Whey protein functionality .............................................................................. 14
1.1.4 Whey protein application ................................................................................. 21
1.1.5 Whey protein analytical methods ..................................................................... 22
1.2 Astringency and mechanisms in various food ........................................................ 26
1.2.1 Terminology of astringency ............................................................................. 26
1.2.2 Mechanisms of astringency in four main groups of astringent compounds ..... 27
1.2.3 Exploration of mechanisms of astringency caused by whey protein ............... 33

vii
1.3 The application of Infrared Spectroscopy (IR) on whey protein. ........................... 35
Chapter 2: Authentication of Whey Protein Powders by Combining Infrared
Spectroscopy and Pattern Recognition Analysis .............................................................. 40
2.1 Abstract................................................................................................................... 40
2.2 Introduction ............................................................................................................ 41
2.3 Materials and methods ............................................................................................ 43
2.3.1 Whey protein sample ....................................................................................... 43
2.3.2 Fourier-Transform Infrared Spectroscopy (FT-IR) .......................................... 43
2.3.3 Multivariate classification analysis .................................................................. 44
2.4 Results and discussion ............................................................................................ 46
2.5 Conclusion .............................................................................................................. 55
Chapter 3: A Novel Application of FT-IR Technique Combined with Chemometrics in
Analyzing and Predicting the Astringency of Acidic Whey Protein Beverages .............. 56
3.1 Abstract................................................................................................................... 56
3.2 Introduction ............................................................................................................ 57
3.3.2 Astringency prediction ..................................................................................... 60
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3.3.3 FTIR Micro-spectroscopy ................................................................................ 60
3.3.4 Portable FTIR Spectroscopy ............................................................................ 61
3.3.5 Multivariate regression analysis ....................................................................... 61
3.4.1 Perceived astringency scores for whey protein beverages ............................... 63
3.4.2 Comparing spectra information from whey protein powder and beverage ...... 65
3.4.3 SIMCA classification of whey protein beverages with fixed pH ..................... 67
3.4.4 PLSR calibration models of whey protein beverages with fixed pH ............... 69
3.4.5 PLSR calibration models of whey protein beverages with various pH ............ 71
3.4.6 PLSR prediction of a separate set of whey protein beverages ......................... 80
3.5 Conclusion .............................................................................................................. 82
Chapter 4 Understanding Factors Influencing Development of Astringency in Whey
Protein Products ............................................................................................................... 83
4.1 Abstract................................................................................................................... 83
4.2 Introduction ............................................................................................................ 84
4.3.2 Astringency prediction ..................................................................................... 85
4.3.3 Fourier-Transform Infrared Spectroscopy (FT-IR) .......................................... 86
ix
4.3.4 Statistical analysis ............................................................................................ 86
4.3.5 Gas Chromatography (GC) analysis ................................................................ 87
4.5 Conclusion .............................................................................................................. 96
4.6 Significance and future work .................................................................................. 96
List of References............................................................................................................. 98
x
List of Tables
Table 1.1. Characteristic information of protein in Whey. ................................................. 2
Table 1.2. Production (in tons) of whey protein products in the United States ................ 11
Table 1.3. Analytical methods for whey protein contents regulated by Code of Federal
Regulations Title 21. ........................................................................................................ 24
Table 1.4. Charges of Whey proteins and salivary proteins in different solutions and their
potential of interaction. .................................................................................................... 34
Table 1.5. A general band assignment of MIR applied to food matrix. ........................... 36
Table 1.6. Recent researches on induced conformational changes of individual whey
protein. ............................................................................................................................. 37
Table 2.1. Interclass Distances generated from whey protein powder calibration model
using bench-top (A) and portable (B) FT-IR Spectroscopy. ............................................ 53
Table 2.2. Number of protein samples used for spectral acquisition using the bench-top
and portable FT-IR Spectrometers ................................................................................... 54
Table 3.1. Sensory astringency scores perceived from whey protein beverage samples
under various pH value .................................................................................................... 64
Table 3.2. The multiple linear regression using pH levels and protein types as predictor
and astringency as response.. ........................................................................................... 65
xi
Table 3.3. Calibration and cross-validation results of multivariate models developed by
whey protein beverages with fixed pH (3.5) using FTIR-ATR Micro-spectroscopy
spectra. ............................................................................................................................. 71
whey protein beverages with various pH using FTIR-ATR Micro-spectroscopy, FTIR-
Reflectance Micro-spectroscopy, or Portable FTIR-ATR Spectrometer spectra. ............ 74
whey protein beverages with fixed and various pH using FTIR-ATR Micro-spectroscopy
spectra. ............................................................................................................................. 79
Table 3.6. Prediction values of astringency in formulated whey protein beverages using
IR spectra and calibration models by FTIR-reflectance Micro-spectroscopy.. ................ 81
Table 4.1. Multiple Linear Regression using pH and whey protein types as predictor and
astringency scores as response for WPI, WPC, and WPH. .............................................. 89
Table 4.2. Calibration and cross-validation results of PLSR models developed by pH of
whey protein beverages using FTIR-Reflectance Micro-spectroscopy spectra................ 90
Table 4.3. Calibration and cross-validation statistic performance of PLSR models
developed by using whey protein powder samples. ......................................................... 93
Table 4.4. Pearson correlation between concentrations of glutamic acid (GLU) and
aspartic acid (ASP) in whey protein powders and the corresponding astringency of
beverages at pH 3.5. ......................................................................................................... 95
xii
List of Figures
Figure 1.1. The amino acids sequence of Bovine β-lactoglobulin. .................................... 3
Figure 1.2. The amino acids sequence of Bovine α-lactalbumin........................................ 4
Figure 1.3. A typical industrial spray drying process. ...................................................... 10
Figure 1.4. Processing of whey protein concentrate......................................................... 12
Figure 1.5. Processing of whey protein isolate................................................................. 13
Figure 1.6. Generalized sequence of proteins-water interaction at increasing ERH. The
actual water content(s) at each Aw varies with the nature and types of protein and the
experimental conditions. .................................................................................................. 15
Figure 1.7. A mouth-feel wheel to classify the sensation elicited by red. ........................ 27
Figure 1.8. The example of hydrolyzable tannins (A) and condensed tannins (B) showing
their molecular structures. ................................................................................................ 28
Figure 1.9. Proposed mechanism for polyphenol-PRPs complex formation .................... 30
Figure 2.1. Mid-Infrared spectra of Whey protein powder from bench-top FT-IR
Spectroscopy (A) and portable FT-IR Spectroscopy (B) with Attenuated Total
Reflectance (ATR) ........................................................................................................... 47
Figure 2.2. Soft independent modeling of class analogy (SIMCA) classification plots for
calibration models using a benchtop (A) and portable (B) FTIR spectrometers. SIMCA
xiii
validation performance for calibration models using a benchtop (C) and portable (D)
FTIR spectrometers based on the infrared spectra of whey protein powders. .................. 49
Figure 2.3. Soft independent modeling of class analogy (SIMCA) discriminating power
based on the infrared spectra of whey protein powder from bench-top FT-IR
Spectroscopy (A) and Portable FT-IR Spectroscopy (B). ................................................ 51
Figure 3.1. Attenuated Total Reflectance (ATR) Mid-Infrared spectra of Whey protein
powder and beverage.. ...................................................................................................... 66
Figure 3.2. Second derivative spectral transformation collected using an Attenuated Total
Reflectance Infrared (ATR-IR) accessory equipped either with a ZnSe crystal plate
(bench-top) or Germanium crystal (Micro-spectroscopy)... ............................................. 67
Figure 3.3. Soft independent modeling of class analogy (SIMCA) classification plot and
discriminating power based on the infrared spectra of whey protein beverage samples.. 68
Figure 3.4. Partial least squares regression (PLSR) plots and loadings based on the
infrared spectra of whey protein WPI, WPC and WPH beverage samples with fixed pH
(3.5) using FTIR-ATR germanium Micro-spectroscopy. ................................................. 70
infrared spectra of whey protein WPI, WPC and WPH beverage samples with various pH
(ranging from 2.2 to 3.9) using FTIR-ATR germanium Micro-spectroscopy. ................. 73
(ranging from 2.2 to 3.9) using FTIR-reflectance Micro-spectroscopy. .......................... 76
xiv
(ranging from 2.2 to 3.9) using portable FTIR-ATR diamond equipment. ...................... 77
(3.5) and various pH (ranging from 2.2 to 3.9) using FTIR-ATR germanium Micro-
spectroscopy ..................................................................................................................... 78
Figure 4.1. The examination of validity using MLR to analyze WPI samples. Residuals vs
Fits for Astringency (A) and Normal plot of Residuals for Astringency (B). .................. 89
Figure 4.2. PLSR plots and loadings based on the infrared spectra of whey protein WPI,
WPC and WPH beverage samples and measured pH using FTIR-Reflectance Micro-
spectroscopy. .................................................................................................................... 91
Figure 4.3. PLSR plots and loadings based on the infrared spectra of whey protein
powder samples (WPI, WPC, and WPH). ........................................................................ 92
xv
Chapter 1: Literature Review
1.1 A Review of whey protein processing and functionality
1.1.1 Whey and whey protein fractions
Whey protein, discovered 3000 years ago, is generally recognized as a value
added key ingredient in modern food industry (Smithers, 2008). It is defined in the
United States Code of Federal Regulations Title 21:
“Whey is the liquid substance obtained by separating the coagulum from milk,
cream, or skim milk in cheese making. Whey obtained from a procedure, in which a
significant amount of lactose is converted to lactic acid, or from the curd formation by
direct acidification of milk, is known as acid whey. Whey obtained from a procedure in
which there is insignificant conversion of lactose to lactic acid is known as sweet whey.
Sweet whey has a maximum titratable acidity of not more than 0.16 percent, calculated
as lactic acid, and an alkalinity of ash of not more than 225 milliliters of 0.1N
hydrochloric acid per 100 grams. The acidity of whey, sweet or acid, may be adjusted by
the addition of safe and suitable pH-adjusting ingredients.”
Whey is a complex solution containing water, proteins, lactose, minerals, milk fat,
and lactic acid (Morr, 1989). Whey proteins, the components that play the most important
role in functional and nutritional properties when used in food products, are composed of
different types of proteins. The proteins involved in whey include α-lactalbumin (α-LA),
1
β-lactoglobulin(β-LG), bovine serum albumin (BSA), immunoglobulins (Igs), lactoferrin
(LF), lactoperoxidase, glycomacropeptides (GMPs) and others (Laetitia M. Bonnaillie
and Peggy M. Tomasula, 2008). The characteristic information of individual protein is
listed in Table 1.1. Whey proteins possess globular structures with high levels of
secondary, tertiary and quaternary structures (Sne ana ovanovi , irol ub ara ,
gn en a e, ). Particular whey fractions, β-lactoglobulins, α-lactalbumins,
enriched fractions containing lactoferrins or glycomacropeptides were mostly reported
whey protein with health benefits and widely used in food products.
Molecular Weight
Protein Isoelectric pH
(kg/mol)
β-lactoglobulin 18 5.4
Major whey protein α-lactalbumin 14 4.4
Bovine serum albumin 66 5.1
Immunoglobulins 150 5-8
Lactoferrin 77 7.9
Minor whey protein
Lactoperoxidase 78 9.6
Glycomacropeptides 8.6 <3.8
Table 1.1. Characteristic information of protein in Whey (Etzel 2004; Kilara and Vaghela
2004).
β-LG is the major protein in whey, with 58% of total whey protein content. The
functional characteristics dominate the functionality of whey protein concentrations and
whey protein isolates. Native β-LG is a small globular protein with nown primary,
secondary and tertiary structure (Sne ana ovanovi , irol ub ara , gn en a e,

2
). The primary structure of bovine β-LG molecule is composed of 178 amino acid
residues and the sequence information is listed in Figure 1.1. The secondary structure is
composed of α-helical (1 %), β-sheet (51%), reverse turn (17%), and aperiodic (17%)
respectively (Creamer et al., 1983). The tertiary structure contains two disulfide bonds
and one sulfhydryl group burying inside native protein that will be exposed and activated
by denaturation (heat, pH, or agents) (Swaisgood, 1986; Galani and Owusu Apenten,
1999; Sawyer et al., 1999; Fox, 2003).
Figure 1.1. The amino acids sequence of ovine β-lactoglobulin (Uniprot, 2014).
α-LA accounts 31% of the total whey protein. It is a compact and acidic globular
protein, with binding ability to metal ions such as Ca2+ and Zn2+ (Permyakov and
Berliner, 2000). The peptide linkages of 142 amino acids residuals, showing in Figure
1. , illustrate the primary structure of bovine α-LA. There are two domains in native α-
LA including a large u-helical domain connected to a small v-sheet domain by a calcium-
3
binding loop. The tertiary structure involves four disulfide bridges, which can stabilize
the protein molecule (Permyakov and Berliner, 2000).
Figure 1.2. The amino acids sequence of ovine α-lactalbumin (Uniprot, 2014).
α-LA is an important protein due to the functionality it can provide. It is one
component of lactose synthase in mammary secretory cells and participates the
interaction with galactosyltransferase to increase the enzyme’s affinity and specificity for
glucose (Hill and Brew, 197 ). With similar amino acid composition to human mil , α-
LA is also considered as the most suitable protein as the main ingredient in infant
formular (Heine et al., 1991). One research conducted on 23 recovered depressed patients
and 20 controls with the dietary supplement rich in whey protein α-LA showed the
enhancing effect on participants’ memory, irrespective of history of depression ( ooi et
al., 6). There have been more studies done to reveal the functionality of α-LA in
cognitive performance improvement, antimicrobial property, and others (Permyakov and
4
Berliner, 2000; Markus et al., ), which indicated that the significance of α-LA was
not limited in nutritional value.
After the development of isolation techniques, minor proteins in whey could be
separated to exhibit their biological potential. LF showed both bacteriostatic and
bactericidal activity against pathogenic microorganisms, including those responsible for
gastroenteric infections, food poisoning, listeriosis, and mastitis, hence attracted high
attention in the application as natural antibiotics and ingredient in infant formulas
(Dionysius et al., 1993). GMP was probably the least known minor protein in whey,
because of no aromatic amino acids, a net negative charge, and the low molecular weight
with only 64 amino acid residuals (Brody, 2000). However, more and more research
focused on GMP has found its promising bioactivities, which made it a prospective
ingredient in functional food and even medicinal application (Martín-Diana et al. 2006;
Nakano et al.2006; Thoma-Worringeretal et al., 2006). The biological activities related to
GMP included binding of cholera and Escherichia coli enterotoxins, inhibition of
bacterial and viral adhesion, suppression of gastric secretions, promotion of
bifidobacterial growth, and modulation of immune system responses (Brody, 2000).
1.1.2 Whey protein processing and production
Liquid whey obtained from the cheese-manufacturing vat was treated by further
processing steps to fulfill the requirement of customers. Liquid whey was traditionally
processed into whey powder and drying was always the final step in manufacturing whey
proteins. In the 1920s four different methods were involved in whey protein processing:
conventional hot roller milk driers; heating until a concentrated liquid was obtained,
5
cooling to solidification, and then extruding in a tunnel; two-stage steam heating; and a
combination of spray drying and rotary drum drying (Gillies 1974). Nowadays there had
been various advanced techniques developed by research beyond the traditional
processing methods, which generated more and more whey protein products with
different functional and biological properties. Each processing step (technique) and the
control of processing conditions (temperature, pH, and others) would alter the protein
properties and enhance the variability of available whey protein products.
Before further processing steps such as concentration, crystallization,
demineralization, and drying, the pretreatments were normally required to prepare the
liquid whey into good condition with less impurities left from cheese manufacturing.
After collected from the cheese vat, whey liquid normally contained small amount of
cheese fines and lipids, which required centrifugation (clarification) as one pretreatment
to remove the impurities (USDEC, 2004); Whey liquid also had starter culture
microorganisms left from cheese manufacturing, which was not expected to be present in
whey product and required pasteurization to inactivate them (USDEC, 2004). High
temperature and short time (72°C, 15sec) pasteurization was used to heat the whey liquid
to inactivate the starter culture microorganisms and kill possible pathogens (USDEC,
2004). Other pretreatments such as shifting pH, addition of phosphates, and addition of
CaCl2 or other ions were also helpful in removing phospholipoprotein complexes (PLPC)
and colloidal calcium phosphate to improve the performance of following processing
steps (Morr, 1993). After these pretreatments, the whey liquid was ready for further
processing.
6
Although Whey proteins had a high nutritional value, mostly because of the high
content of essential amino acids, especially sulfur-containing ones, whey powders dried
directly from fresh whey liquid was not a value-added product, due to the high lactose
and ash contents (Wit, 1998; USDEC, 2004). Many techniques have been developed to
selectively concentrate and isolate whey proteins and membrane technology and ion
exchange had been recognized as value-added process.
There have been four classes of membranes used to concentrate whey: reverse
osmosis (RO), nanofiltration (NF), ultrafiltration (UF) and microfiltration (MF) (USDEC,
2004). Basically these techniques involved passing whey liquid across a semi-permeable
membrane, while a pressure gradient was created by a combination of pumps and valves
(USDEC, 2004). When whey liquid flowed by the membrane with designed pore size, the
smaller molecules would permeate the membrane, and so called permeate; the larger
molecules and particles would not be able to cross the membrane and hence be
concentrated on the other side of the membrane, which were referred to as retentate
(USDEC, 2004).
As one of the most popular used membrane technique, UF can produce various
whey protein products with different nutrition ratio of the retentate and permeate, based
on the retention characteristics of the membranes used (Atra et al., 2005). In the retentate
part, particles with bigger molecular weight (size) such as protein and fat fraction were
kept and concentrated, while particles with smaller molecular weight (size), such as
lactose, minerals and vitamins, were penetrated and separated as permeate (Hinrichs,
2001; Kessler, 2002). UF was able to separate and concentrate substances with a
7
molecular weight between103 and 106 Da, which were much bigger than NF’s molecular
weight capacity, ranging from 100 and 500 Da (Atra et al., 2005). With this small pores
sizes in membrane, NF membranes could only allow the penetration of some monovalent
ions and water, hence it could be used to concentrate of the permeate produced during UF
of whey, which mostly consisted of lactose in the same concentration as in the water
phase of the original fluid (Atra et al., 2005). MF had the largest membrane pores sizes
among all four classes mentioned above, and could permeate smaller soluble proteins,
peptides, lactose, minerals, non-protein nitrogen components, and water easily (USDEC,
2004). The retained part was mainly fat globula, and it was considered extremely
effective for removing the PLPC at all pH and temperature conditions. Hence it was
either considered as an alternative process to pretreatment to remove PLPC, or as an
amendment method to remove a small amount of fat that was not recovered through
centrifugation during pretreatment (Morr, 1993; USDEC, 2004). RO had the smallest
membrane pores sizes and only water can permeate through the membrane, hence it could
filter the whey liquid in the way that the concentrated solids contained the same ratios as
before (USDEC, 2004).
There had been two major ion exchange processes available, including the Vistec
process and the Spherosil process (Morr, 1989). They were different in their basic
operation: the former one used a stirred-bed ion exchange reactor column, whereas the
Latter one used a fixed-bed ion exchanger (Morr, 1989). Since the stirred-bed process is
much superior, it had been used as a more popular method to produce commercial Whey
protein products (Morr, 1989). In processing, liquid whey was firstly adjusted to acidic
8
pH to obtain the positive charged protein molecules, and then it was pumped into a tank
(ion exchange tower) that contained negatively charged resin beads. The positively
charged protein was retained in the negatively charged resin, and other components such
as fat, lactose, and mineral were eluted out of the tower and separated from the proteins
(Huffman, 1996). Once the resin was fully loaded by proteins, a new mobile phase was
added to adjust the pH of the tank to alkaline and detach the protein from the resin for
following processes (Huffman, 1996).
Diafiltration (DF) was used to further process the retentate after UF of whey
liquid, and produce whey protein products with a higher protein content. With the
addition of DF volumes (normally wash-water), the retentate macromolecules were
further purified by eliminating more lactose and minerals (Román et al., 2012).
Crystallization was used to process permeate produced from membrane filtration
step, hence it was considered as a further processing or economical disposal of the
remaining lactose stream (Jelen, 1979). The lactose in permeate was supersaturated to
crystallize and high quality lactose could be utilized in the sweetener market (Jelen,
1979).
Spray drying was normally the last step in processing whey protein powders.
Typical drying operations consisted of evaporation in multistage vacuum evaporators,
followed by spray drying (Jelen, 2011). As one of the mostly used drying techniques,
spray drying was applied to not only whey powder, but also instant coffee, milk powder,
tea and soups, as well as healthcare and pharmaceutical products, due to the advantages
such as continuous operation, suitable to heat sensitive materials, short processing time,
9
and automatic control (Chegini and Taheri, 2013). The quality of whey powder processed
through spray drying was affected by operating conditions and process variables: the
method and conditions of atomization, the type of spray/air contact, drying air
temperature and feeding concentration, feeding temperature and the degree of feed
aeration were all important parameters (Chegini and Taheri, 2013). A typical industrial
spray drying process is showed in figure 1.3.
Figure 1.3. A typical industrial spray drying process (Chegini and Taheri, 2013).
10
With various processing techniques, specific whey protein products could be
provided based on the requirement of customers. There have been large amount of whey
products available in the U.S. market as shown in Table 1.2.
2004 2005 2006

Reduced lactose and mineral whey 38507 44620 41547
Whey protein concentrate 25-49.9 123266 125363 134928
Whey protein concentrate 50-89.9 38147 48783 59083
Whey protein isolate 12544 12517 13913
Lactose 301921 323854 335049
Table 1.2. Production (in tons) of whey protein products in the United States (USDA
National Agriculture Statistics Services)
The United States Code of Federal Regulations Title 21 defined Whey protein
concentrate (WPC) as:
“Whey protein concentrate is the substance obtained by the removal of sufficient
nonprotein constituents from whey so that the finished dry product contains not less than
25 percent protein. Whey protein concentrate is produced by physical separation
techniques such as precipitation, filtration, or dialysis. As with whey, whey protein
concentrate can be used as a fluid, concentrate, or dry product form. The acidity of whey
protein concentrate may be adjusted by the addition of safe and suitable pH-adjusting
ingredients.”
WPC in the market was mostly available as 35% or 85% protein contents, and
85% WPC was used more as human food ingredients (Foegeding and Luck, 2011). The
11
processing steps were listed in figure 1.4.
Figure 1.4. Processing of whey protein concentrate (USDEC, 2004).
Whey protein isolate (WPI) contained larger than 90% of protein and 4–6%
water, the remaining 4–6% of the ingredient was a combination of fat, lactose, and ash
(Foegeding and Luck, 2011). The processing steps were listed in Figure 1.5.
12
Figure 1.5. Processing of whey protein isolate (USDEC, 2004).
Whey protein hydrolysate (WPH) was obtained by hydrolyzation of WPC or WPI
using enzymes, acids or alkali reagent, cleaving the peptide bonds in whey protein
molecules producing peptides with different sizes and free amino acids, which could
reduce allergenicity, achieve specific dietary requirements, or to improve functional
properties (Adler-Nissen, 1986; Farrell et al., 1987; Asselin et al., 1988; de Freitas et al.,
1993; Cordle, 1994; Frokjaer, 1994; Nielsen, 1997; Clemente, 2000; Sinha et al., 2007;).
Compared to acid and alkaline hydrolysis, which were hard to control and tend to reduce
13
the nutritional qualities of WPH, enzymatic hydrolysis (either acid or neutral) has
become a better choice to gain biologically active components for food application (Sinha
et al., 2007). The degree of hydrolysis and the molecular weight distribution of the
constituent peptides were normally used to characterize WPH, which could affect the
functional and sensory properties (van der Ven et al., 2002).
1.1.3 Whey protein functionality
The functionality has been defined as “any property of a food or food ingredient,
except its nutritional ones, that affects its utilization (Pout-El, 1981).” When tal ing
about the functionality of whey protein, water binding, gelation, emulsification, and
foaming were those frequently required in food production and attracted more attention
from the researchers.
Protein-water interactions in food system were always the first considered
parameter, because of its wide influences in solubility, gelation, emulsifying, foaming
capacity and other functionalities. Kuntz and Kauzman (1974) defined protein-water
interaction at increasing water activities based on changes of water molecules associated
with proteins: “The initial molecules of water that associate with, sorb, or bind to a dry
protein display altered physical properties, e.g., reduced vapor pressure, enthalpy,
entropy, volume, chemical potential; decreased translational/rotational frequencies; and
increased specific gravity, heat of vaporization, and heat capacity; The bound water was
not freezable at normal temperatures or even as low as -40°C. As the equilibrium relative
humidity (ERH) is increased, additional water molecules progressively associate with
protein in less structured layers and as capillary-held water. These water molecules show
14
a gradual continuum in physical properties from the highly structured initial layers to
those typical of normal bulk liquid water” (Figure 1.6).
Dry protein
Sorption at Aw 0.05-0.4
Protein covered by monolayer of

adsorbed water molecules
20-30 g H 20/100 g protein
H2O at Aw 0.5-0.75
Protein with multilayers of adsorbed water

30-40g H2O/100g protein Swelling of protein “glassy” state
H2O at Aw 0.98
Hydrated protein with multilayer and capillary condensed water Swollen, solvated,
40-50g H2O/100g protein Partly solubilized protein
H2O at Aw <0.99
Swelling
Solvation
Dispersion
Dissolution
Figure 1.6. Generalized sequence of proteins-water interaction at increasing ERH. The

actual water content(s) at each Aw varies with the nature and types of protein and the
experimental conditions (Kinsella et al., 1986).
The factors affecting water binding of protein involved both interior nature of
proteins themselves and exterior conditions of the environments. As stated by Kinsella
15
and others (1986), amino acid composition, protein conformation, surface
polarity/hydrophobicity, ionic concentration, ion species, pH, and temperature were
important factors in the water binding of proteins. Amino acids with electrical charged
side groups or polar side groups tended to associate with more water molecules by either
ion-dipole or dipole-dipole interactions, on the other hand, nonpolar amino acids tended
to be involved in the minimal water interaction. Similarly, when more polar groups were
exposed to the aqueous phase, which was possibly based on the protein conformational
structure and surface area, the water binding would increase accordingly (Mangino,
1984). Salts exhibited different abilities when introduced to protein-water interaction,
which was recognized as salting in and salting out: less amount of salts could interact
with both charged groups on the protein surface and water molecules, and hence increase
the water-binding ability, whereas high amount of salts would compete with protein for
water molecules and decrease the water-binding ability (Mangino, 1984). pH was able to
affect the net charge and conformation of protein: when pH was away from the isoelectric
point, the solubility tended to increase due to enhanced water-binding ability of ionized
amino acids side groups, and when pH approached the isoelectric point, the protein was
prone to precipitate (Kuntz, 1971). Normally water sorption decreased while the
temperature increased and hydration around polar groups decreased too (Noguchi, 1981).
In general, whey protein was classified as heat induced gelation, and formed a
continuous network of macroscopic dimensions immersed in a liquid medium exhibiting
no steady-state flow. The gelation process was suggested to follow a two-stage process:
The initial reactions in the gelation process involved the denaturation of a protein
16
molecule by weakening and breaking of hydrogen and disulfide bonds in secondary and
tertiary structure to disrupt native protein conformational structures. The second reaction
involved polymerization or aggregation of the denatured protein molecules to produce a
three-dimensional protein structure and immobilized a large amount of water molecules
to form a gel (Schmidt, 1981; Brandenburg et al., 1992; Mangino, 1992). A proper
balance between repulsive and attractive protein-protein and protein-water interaction
was required in forming and maintaining the protein gel structure (Schmidt, 1981).
Properties of protein gels were also affected by intrinsic and extrinsic factors, which
included but not limited to the composition and concentration of the proteins, heating
temperature, pH, ionic strength ， calcium concentration, and free sulfhydryl
concentration (Schmidt, 1981; Damodaran, 1989; Foegeding, 1989). A lot of researchers
have illustrated the significant influences of minerals on the characteristics of whey
protein gels, and they also have proved that salt levels and salt types had various impacts.
Schmidt and others (et al., 1978,1979) found that dialyzed WPC obtained the maximum
gel hardness values when protein suspensions contained 200mM NaCl or 11.1mM CaCl2,
and hardness decrease with increasing salt concentrations. Mulvihilland and Kinsella
(1988) confirmed these observations with similar results and further suggested that CaCl2
appeared to be far more effective than NaCl because much lower concentration of CaCl2
was required to produce similar increases in hardness. Kuhn and Foegeding (1991)
conducted the experiment to investigate the influence of monovalent and divalent salts of
on the rheological properties of WPI gels. They found that the charges of salts had
obvious impact on the shear strain values, and WPI gels formed with addition of
17
monovalent salt showed significant difference from WPI gels formed with divalent salts
(Kuhn and Foegeding, 1991). Protein concentration was also an important factor in heat
induced gel formation and the properties of the gel structure. A critical level of protein
concentration was required to establish the gel formation that should be high enough to
facilitate the protein-protein interaction, and this critical level might be different based on
the protein used. While the concentration was increased higher than the critical level, a
firmer gel might be formed (Schmidt, 1981). The temperature and pH value had great
effects on protein denaturation, hence were able to affect gel formation in various ways.
Emulsions are heterogeneous systems consisting of two immiscible liquids that
were noticed as dispersed phase and continuous phase, and the emulsifiers functioned to
reduce the interfacial energy and facilitate the dispersion of the dispersed phase (Kinsella
and Whitehead, 1989). When a protein was used as an emulsifier, both nutritional reasons
and functional nature of the proteins made them good candidates in food applications.
Proteins are macromolecules containing both hydrophilic and hydrophobic groups. To act
as an emulsifier, protein needed to firstly approach the interface of two phases and
partially unfold to expose the hydrophobic groups, which could happen as random
fluctuation at either solvent phase or water-oil interface (Mangino, 1984). To lower the
free energy in the two phases system, the protein had the chance to insert its exposed
hydrophobic groups into the oil phase and leave the hydrophilic groups inside the water
phase. The rigidity of the protein structure and the number and location of hydrophobic
groups were important factors that could affect the unfolding and hence the emulsifying
18
ability (Mangino, 1984). In addition, pH, salts, protein concentration, and temperature
could also influence the insertion of whey protein into oil phase (Yamauchi et al., 1980).
Singh and Dalgleish (1998) conducted experiments to investigate the emulsifying
properties of WPH with degrees of hydrolysis (DH) varying from 8 to 45%, and to
determine the minimal length of peptides that could be used to produce a stable oil-in-
water emulsion system. They observed that a minimum length of peptide, which was the
product of hydrolyzed total whey protein, was required for effective stabilization of
emulsions, and the mean length of the effective peptides was about 5 amino acids (Singh
and Dalgleish, 1998). They also suggested that the enzymes used to hydrolyze total whey
protein played important roles in the functional behavior of WPH (Singh and Dalgleish,
1998).
After establishment of the emulsion, the two phases system was
thermodynamically unstable, and would finally collapse. Interfacial tension between the
two phases, characteristics of the adsorbed film in the interface, magnitude of the
electrical charge on the dispersed globules, size and surface-volume ratio of the dispersed
globules, and viscosity of the dispersing phase were all factors influencing the stabilizing
of emulsion (Morr and Ha, 1993).
Food foams were most commonly formed by mechanically dispersing air into a
solution, with foaming agents such as protein, emulsifier, and polysaccharide food
stabilizer gum involved in the system to lower the high surface tension and high surface
energy (Mangino, 1984; Morr and Ha, 1993). Foam formed and stabilized by protein
shared quite similar mechanism as emulsion system involving protein molecules. The
19
protein molecules needed to firstly diffuse to the interface of air and solution, unfold
themselves and surround the nonpolar air phase to lower down the interfacial tension
(Mangino, 1984).
The stability of the foam was dependent upon factors such as total solids, protein
and carbohydrate concentration, pH, the concentration of calcium and other ions, and
whipping method (USDEC, 2004). Phillips and others (1991) reported that addition of
salts (Na2SO4, NaCl, and NaSCN) with low concentrations (0.1M) reduced maximum
foam expansion values of WPI solutions, however addition of Na2SO4 with high
concentration (1M) increased the maximum foam expansion compared to the control
WPC solution. More research revealed the effect of thermal treatment on the physical
properties of whey protein foams. Temperature of heat treatment and pH were claimed to
have major influence on the protein denaturation and protein solubility, hence were able
to impact foam stabilized by protein molecules. Nicorescu and others (2009)
demonstrated that foam overrun of a commercial WPI solution slightly decreased while
the temperature of thermal treatment upon protein was increased, they also found that the
temperature of thermal treatment at 80°C was best for stability against drainage, and
increasing the temperature above 80°C did not improve the functional properties of the
proteins. Richert and others (1974) also found that severe heat treatments generally
impaired the foaming characteristics of WPC dispersions. The optimum pH value to
stabilize foam was detected to be different based on the whey protein used: maximum
stability of foam involving WPC solution was reported at approximately pH 7 (Richert et
20
al., 1974), while the maximum foam stability for unheated WPI was obtained at pH 5.0
(Phillips, 1990).
1.1.4 Whey protein application
As mentioned in section 1.2, nowadays there is abundance of commercially
available whey protein products: WPC, WPI, and WPH were the major supplies, while
reduced lactose and mineral whey, whey powder, whey permeate, demineralized whey
and whey protein fraction were also significant components in food industry. These whey
derivatives differ not only in the amount of protein content, but also in the molecule
interaction that is related directly to the control of processing methods and parameters
(Jelen, 2011; Foegeding and Luck, 2011).
Hence whey protein products, especial commercial available products, generate
diverse and specific functions when introduced to the food application. The protein
content in WPC can range widely from 25% to 89%, the lower level (35%) and higher
level (80%) were normally used in different food products due to the nutritional value,
price, and functionality. WPC with 35% protein content could be used in bakery products
to form a heat and shear-induced produc; another usage was to put in stews and sauces
because of their thickening properties; possible application also included luncheon meats
and meat patties (Morr and Ha, 1993; Foegeding and Luck, 2011). All of these
applications are the outcomes of combining protein, lactose, and minerals (Morr and Ha,
1993; Foegeding and Luck, 2011). WPC with 80% protein content was normally
designed for gelation, emulsification, and foam formation functions in food products
since its higher protein composition, for example the high gel strength and water-binding
21
properties made WPC with 80% protein content a nice candidate for meat products
(Foegeding and Luck, 2011). WPI has even higher level of protein composition with a
minimum of 90%, which is obtained from the additional processing steps (either ion
exchange chromatography or microfiltration) (Tunick, 2008). By removing the impurities
from WPI by ionic charge, this ingredient gains excellent water binding, gelling,
emulsifying, and foaming abilities and is broadly used in nutritional supplements, sports
and health drinks, and protein-fortified beverages (Foegeding and Luck, 2011). In
contrast to WPC and WPI as the intact protein, WPH is produced by enzymatic
hydrolysis and hence contains peptides that are going to enhance the absorbance and
lower the allergenicity (Foegeding and Luck, 2011). In addition to these properties, WPH
also performs nicely in improving heat stability, producing bioactive peptides, tailoring
amounts and size of peptides for special diets, and altering the functional properties of
gelation, foaming and emulsification (Nielsen, 1997; Foegeding et al., 2002).
Consequently it is easy to find WPH as an important ingredient in infant formula and
enhanced-performance products (Sinha, 2007; Foegeding and Luck, 2011). Actually, the
differences of functionalities are not only seen among WPC, WPI and WPH, different
commercial whey protein products generated distinct properties and functionalities,
because variation present during whey protein manufacturing and processing from
original products (milk) will result in diverse composition and functional properties.
1.1.5 Whey protein analytical methods
The analysis of whey protein concentrates was described in Code of Federal
Regulations Title 21, which included the analytical methods for all major contents
22
present in whey protein powders (Table 1.3). In addition, various analytical methods to
determine the whey protein composition were developed, because the composition was
more important in the prediction of whey protein nutritional value, functionalities and
biological activities. Researchers had explored electrophoresis and liquid
chromatography as the analytical methods to fractionate whey proteins, and High
performance liquid chromatography (HPLC) has became the most popular method due to
its versatility, short analysis time, high resolution, and superior column performance
(Elgar et al., 2000). Electrophoresis had been traditionally adopted in the research of
proteins: native polyacrylamide gel electrophoresis (PAGE) separated the proteins by
their different charges and sizes, whereas sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE) worked by molecular mass differences only (Kinghorn et
al., 1995). Kinghorn and coworkers (1995) measured the major proteins in both acid
whey and WPC samples by native PAGE and SDS-PAGE, and compared the separation
achieved by both methods. They demonstrated that native PAGE could be used to
determine levels of α-Lac, β-Lg A, β-Lg B and BSA, wheress IgG proteins can not be
quantitated. SDS-PAGE could be used to quantitate BSA and IgG (using the heavy chain)
while the presence of glyco-α-Lac might interfere with the determination of α-Lac and β-
Lg levels. The accuracy of both PAGE methods was dependent on staining/de-staining
protocols and on reproducible densitometry (Kinghorn et al., 1995).
23
Criteria Analytical method Section Heading
Protein content >25% Total Nitrogen-- 16.036 Total Solids
Officials (Liquid sample)
Final Action
Kjeldahl Method 16.193 Protein--

(Dry sample), Official Final
Action
Fat content 1-10% Reese-Gottlieb Method 16.059 Fat
[Reference Method] (Liquid sample)
(11)--Official Final
Action
16.199 Fat in Dried
(Dry sample) Milk(45)--
Official Final
Action
Ash content 2-15% Ash (5)--Official Final 16.035 Total Solids
Action (Liquid sample)
Ash--Official Final 16.196 Dried Milk,
Action (Dry sample) Nonfat Dry
Milk, and
Malted Milk
Lactose content ≤6 % Gravimetric Method-- 16.057 Lactose
Official Final Action (Liquid sample)
Lane-Eynon General 31.061 Lactose--
Volumetric Method (Dry sample) Chemical
Methods--
Official Final
Action
Moisture 1-6% Moisture (41)--Official 16.192 Dried Milk,
content Final Action Nonfat Dry
Milk, and
Malted Milk
Solids content Method I--Official 16.032 Total Solids
Final Action
Titratable Acidity (2)--Official 16.023 Milk
Acidity Final Action
Equivalent
potentiometric method
Table 1.3. Analytical methods for whey protein contents regulated by Code of Federal
Regulations Title 21.
24
Chromatography technique was utilized as the main method in analytical and
preparative separation of whey proteins. Ion exchange chromatography, reversed phase
chromatography, size-exclusion chromatography, hydrophobic chromatography, and
affinity chromatography have been explored in the separation of major or minor whey
proteins and each method had its own merits and sensitivity. For instance, Elgar and
coworkers (2000) developed a reversed phase-HPLC method to simultaneously separate
and quantify the α-lactalbumin, β-lactoglobulin, bovine serum albumin, proteose peptone,
caseinomacropeptide, and immunoglobulin G in bovine whey samples including WPC
and WPI. The α-lactalbumin detected by reversed phase-HPLC showed similar results as
literature value and their reference value using sodium dodecyl polyacrylamide gel
electrophoresis (SDS–PAGE), whereas β-lactoglobulin showed lower level compared to
SDS-PAGE due to the solubility interference; Bovine serum albumin and
immunoglobulin G detected using reversed phase-HPLC also correlated well with their
reference value measured by SDS-PAGE, and proteose peptone and caseinomacropeptide
were within reasonable range of literature values or reference values separately (Elgar et
al., 2000). Doultani and coworkers (2003) conducted the ion exchange chromatography
to recover the major proteins in mozzarella cheese whey. Both cation exchange
chromatography packed with SP Sepharose Big Beads and anion exchange
chromatography packed with Q Sepharose Big Beads were used in the recover process,
because glycomacropeptide could not bind to the S-beads and pass through into the
effluent solution of cation exchange chromatography. The results turned out to be that
92% of the major whey proteins (α-lactalbumin, β-lactoglobulin, bovine serum albumin
25
and immunoglobulin G) and 96% of the glycomacropeptide were successfully recovered
with high throughput (Doultani et al., 2003). Hence it needed to be noticed that the
increasing robustness of HPLC with the development of efficient columns, buffers,
solvents, sensitive detector and higher instrument automation made it a more
comprehensive and reliable analytical method in the analysis of whey protein
compositions.
1.2 Astringency and mechanisms in various food
1.2.1 Terminology of astringency
Astringency is a complex phenomenon and it incorporates multiple sensations
involving the drying and roughness of oral surfaces with the additional perception of
tightening, or puckering of the oral mucosa and muscles around the mouth (Lee and
Lawless, 1991). There have been a lot of attempts to describe this complex terminology
and argument about sub-qualities such as pucker, sourness and bitterness were under
discussion for a long while (Lee and Lawless, 1991; Green, 1993). The American
Society for Testing and aterials (AST ) defined astringency as “the complex of
sensations due to shrinking, drawing or puckering of the epithelium as a result of
exposure to substances such as alums or tannins” (ASTM, 2004). Gawel et al. (2000)
identified a range of oral sensations from red wines by a descriptive panel and defined
astringency. The related terms were shown in figure 1.7.
26
Figure 1.7. A mouth-feel wheel to classify the sensation elicited by red wine (Gawel et
al., 2000).
1.2.2 Mechanisms of astringency in four main groups of astringent compounds
Astringency was identified in an abundance of food and beverages such as wine,
tea, cranberry juice, unripen fruits, organic acids, and others, which could be classified
into four “true” groups of astringent compounds: salts of multivalent metallic cations
(particularly aluminum salts), dehydrating agents (ethanol and acetone), mineral and
organic acids, and vegetable tannins (Joslyn and Goldstein, 1964).
The mechanisms of astringency caused by vegetable tannins have been well
studied and was thought to result from the interaction of tannins with specific salivary
proteins to form protein-tannin complexes, which would diminish saliva’s ability to
27
lubricate the mouth by subsequent aggregation and precipitation (Luck et al, 1994; Baxter
et al., 1997; Dinnella et al., 2009).
Tannins, commonly referred to as polyphenols, were categorized as condensed
tannins or hydrolyzable tannins as shown in figure 1.8 (Haslam et al., 1988; Bennick,
2002). Tannins in plants were recognized to have biological effects or defense effects,
which could protect plant leaves and unripe fruits from the browsing by their astringency
properties (Bennick, 2002). Traditionally, polyphenols triggering astringency have been
defined with molecular weights between 500 and 3000 Da (Bakker, 1998; Lesschaeve
and Noble, 2005), and it was generally accepted that the degree of polymerization and
molecular weight of polyohenol was positively related to its precipitating ability and
perceived intensity (Bate-Smith, 1973; Arnold et al., 1980; Peleg et al., 1999).
Figure 1.8. The example of hydrolyzable tannins (A) and condensed tannins (B) showing
their molecular structures.
28
It was generally in agreement that salivary proteins were comprised of proline-
rich proteins (PRPs), histidine-rich proteins (histatins or HRPs), α-amylase, and
mucinglycoproteins (de Freitas and Mateus, 2001; Charlton et al., 2002; Condelli et al,
2006; Bajec and Pickering, 2008), and PRPs and HRPs counted for the major proteins
responsible for tannins binding. The extended and unfolded structure of PRPs and HRPs
provided plenty of binding sites that were easily accessed by tannins (Charlton et al.,
1996; de Freitas and Mateus, 1 bstl et al., Dangles and Dufour, 6 Croft
and Foley, 2008; Canon et al, 2009).
Charlton et al. (2002) proposed a 3-stage model describing the binding and
precipitation of PRPs by polyphenols, which was confirmed and further modified by
bstl and cowor ers ( ) (Figure. 1.9). In the first stage, polyphenols interacted with
salivary proteins and bound to multiple sites, causing the previously randomly coiled
protein to coil around the polyphenol and compact. In this stage, hydrophobic interactions
and hydrogen bonding were both involved in the interaction between polyphenols and
PRPs (McRae and Kennedy, 2011). In the second stage, polyphenol bridges were formed
via cross-linking of protein-phenol complexes and polyphenol-PRPs dimers were yielded.
In the last stage, the dimers aggregate to form large complexes and precipitate (Charlton
et al., bstl et al., ). In the last two stages, hydrogen bonding was shown to be
involved (Hagerman et al., 1998; Simon et al., 2003; Cala et al., 2010). Potential
interactions between polyphenols and PRPs could include covalent bonds, ionic bonds,
hydrophobic interaction, or hydrogen bonding. However there was little evidence that
29
prove the presence of covalent or ionic bonds towards these interactions (Haslam et al.,
1991; Hagerman and Butler, 1978).
Figure 1.9. Proposed mechanism for polyphenol-PRPs complex formation ( bstl et al.,
2004).
Factors modulated astringency caused by vegetable tannins have been reported
separately for individual food or beverage. Beverage pH, viscosity, temperature,
sweetener and ethanol concentration were mostly studied and showed diverse relationship
toward astringency. It is generally understood that pH has great impact on the tannins
astringency. Kallithraka and coworkers (1997) found that lowering the pH of model
solutions and red wines resulted in the increase of the astringency intensity. Fontoin and
others (2008) showed similar relationship in model wine solutions. Experiments were
also conducted to reveal the relationship between pH and astringency in cranberry juice,
and results illustrated that juices with low pH were significantly higher in astringency,
regardless of sample temperature or viscosity. High viscosity might contribute to reduced
astringency, and possible mechanisms were suggested to be: (1) the lubricating ability
30
caused by thickening agent such as carboxymethylcellulose (Peleg and Noble, 1999); (2)
the complex formed by hydrocolloid gums and tannins which could reduce tannins
binding efficiency with proteins (Taira, Ono & Matsumoto, 1997). In addition, the
increased viscosity of the model wine may explain the association between high sucrose
concentrations and low astringency (Smith et al., 1996). Surprisingly the addition of
sweetener (aspartame) in grape seed tannins had no impact on the astringency parameter
(Smith et al., 1996). Another important modulating factor normally discussed in red wine
was proposed to be the concentration of ethanol, and higher concentrations were related
to lower intensity of perceived astringency (Vidal et al., 2004; Fontoin et al., 2008). The
recommended mechanisms contained: (1) the conformational changes of tannins in
higher ethanol wines, which could reduce the binding abilities of tannins, the self-
association of bound tannins, and the formation of protein aggregates (Fontoin et al.,
2008). (2) greater ethanol concentrations may increase the lubricity of the oral cavity and
reduce the perception of roughness (Demiglio and Pickering, 2008; Fontoin et al., 2008).
Although the mechanisms caused by salts of multivalent metallic cations were not
fully understood, aluminum sulfate, aluminum ammonium sulfate and aluminum
potassium sulfate have been defined to be astringent agents (Jellinek, 1985; Lee and
Lawless,1991; Breslin et al, 1993). Besides, researchers also stated that zinc was able to
induce astringency as a primary sensation (Keast, 2003; Lim and Lawless, 2005), while
iron, copper, and the minerals magnesium and calcium could induce astringency as the
secondary sensation (Lim and Lawless, 2005; Lawless et al., 2004). Current
understanding about the mechanisms of astringency triggered by metal cations focused
31
on the binding ability of aluminum toward some proteins and the strong binding ability
toward water (Trapp, 1985; Haslam et al., 1988; Khalil-Manesh et al, 1989).
Contrary to the effect of pH on phenols, addition of acid suppressed the
astringency of alum cations. Lawless et al. (1994) reported alum-acid mixtures (alum-
gallic acid or alum-citric acid) showed less intensity in astringency than the unmixed
components, and aluminum cations may act as Lewis bases, binding with atoms with
unshared pairs of electrons, such as carboxylate ions. Other researchers speculated that
chelation of the aluminum cation upon acid reduced the binding ability of aluminum ions
with salivary proteins (Peleg et al., 1998). The viscosity was also indicated as one of the
parameters regulating astringency caused by alum cation. The astringency was detected
to be at lowest level while the viscosity of aluminmum sulfate was increased from
unthickened station to 5-7cP by methylcellulose (Smith and Noble, 1998). The authors
considered that the rising viscosity was able to restore lubrication lost by salivary
proteins and possibly lower diffusivity of the astringent stimuli (Smith and Noble, 1998).
Organic acids such as acetic, fumeric, quinic, adipic, lactic, malic, tartaric, and
citric acid (Martin and Pangborn, 1971; Hyde and Pangborn, 1978; Rubico and
McDaniel, 1992; Hartwig and McDaniel, 1995; Sowalsky and Noble, 1998), can induce
sensations of astringency. The astringency elicited by organic acid is solely a function of
pH, in a pattern of inverse relationship (Hartwig and McDaniel, 1995; Sowalsky and
Noble, 1998; Lawless et al., 1996). No influence from either specific anion or acid
concentration was observed in modulating astringency caused by organic acid (Sowalsky
and Noble, 1998).
32
Dehydrating agents such as ethanol were considered to be astringent, and were
generally used in cosmetic and medication products, however there is no direct evidence
proving the astringency as an oral sensation.
1.2.3 Exploration of mechanisms of astringency caused by whey protein
While whey protein ingredient was attracting more attention from the food
industry with its versatile functionalities and applications, the main problem, astringency,
generated as whey protein beverage formulated under acidic pH has emerged into the
view of researchers. The mechanisms and modulating factors related to astringency
caused by whey protein beverages were still unclear and under great debate.
Recently it was generally in consensus that whey protein itself induced the
astringency. Sano and coworkers (2005) conducted an experiment to compare the sodium
phosphate buffer solution at pH 3.5 as control with whey protein dissolved in the same
buffer solution, they detected no astringency from buffer but found astringency from the
latter solution. This experiment indicated that astringency in acidic whey protein
beverages was resulted from whey protein instead of acid, which was further confirmed
by the dependent relationship between astringency and whey protein concentrations
(Sano et al., 2005; Kelly et al., 2010). However opposite conclusion was brought by other
scientists stating that the astringency of acidic whey protein solutions appeared to be
caused by their high acidity and not directly by the whey proteins (Lee and Vickers,
2008). In this experiment, the total acidity of the solutions was controlled in both control
solutions and whey protein solution, and comparison between both samples did not
support the results gained by Sano and coworkers (Lee and Vickers, 2008).
33
Sano and coworkers also proposed the mechanisms of whey protein beverages.
When acidic WPI solution (pH 3.5) was mixed with salivary protein (pH 7) in the oral
cavity, the pH solution could reach a pH of about 5, which was approximately the
isoelectric point of whey protein. Whey protein would precipitate at their isoelectric
points in the mouth (Sano et al., 2005). Latterly Beecher and coworkers (2008) suggested
another mechanism from their evaluation of four groups of whey protein solutions at pH
6.8, 3.4, 3.0 and 2.6. Based on their observations (Table 1.4), they concluded that the
astringency of acidic whey protein solution could be attributed to the interactions
between the positively charged whey proteins (pH < pI) and negatively charged saliva
proteins.
The pH of whey Charge of whey Charge of salivary Potential for

protein solution proteins proteins aggregation
6.8 - - Less likely
3.4 + - Strongest
3.0/2.6 + + Decreased
Table 1.4. Charges of Whey proteins and salivary proteins in different solutions and their
potential of interaction.
Vardhanabhuti and coworkers (2010) further supported this mechanism by
examining the astringency caused by a commercial lactoferrin at pH 3.5, 4.5, and 7.0.
Lactoferrin had isoelectric point equal to 8.8; hence it remained positively charged at all
pH mentioned above. Their results showed that lactoferrin was astringent at all pH values
34
(3.5, 4.5, and 7.0), indicating that interactions between positively charged lactoferrin and
salivary proteins played a role in astringency (Vardhanabhuti et al., 2010).
1.3 The application of Infrared Spectroscopy (IR) on whey protein.
Infrared spectroscopy is based on the absorption of electromagnetic energy of
vibrations between atoms in a molecule and generation of spectrum information about the
chemical composition and conformational structure of the matrix (Willard et al., 1981).
Those vibrations between atoms are determined by the mass of the constituent atoms, the
shape of the molecule, the stiffness of the bonds, and the periods of the associated
vibrational coupling (Karoui et al., 2010). The mid-infrared spectroscopy (MIR)
(wavenumber range 400 to 4000 cm−1) became especially attractive since measurements
in this range provide direct information concerning the specific constituents in the
sample, as well as their characteristic molecular structure (Etzion et al., 2004). The MIR
characteristic absorption bands generated are generally related to the major components
in food matrix (Table 1.5). IR has been widely used in food analysis with advantages as
excellent signal-to-noise ratio, simultaneous detection of all frequencies, reduced scan
time without loss of resolution, high energy throughput, superior wavelength resolution,
and superior wavelength accuracy obtained through the use of an internal laser to
maintain wavelength calibration (Griffiths and Haseth, 1986). The development of
attenuated total reflectance (ATR) technology has made the application more successful
as it provided a simple and reproducible method to handle liquids and paste samples
(Kuehl and Crocombe, 1984). Research on whey protein products, whether solid or
35
liquid, have been facilitated with the aid of IR and covered the areas including
conformational analysis, quality authentication, and compositional quantitation.
Vibration description and Approximate

Food Component
Chemical bonds Frequency (cm-1)
OH-stretching 3300
Water
OH-bending 1640
Amide I (C=O stretching) 1653
Amide II (N-H deformation and
1567
Protein C-N stretching)
Amide III (CN stretching, NH
1 9−13 1
bending)
Acyl chain C-H 3000~2800
C=O group 1750
Fat
Triglyceride ester linkage C-O 1175
Trans double bonds 966
Aqueous sugar C-O and C-H stretching 1100~1000
Cellulose 1170-1150, 1050, 1030
Pectin 1680-1600, 1260, 955
Table 1.5. A general band assignment of MIR applied to food matrix (Finch and
Lippincott, 1956; Lin and Brown, 1992; Van de Voort et al., 1992; Libnau et al., 1994;
Safar et al., 1994; Dupuy et al., 1996; Michell and Schimleck , 1996; Mascarenhas et al.,
2000; Yang and Irudayaraj, 2000; Ripoche and Guillard , 2001; McClure and Standfield,
2002; Sørense et a., 2003; Kong and Yu, 2007; Hashimoto and Kameoka, 2008; Huang et
al., 2008).
There have been a lot of approaches toward the whey protein conformational
change induced by factors such as pH, heat, interactions with other molecules and more.
Researchers adopted IR as a major technique to assist the identification of the molecular
changes. Table 1.6 showed some of the research done by controlling various factors.
36
Protein Factors induced Conformational change Reference
conformational change
β-lactoglobulin Heating temperature, Intermolecular Allain et
pH, and high pressure hydrogen-bonding β- al., 1999
homogenization of sheet structure
gelling conditions (1614 cm−1)
β-lactoglobulin Heated at 90 °C for 30 Heat increased β-sheet Ngarize et
min and high-pressure- structures at the expense al., 2004
treated at 600 MPa for of α-helix and high-
20 min pressure increased
random coil;
Intermolecular β-sheet
(1625 and 1680 cm-1)
BSA Heat ( −9 °C) Formation of an Murayama
irreversible and
intermolecular β-sheet Tomida,
on heating above 70 °C; 2004
Short-segment chains
connecting α-helical
segments (1630 cm-1)
BSA NaCl, lactose, sucrose, Loss of α-helical Boye et
-1
glucose, cysteine, urea, structure (1654 cm ) and al., 1996
NEM, and SDS, and pH, the rise of the ordered
in gelling non-native β-sheet
structure
(1616 and 1684 cm-1)
α -lactalbumin and β Interaction with phenolic Shift of Amide I and II Zhang et
-lactoglobulin acids peaks; Changes of al., 2014
percentage content of
component bands;
Reduction in the amount
of protein α -helix
structure
Table 1.6. Recent researches on induced conformational changes of individual whey
protein.
One of the most important applications of IR related to whey protein is the
detection of milk adulteration. Milk can be adulterated by addition of water, neutralizers,
salt, sugar, high solid contents, whey, or hydrogen peroxide (Kartheek et al., 2011),
37
which could bring economic problems and health concerns. Whey protein could be added
to milk as an inexpensive protein source, which would be hard to detect due to structure
similarity. Mendenhall and Brown (1991) examined 135 non-fat dry milk samples
containing various concentrations of WPC (1.26 to 33.00%). A strong correlation (Y>
0.99) between the actual concentrations and the values predicted by the Fourier transform
infrared spectroscopy (FTIR) method was found through the utilization of partial lease
square regression (PLSR), and detection limit was identified as 5.6% whey protein
concentrate (Mendenhall and Brown, 1991). Santos and coworkers (2013) spiked the
control milk samples with known levels of whey ranging from 1.87 to 30 g/L. A
classification model built using MIR spectra and multivariable chemometrics to
discriminate control milk and adulterated milk samples was successfully built with large
interclass distances (7.7 between control and whey), and the detection limit was identified
as 7.5 g/L. The authors further quantified the adulterant levels of whey in milk using the
same MIR spectra and PLSR and obtained satisfactory results for both calibration and
prediction models (r = .96). They concluded “MIR could provide the dairy industry with
a simple, rapid and non-destructive technique for detection and quantification of milk
adulteration” (Santos et al., 13).
Since the value and functionality of whey protein products were largely affected
by their composition, the market required a convenient and accurate analytical method.
IR combined with statistical analysis has been found to be a potential method in the
prediction of the major compositions. A study aimed to determine the potential of near
infrared spectroscopy in estimating whey protein products’ compositions was carried out
38
based on 47 whey powders (Baer et al., 1983). The calibration and prediction models
were developed individually for moisture, protein, fat, and lactose to evaluate the
feasibility of IR in correlating the spectra information and reference values. Good
correlation was obtained with high R-value and low standard error of prediction.
Researchers also made their effort to predict whey protein fractions such as α-lactalbumin
and β-lactoglobulin using FTIR spectra (De Marchi et al., 2010; Rutten et al., 2011).
They proved it was possible to use FT-MIR as a routinely assessment in practice,
however these application need to be cautious because the results might not be accurate
enough when variation of genetic levels was involved (De Marchi et al., 2010; Rutten et
al., 2011).
39
Chapter 2: Authentication of Whey Protein Powders by Combining Infrared
Spectroscopy and Pattern Recognition Analysis
2.1 Abstract
Whey proteins are attractive ingredients to the food industry because of their high
nutritional value and wide functionality. Whey protein powders are available from
different suppliers using various processing methods and conditions, resulting in
variability in their component make-up, levels and protein structure, having an impact in
their functionality. There are three major types of whey protein including whey protein
isolates (WPI), whey protein concentrates (WPC) and whey protein hydrolysates (WPH),
which provided diverse functionality to food applications. Our objective was to develop a
simple and rapid method to differentiate whey protein types by combining a portable
infrared spectrometer with pattern recognition analysis. Whey protein powders including
WPI (n = 56), WPC (n = 32) and WPH (n = 23) were obtained from different suppliers
and their spectra collected using a portable infrared spectrometer by pressing the powder
onto an ATR-IR diamond crystal with a pressure clamp. Spectra were analyzed by soft
independent modeling of class analogy (SIMCA) generating a classification model
showing excellent ability to differentiate whey protein types by forming tight clusters
with interclass distance values of >3, considered to be significantly different from each
other. The major band centered at 1393 cm-1 was responsible for separation and can be
associated with carboxylic acid side chains of amino acids present in whey proteins.
40
Portable IR units was able to quickly assess the quality of the incoming raw material
allowing for timely corrective measures during manufacturing. Portable systems are
simple to use and require minimal or no sample preparation, thus reducing assay time and
helping to streamline the analytical procedure so that it is more applicable for field-based
screening and higher sample throughput.
2.2 Introduction
Whey proteins are valuable food ingredients providing functional properties
including gelation, high solubility, water-holding capacity, foaming/emulsification and
sensory characteristics enabling their use in numerous food applications (Lee et al., 1992;
Bouaouina et al., 2006; Dissanayake et al., 2010). Commercial products include whey
protein concentrates (WPC), isolates (WPI) and hydrolysates (WPH) differing on protein
concentration and composition related to processing methods (Foegeding and Luck,
2011; Jelen, 2011). Separation techniques and thermal conditions are the most significant
factors in the variability of whey protein products, not only affecting the amount of
protein and non-protein components but also modifying protein structure ultimately
imparting functionality to food products (Foegeding and Luck, 2011; Jelen, 2011).
Advances in processing technology, including ultrafiltration, microfiltration, reverse
osmosis, and ion-exchange, have resulted in development of several different finished
whey products: whey protein concentrates (WPC) containing between 50 - 85% protein
on a dry basis, whey protein isolate (WPI) containing between 90-98% protein and very
small amounts of lactose and fat, reduced lactose whey, demineralized whey and
41
hydrolyzed whey (Huffman, 1996). The higher level of protein in WPI has found
applications in nutritional supplements, sports and health drinks, and protein-fortified
beverages (Foegeding and Luck, 2011). WPH is produced by enzymatic hydrolysis
containing peptides that enhances digestibility, reduces allergenicity, improves heat
stability, produces bioactive peptides, and alters the functional properties of gelation,
foaming and emulsification becoming an important ingredient in infant formula and
enhanced-performance products (Foegeding et al., 2002; Foegeding and Luck, 2011).
While the production of whey powder has steadily increased (Foegeding and
Luck, 2011), the market demand far exceeds supply resulting in premium prices for whey
protein powders. Ingredient authentication of commercial whey products would limit
fraud but also help ensure manufacturers achieve the desired functionality in their
product. Ion exchange chromatography, electrophoresis, gel-permeation or size exclusion
chromatography, and HPLC techniques have been proven to be effective approaches
towards characterization of individual protein type within complex mixtures of whey
products (Kilara, 2008). In protein research, infrared (IR) spectroscopy has been applied
for the qualitative or quantitative determination of whey protein ingredients (Baer et al.,
1983; Marchi et al., 2009) and to study secondary structure of proteins (Curley et al.,
1998). van der Ven and others (2002) reported the combination of mid-infrared
spectroscopy and multivariate data analysis in protein hydrolysate characterization. Their
findings showed that infrared spectra correlated to various functional properties of whey
and casein hydrolysates to speed up product development (van der Ven et al., 2002).
However there is still a need to establish a fast and efficient method to easily characterize
42
whey protein products that are not limited to a single type to benefit the dairy industry to
support quality control. The objective of this research was to explore the potential of
infrared spectroscopy in authentication of whey protein powders by characterizing
commercial whey protein products (WPI, WPC, and WPH) through pattern recognition
analysis.
2.3 Materials and methods
2.3.1 Whey protein sample
Different commercial whey protein powder products coded with 3 digit number,
including WPI (n=56), WPC (n=32) and WPH (n=23) were obtained from different
manufacturers. Sample information, including moisture, protein, ash, fat, sugar and pH,
was also provided for each powder from individual manufacturer. The pH of powder was
measured by dissolving 5g of each powder into 8oz of deionized water. In addition, a soy
protein powder and gelatin powder were included for validation of the model. Protein
levels and pH info were available.
2.3.2 Fourier-Transform Infrared Spectroscopy (FT-IR)
Spectra measurements were performed using bench-top (Excalibur FTS 3100GX;
Varian, Palo Alto, CA) and portable (Cary 630, Agilent Technologies, Santa Clara, CA)
spectrometers. The bench-top included a dynamically aligned Michelson interferometer,
potassium bromide beam splitter, and a deuterated triglycine sulfate (DTGS) detector.
Spectra were observed using commercial software (Win-IR Pro, Version 3.4.2; Varian
Inc.). IR spectra were collected from 4000 to 700 cm-1 with resolution of 8 cm-1, co-
43
adding 64 scans to increase signal-to-noise ratio. Measurements were performed on an
attenuated total reflectance (ATR) accessory, equipped with a diamond crystal, with
refractive index of 2.5 and an incidence angle of 45o, yielding three internal reflections.
The instrument was calibrated before measurements using the crystal alone (air) as
background. The powder samples were pressed onto the ATR-IR crystal using a high-
pressure clamp (PIKE Tech, Madison, WI). Spectral collection was done in duplicate for
each powder sample.
The portable spectrometer was equipped with a temperature-stabilized DTGS
detector, ZnSe beam splitter and spectrum was collected using a single-bounce diamond
ATR accessory. The powder samples were pressed onto diamond crystal using a pressure
clamp with a slip clutch press providing consistent pressure. The IR spectra were
collected using MicroLab software (Agilent Technologies, Santa Clara, CA) operating in
the wavenumber ranges from 4000 to 700 cm-1 with resolution of 4 cm-1, and 64 scans
were co-added to increase signal to noise ratio. Replicate spectra were also obtained for
each powder sample.
2.3.3 Multivariate classification analysis
The spectra collected were analyzed using the supervised pattern recognition
discriminant analysis: soft independent modeling of class analogy (SIMCA) (Wold,
1976). SIMCA is a classification algorithm based on principal component (PC) analysis
that is applied to each category of interest separately generating a “training set” to build
the classification model (Branden and Hubert, 2005). Spectral data were vector-length
normalized and transformed to their second derivative using a 25-point Savitzky-Golay
44
polynomial filter. A cross validation algorithm was used to determine the number of
principal components describing the systematic variation from spectral data (Wold 1978;
Wold et al. 1983), thus, avoiding over-parametrization or modeling of the noise (Bro et
al., 2008). Sample residuals and Mahalanobis distances were used to determine outliers
(Hruschka, 2001). SIMCA models were evaluated in terms of the matrix of scores and
loadings, discriminating power and interclass distances (Albano et al., 1978; Branden and
Hubert, 2005). Scores-plot projections of the original data onto principal components
axes were used to visualize sample clustering (patterns, groupings, or outliers) while the
loadings plot shows the impact of the variables (wavenumbers) on each vector. Interclass
distance is a critical parameter to determine the separation between classes, which is
measured as the distance by taking along a line connecting the their geometric centers
(Albano et al., 1978), and this is conducted by the comparison of the F-statistic with a
given confidence interval, which is normally 9 % ( eza- rquez et al., 1 ). When F
value is larger than the critical F value, which means the probability is smaller that
critical value (α), the interclass distance can be identified as significantly large and hence
the un nown spectrum does not belong to the nown class, either it is an outlier, or it
comes from a class not represented in the data set ( eza- rquez et al., 1 ). It is also
well known to use external validation samples sets for the evaluation of established
calibration model. The final step in modeling was testing the predictive ability of the
SI CA model by using “validation” dataset. SIMCA model enables positive
identification of new samples to the class only if it falls within the class borders, which
are 95% probability clouds defined around the samples of each class. If it falls outside the
45
class border, it is considered as an outlier (Infometrix, 2008). A chemometrics modeling
software, Pirouette (v4.0, Infometrix Inc., Woodville, WA), was used to perform the
multivariate classification. For analysis, spectra were imported into Pirouette as .dx files
and then analyzed by SIMCA.
2.4 Results and discussion
The spectrum of commercial whey protein powders was collected by attenuated
total reflectance using a benchtop (Figure 2.1A) and portable (Figure 2.1B) infrared
spectrometer, allowing optimization of the contact of the powders with the ATR crystal
surface and minimizing noise interference. Whey protein powders (WPI, WPC, and
WPH) showed fairly narrow bands arising from functional group vibrations in the
information-rich region of 1800 to 900 cm-1, with very similar spectral patterns obtained
by using the bench-top and portable FT-IR units (Figure 2.1). The large broad bands in
the raw spectrum centered at approximately 1640 cm-1 were associated with amide I (α-
helix and β-sheet structures of proteins) while the signal at 1520 cm-1 corresponded to
amide II (combined δ(N-H) and ν(C-N)), contributing to the peptide bond group
vibrations of proteins (Kong and Yu, 2007). The band at approximately 1450 cm-1
resulted from the deformation bending of C-H in the >CH2 groups and the band at 1390
cm-1 corresponded to the stretching of C=O in the COO- groups (Coates, 2000). A
distinct feature in the spectra was associated with the broad peak band ~ 1250 cm-1
corresponding to the combined signal from the amide III groups and P=O stretching in
phosphodiesters (>PO2-) (Coates, 2000; Kong and Yu, 2007). The bands between 1200
46
and 900 cm-1 were associated with vibrations of C-O-C and C-O, attributed to sugars,
acids and/or polysaccharide components of the samples (Shiroma and Rodriguez-Saona,
2009).
CH groups of lipids
A
Amide II
Amide I
υC-O-C of
acyl chain of TAG

C-H stretching of
C=O groups of lipids

polysaccharides
O-H groups
Absorbance
WPH
WPC
WPI
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
Wavenumber (cm-1)
B
CH groups of lipids
Amide II
Amide I
υC-O-C of
acyl chain of TAG
C-H stretching of
polysaccharides
O-H groups
Absorbance
WPH
WPC
WPI
3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1800 1600 1400 1200 1000 800
Wavenumber (cm-1)
Figure 2.1. Mid-Infrared spectra of Whey protein powder from bench-top FT-IR
Spectroscopy (A) and portable FT-IR Spectroscopy (B) with Attenuated Total
Reflectance (ATR)
47
The spectral complexity required the application of mathematical filter functions
(normalization and second derivative), as well as, selection and combination of certain
spectral regions (1800-1000 cm-1) to enhance spectral resolution and model performance.
Prior to data analysis, each spectrum was vector-length normalized to minimize the
effects of potentially variable concentrations in the samples, and to reduce the systematic
error in the data (Zeaiter et al., 2005; Rinnan et al., 2009a). Second derivative
transformation of the spectra was performed to resolve spectral bands from the raw
spectrum (Zeaiter et al., 2005).
The SIMCA classification plots (Fig. 2.2) showed well separated clustering
among the powder samples, whose orientation in the 3D space correlated with whey
protein type (i.e. WPC, WPI and WPH). However, Figure 2.2 showed the existence of 7
clusters defined by pH and protein level of the powders associated with WPI (3 classes)
and WPH (3 classes) while all WPC samples were classified in a single cluster. A similar
clustering pattern was obtained using the bench-top (Figure 2.2A) and portable (Figure
2.2B) infrared units. High protein concentration (>65%) WPC are produced by
ultrafiltration and diafiltration while WPI and WPH are manufactured using various
technologies such as ion exchange, electrodialysis, nanofiltration, controlled enzymatic
hydrolyzation (Jelen, 2011), having a strong effect on the composition of the final
powder. van der Ven and others (2002) reported the ability of FTIR spectra to distinguish
protein hydrolysates prepared from different protein (casein or whey) sources and
proteolytic enzymes (acidic or neutral/alkaline).
48
A WPI (II) B WPI (III) WPI (II)
WPI (III)
PC2
PC2
WPC
WPI (I) WPI (I) WPC
PC3
PC1 PC1
PC3
WPH (I)
WPH (II) WPH (I)
WPH (II)
WPH (III)
WPH (III)
C Soy protein D WPI (III) Soy protein

WPI (II)
WPI (II)
WPI (III) PC2
PC2
WPI (I)
WPC
Gelatin WPC
PC3
WPI (I) Gelatin
PC1
PC1
PC3
WPH (II) WPH (I) WPH (I)

WPH (II)
Figure 2.2. Soft independent modeling of class analogy (SIMCA) classification plots for
calibration models using a benchtop (A) and portable (B) FTIR spectrometers. SIMCA
validation performance for calibration models using a benchtop (C) and portable (D)
FTIR spectrometers based on the infrared spectra of whey protein powders. WPI (I): pH
of protein powder ranging from 5.8-7.5, WPI (II): pH ranging from 2.8-3.5, WPI (III): pH
ranging from 3.8-4.0, WPH (I): protein levels were 80%-85% and pH ranging from 6.0-
9.0, WPH (II): 90% and pH ranging from 7.2-7.8, WPH (III): CGMP. pH was measured
by dissolving 5g of whey powder in 8 oz of deionized water.
49
SIMCA showed that WPI grouping was based on their powder pH level with
classes discriminated having pH ranges of 5.8 to 7.5 (WPI - I), 2.8 to 3.5 (WPI – II) and
3.8 to 4.0 (WPI – III), most likely associated with the type of technology used to
manufacture the WPI powder. A different situation was observed for WPH, with samples
clustering based on protein levels of 80% to 85% (WPH – I) and 90% (WPH – II). A
unique case was WPH - III samples that clustered furthest from the rest and were
associated with containing casein glycomacromolecule protein (CGMP).
Examination of the discriminating power plot provides information on spectral
frequencies that were most important for separating the different classes. Figure 2.3A
showed that the SIMCA model, generated using spectra collected using the benchtop
FTIR unit, had a major discriminating band 1390 and 1570 cm-1 corresponding to the
deprotonated carboxylate group for the symmetric and antisymmetric stretching vibration
(Barth, 2000). This band shifts upon cation chelation, and the effects depend upon the
mode of chelation and have been used to study several Ca2+ binding proteins (Fabian et
al., 1996; Mizuguchi et al., 1997). Thus, the band observed at 1339 cm-1 most likely can
be attributed to the carboxylate vibrations of Asp groups due to protonation change
(Gerwert et al., 1989).
50
1578 1636
A
1339
Discriminating Power
300
1389
200
1446
1200
100 1523
0
1080 1273 1466 1659
600
B 1351
Discriminating Power
1387 1570
1627
400
1426 1510
200
0
1000 1200 1400 1600 1800
Wavenumber (cm-1)
Figure 2.3. Soft independent modeling of class analogy (SIMCA) discriminating power
based on the infrared spectra of whey protein powder from bench-top FT-IR
Spectroscopy (A) and Portable FT-IR Spectroscopy (B).
The amide I region of the spectrum (~1630 cm-1) in figure 2.3 was also associated
with the discrimination of whey protein types. The absorbance in amide I band region is
predominantly due to the stretching vibration of the carbonyl peptide bond, whose
frequency is highly sensitive to hydrogen bonding and thus to protein secondary structure
51
(Kong, 2007). The amide I band with a maximum wavenumber centered near 1633 cm-1,
is characteristic of β-sheet (Farrell Jr., 2001). The aliphatic moieties of amino acid side
chains give rise to vibration absorbance bands at 1465 and 1450 cm-1 associated to δ
(CH3) and δ(CH2) group frequencies while the ν(CN) band near 1 3 cm-1 has been
related to backbone conformation (Barth, 2000). The discriminating power plot for the
model created using spectra from the portable FT-IR unit (Figure 2.3B) also showed the
importance of the carboxylate groups of acidic amino acids (1350, 1387 and 1570 cm-1)
in the classification of the powders.
SIMCA classification models showed large interclass distance values (Table 2.1)
for discrimination among WPI, WPC and WPH samples, evidencing the ability of
infrared spectroscopy combined with chemometrics to achieve reliable resolution of
unique spectral markers. Interclass distance is the distance between the geometric centers
of two clusters; it represents the performance of separation with larger interclass distance
resulting in better separation (Albano et al., 1978). Class distances greater than 3 between
clusters are considered significant for identification of data points as members of a group
(Kvalheim and Kratang, 1992). All of our interclass distances obtained using spectra
from both bench-top and portable FT-IR spectrometers had values larger than 3,
evidencing the excellent discrimination among all classes from the view of statistical
significance (p < 0.05). Interestingly class 5 (WPH-III) achieved large interclass
distances from the rest of the classes (9.42-49.92), which indicated that the CGMP
powder had marked different compositional characteristics from others. Most whey
powder samples fall in classes WPC, WPI (I) and WPH (I), showing interclass distances
52
of 3 (WPC vs WPI (I)), 6 (WPI (I) vs WPH (I)) and 5.5 (WPC vs WPH (I)). The protein
levels of WPH (I) and WPH (II) groups averaged 81.82±2.13% and 90%, respectively,
exhibiting an interclass distance of 4 reflecting differences in composition associated
with levels of protein isolation. Overall, the interclass distances showed excellent
predictive ability of the model for discriminating among the different commercial whey
protein powders.
A
CS1@3 CS2@3 CS3@3 CS4@3 CS5@3 CS6@3 CS7@3
CS11 0.00
CS2 3.33 0.00
CS3 6.3 . 8 0.00
CS4 7.9 9.3 . 9 0.00
CS5 1 .83 1 . 1 .1 16.1 0.00
CS6 7.3 6.78 1 .77 19. 3 .8
CS7 . 7 6. 11.7 1 .3 6. 8 5.31 0.00
CS1@3 CS2@3 CS3@3 CS4@3 CS5@3 CS6@3 CS7@3

CS1 0.00
CS2 3. 0.00
CS3 6. .69 0.00
CS4 1 . 1 . 8 . 8 0.00
CS5 16.18 16.37 9. 3 .1 0.00
CS6 7. 7 8. 1 .3 8. 9 9.9 0.00
CS7 .6 7.73 11. 9 31.3 3 . 8.02 0.00
Table 2.1. Interclass Distances generated from whey protein powder calibration model
using bench-top (A) and portable (B) FT-IR Spectroscopy.
1
CS: CS indicated class assigned to individual category of whey powder products.
Classes were assigned as: Class 1: WPI (I), pH of protein powder ranging from 5.8-7.5,
class 2: WPC, class 3: WPH (I), protein levels were 80%-85% and pH ranging from 6.0-
9.0, class 4: WPH (II), 90% and pH ranging from 7.2-7.8, class 5: WPH (III), CGMP,
class 6: WPI (II), pH ranging from 2.8-3.5, class 7: WPI (III), pH ranging from 3.8-4.0.
pH was measured by dissolving 5g of whey powder in 8 oz of deionized water.
CS1@3: this indicated the number of factor used in SI CA model.
53
Finally, the predictive ability of the model was tested using a validation set of
samples that were not included in the calibration set. The number of test samples used
was 30% of the total samples evaluated for each class (Table 2.2). However, WPI (II) and
WPH (II and III) samples were not included in the validation because of its limited
numbers (3-5 samples). We obtained 100% accurate predictions for all whey protein
powders (Figure 2.2C and 2.2D) that were assigned to its correct class. Furthermore, we
included a soy and gelatin protein to test the model performance in predicting proteins
from different sources. The results showed that these “confounding” proteins were
reliably predicted as not belonging to any of the classes by using the models generated
from spectra by bench-top (Figure 2.2C) and portable (2.2D) spectrometers.
Number of Sample Involved

Protein Powder Type FT-IR Benchtop Portable FT-IR
Spectroscopy Spectroscopy
Training Validation Training Validation
WPC WPC 24 8 10 5
WPI (I) 34 16
WPI (II) 12 7
WPI
WPI (III) 10 4
Total 44 12 20 7
WPH (I) 16 16
WPH (II) 5 2
WPH
WPH (III) 3 3
Total 19 5 16 5
SOY PROTEIN 1 1
GELATIN 1 1
Table 2.2. Number of protein samples used for spectral acquisition using the bench-top
and portable FT-IR Spectrometers
54
2.5 Conclusion
FT-IR spectroscopy combined with pattern recognition chemometrics had the ability to
authenticate commercial whey protein products, clustering powders according to different
manufacturing processing, reflected by differences in powder pH (WPI) and protein
levels (WPH). Portable FT-IR spectroscopy proved its robustness by producing models
with high spectral resolution and large interclass distances resulting in accurate prediction
ability, which made this technology a perfect choice for “in-field” authentication.
55
Chapter 3: A Novel Application of FT-IR Technique Combined with Chemometrics
in Analyzing and Predicting the Astringency of Acidic Whey Protein Beverages
3.1 Abstract
Formulating whey protein beverages at an acidic pH, in order to obtain better
clarity, normally develops an unpleasant and astringent flavor. ur ob ective was to
explore the application of infrared spectroscopy in predicting the astringency of acidic
whey protein beverages. Three types of whey protein powders including whey protein
isolate (WPI), whey protein concentrate (WPC), and whey protein hydrolysate (WPH)
from different manufacturers were used to formulate beverages at pH range . - 3.9.
Each sample was sensory tested through trained panelists by the SpectrumT method of
descriptive analysis for generation of quantitative astringency data. Three Fourier
transform infrared spectroscopy (FTIR) techniques, including FTIR- attenuated total
reflectance (ATR) icro-spectroscopy, FTIR-Reflectance icro-spectroscopy and
Portable FTIR-ATR spectrometer were used for spectra collection. Spectra were analyzed
by multivariate regression analysis (Partial least squares regression, PLSR) to build
calibration models with the combination of sensory astringency scores obtained from
trained panelists. The PLSR analysis showed a strong relationship between the reference
astringency values and the IR predicted values. PLSR models based on different protein
types (WPI, WPC, and WPH beverages) were generated for astringency determination
with standard error of cross validation (SECV) less than . 7 and correlation coefficient
56
larger than .7 . a or characteristic absorption bands related to astringency were
-
associated with the C groups and amide I & II regions. A rapid and reliable technique
to accurately and precisely predict the astringency of acidic whey protein beverages was
developed, which can contribute to improve sensory characteristics of whey-containing
products and provide a novel tool for timely corrective actions during processing, thus
decreasing manufacturing cost.
3.2 Introduction
The use of whey proteins in beverages in food industry is attractive because of
their high nutritional value and wide functional versatility. Also the consumption of whey
protein beverages is appealing due to weight control, muscle building and health benefit.
Accompanying the increasing popularity of whey protein beverages, the sensory
challenge of astringency has developed (Sano et al., eecher et al., 8) when
they are formulated at low pH to improve beverage clarity and heat stability ( iller,
7). This astringency limits consumer acceptance of these products.
Astringency is described as a drying, puc ering sensation which is typically
caused by four ma or types including vegetable tannins, dehydrating agents (alcohol),
salts of multivalent metallic cations (alum), and acids ( ate-Smith, 19 , Siebert and
Chassy, ). Astringent foods such as wines, teas, fruits, and soy-based products are
one of the most widely studied categories, and the mechanism of astringency has been
attributed to polyphenolic compounds (Haslam et al., 1988) that bind and precipitate
specific salivary proteins ( axter et al., 1997 Kallithra a et al., 1998), diminishing
57
saliva’s ability to lubricate the mouth (Clifford, 1996). Further studies included whey
protein beverages as a new member in astringency food family (Sano et al., ),
however, the exact cause of astringency in acidic whey protein beverages is un nown.
Recent studies that have investigated the astringency of whey protein beverages have
proposed that the whey proteins are directly responsible for the astringency. eecher and
others ( 8) suggested that at low pH, positively charged whey proteins are capable of
binding with and aggregating salivary proteins, causing astringency. Whereas Lee and
Vic ers ( 8) suggested that the astringency of acidic whey protein beverages was
possibly caused by their high acidity.
Currently, astringency of whey protein beverages is determined by trained sensory
panelists, a benchmar in qualifying and quantifying sensory properties, however, it is
laborious, expensive and time consuming. With the demand for protein fortified foods
and beverages increasing steadily, a quic and reliable method for predicting astringency
to improve overall flavor is important. Time and money can be saved through analysis of
a few variables whereby quality control of the product could be established early in the
manufacturing process. Infrared spectroscopy ( -7 cm-1) is an attractive technology
for the rapid, sensitive, and high-throughput analysis of food components, producing a
molecular "fingerprint" spectrum that represents the absorption bands corresponding to
frequencies of vibrations between the bonds of the atoms ma ing up the material.
Furthermore, specific fairly narrow bands arising from group vibrations may be assigned
to nown specific chemical entities in most cases. Developments in the field of
multivariate techniques have been prompted by the need of reliable, accurate, robust and
58
simple methods for routine analysis of spectroscopic data (Udelhoven et al., ).
Principal component regression (PCR) and partial least-squares regression (PLSR) are
factor analysis methods that have the potential to estimate the component concentration
from the infrared spectra (Haaland and Thomas, 1988). ur ob ective was to develop an
astringency predictive model for acidic whey protein beverages based on the relationship
between the perceived intensity of the selected sensory stimuli and unique infrared
spectral fingerprints.
Whey protein beverages were made from whey protein isolate (WPI), whey
protein concentrate (WPC) or whey protein hydrolysate (WPH) powder obtained from
different manufacturers. Sucrose was added in each formulation, and phosphoric acid
( %) or Potassium carbonate ( %) was used to ad ust the pH of individual beverage
sample to target value. Two batches of beverage samples were prepared separately. The
first batch was controlled under uniform pH value (pH=3. ) after formulation from
different powders, including WPI (3 ), WPC (18), and WPH (1 ) powders. In the second
batch, beverage formulated by each whey protein powder was further ad usted to
different pH values (ranging from . to 3.9). WPI (9), WPC (6), and WPH ( ) powder
samples from different manufacturers were used in the second batch. Each sample was
coded with 3 digit number and vacuum pac ed in a glass bottle. everage samples were
maintained at ºC until spectra collection using FT-IR.
59
A separate batch of samples was made from pre-selected powders, including
whey protein powders and 1 gelatin powder to evaluate the model performance. Each
whey protein solution was formulated from 1 type of powder along with sucrose, and
dissolved in water. After measuring pH of each fresh solution, phosphoric acid ( %) or
potassium carbonate ( %) was used to ad ust the pH to , 3, , , 6 and 7. Solids
measurement was ta en before and after pH ad ustment to verify how the concentration
may have changed.
3.3.2 Astringency prediction
Quantitative sensory test on all whey protein samples mentioned above was
conducted at the Sensory Service Center at North Carolina State University, which is an
internationally recognized program in Dairy products. Sensory evaluation was conducted
by panelists trained in the Spectrum method of descriptive analysis for generation of
quantitative data. A to 1 -point universal intensity scale was utilized to describe
astringency score, where meant no intensity of astringency perception and 1 indicated
highest intensity of astringency possible.
3.3.3 FTIR Micro-spectroscopy
To perform spectra collection from each beverage sample, a Varian 6 U A IR
microscope (Varian, Randolph, A) interfaced with an Excalibur series 31 GX FT-IR
spectrometer (Varian, Walnut Cree , CA) was used for rapid data scan. The FTIR
microscope was equipped with a dynamically aligned ichelson interferometer, a
motorized x-y stage, a potassium bromide beam splitter, and a broadband mercury-
cadmium-telluride ( CT) detector. Spectra were observed using Resolution Pro Software
60
(Version . , Varian). IR spectra were collected from to 7 cm-1 with co-adding
1 8 scans to increase signal to noise ratio. easurements were performed by two
approaches: using a slide-on attenuated total reflectance (ATR) germanium ob ective
(Varian 6 U A, Palo Alto, CA) or FT-IR reflectance. In both methods, aliquots ( . ul)
of the beverage samples were placed on a microscope slide, and vacuum dried for
minutes to form a thin film. The slide was placed directly onto the stage of Varian 6
U A IR microscope. Resolution of 8 cm-1 was used in the ATR approach, and Resolution
of cm-1 was used in the reflectance approach.
3.3.4 Portable FTIR Spectroscopy
easurements were also performed in a portable system (Cary 63 FTIR
Spectrometer, Agilent Technologies, Santa Clara, CA) coupled with a diamond ATR
accessory, a temperature-stabilized DTGS detector, and ZnSe beam splitter. In every
spectrum collection step, . ul of the beverage sample was placed on the center of
diamond crystal then water vacuum was applied for 1 minute to get rid of the ma ority of
the water in the beverage. Finally, a nice film was formed on top of diamond crystal and
it was the moment to ta e spectrum measurement. The IR spectra were collected using
icroLab software (Agilent Technologies, Santa Clara, CA) operating in the
wavenumber ranges from to 7 cm-1 with resolution of cm-1, and 6 scans were
co-added to increase signal to noise ratio.
3.3.5 Multivariate regression analysis
The astringency scores of whey protein samples were used to test the ability to
predict the sensory attributes based on their IR spectra in a multivariate model.
61
ultivariate regression analysis (Partial least squares regression, PLSR) can analyze data
with strongly correlated, noisy, and numerous X-variables, and also simultaneously
model responser variables (Wold et al., 1). In this experiment, PLSR was performed
to build calibration models to simultaneously correlate the reference sensory astringency
score to the infrared spectral information. PLSR is a bi-linear regression analysis method
that extracts a small number of orthogonal factors that are linear combinations of the
spectral (X) variables, and uses these factors as regressors for the analyte’s concentration
(Y-variable). These orthogonal factors (latent variables) explain as much as possible of
the covariance of the X and Y variables. PLSR has the potential to estimate the
component concentration and chemical and physical properties (loading vectors, vector
of final calibration regression coefficients, and spectral residuals) from the spectra. PLSR
has been particularly successful in developing multivariate calibration models for the
spectroscopy field because it uses the concentration information (Y-variable) actively in
determining how the regression factors are computed from the spectral data matrix (X),
reducing the impact of irrelevant X-variations in the calibration model. This capability
provides a more information-rich data set of reduced dimensionality and eliminates data
noise which results in more accurate and reproducible calibration models. (Wold et al.,
1). efore analysis, pre-processing methods such as standard normal variate
transformation (SNV) and Savitz y–Golayand second derivative were used to remove the
slope variation from spectra caused by scatter and variation of particle size ( arnes et al,
1989 Candolfi et al., 1999), to resolve overlapping bands, and to correct for baseline drift
(Rinnan et al., 9b). PLSR models were evaluated in terms of loading and score
62
vectors, standard error of calibration (SEC), standard error of cross validation (SECV),
coefficient of determination (r-value) and outlier diagnostics. The PLSR calibration
model was cross validated (leave-one-out approach) to validate the prediction ability of
new data and estimate the chance to obtain a good fit of random sample (Wold et al.,
1).
3.4.1 Perceived astringency scores for whey protein beverages
Whey protein beverages manufactured with a fixed pH (3. ) ranged in astringency
scores from 1.9 to 3.7 for WPC, . to . for WPI and .1 to .3 for WPH, indicating the
importance of protein source in the astringency sensory response. Furthermore,
astringency scores of whey protein beverage samples manufactured at various pH levels
(Table 3.1) showed that whey protein beverage samples exhibited different astringency
patterns when their pH values decreased from 3.9 to . : Statistical analysis ( ultiple
Linear Regression, Table 3. ) showed that astringency scores were explained by pH and
protein source (type and processing) (p-value < . ) in acidic whey protein beverages,
revealing the complexity of astringency development in acidic whey protein beverages in
the pH ranges of . -3.9. eecher and others ( 8) also evaluated the effect of pH on
astringency development in whey protein solutions and found that whey protein solutions
exhibited higher astringency at acidic pH (~3. ) than neutral pH (6.8), indicating the
importance the pH levels to explain the astringency. They also found a decrease in
astringency between pH 3. and .6 that coincided with an increase in sourness ( eecher
63
et al., 8). ur results indicated that differences in astringency of whey protein
beverages at acidic pH might also be associated to the type of processing of the whey
proteins during manufacture of the powders.
Whey Protein Type

Whey Protein Concentrate Whey Protein Isolate Whey protein Hydrolyzate
Sample pH Astringency RSD Sample pH Astringency RSD Sample pH Astringency RSD
2.59 3.8 5.69 2.40 2.6 2.41 2.5 16.84
2.89 3.7 7.49 2.48 3.5 2.74 2.4 12.52
WPC1 3.53 2.9 6.75 WPI1 2.49 2.3 WPH1 3.19 2.1 13.53
3.34 3.4 7.87 3.29 3.0 3.43 2.0 22.07
3.87 3.3 8.36 3.70 2.6 3.78 1.9 20.21
2.56 3.7 5.96 2.43 2.9 2.52 3.8 7.91
2.86 3.6 6.86 2.65 3.1 2.82 3.6 6.41
WPC2 3.21 3.6 6.55 WPI2 3.08 2.6 WPH2 3.34 3.8 6.14
3.57 3.0 7.96 3.45 3.0 11.68 3.58 3.3 8.08
3.91 3.7 6.38 3.72 3.3 10 3.95 2.9 8.54
2.58 3.4 6.82 2.25 2.8 2.51 3.5 5.99
2.85 3.7 5.55 2.53 2.9 2.77 3.7 6.2
WPH3
WPC3 3.30 3 7.47 WPI3 2.86 2.9 3.24 3.0 9.89
3.58 3.1 6.78 3.29 3.2 3.65 2.9 6.51
3.85 3.5 5.52 3.56 2.8 3.96 2.5 9
2.56 3.7 6.46 2.55 3.4 2.44 2.9 6.32
2.86 3.2 7 2.82 3.4 2.76 3.6 5.26
WPC4 3.28 2.7 14.05 WPI4 3.24 2.9 WPH4 3.27 3.0 7.12
3.56 2.9 4.14 3.50 2.7 3.47 3.0 5.01
3.89 2.5 8.08 3.77 2.3 3.87 3.0 6.3
2.61 3.6 7.17 2.59 3.2
2.89 3.5 5.47 2.87 2.9
WPC5 3.26 3 9.1 WPI5 3.31 2.9
3.53 2.9 5.89 3.56 2.4
3.83 3.1 7.47 3.87 2.8
2.63 3.8 4.64
2.91 3.5 8.06 2.62 3.6 7.51
WPC6 3.34 3 6.73 WPI6 2.93 3.2 9.47
3.63 3.2 4.48 3.31 3.1 9.58
3.92 3.1 5.86 2.38 3.0 9.93
2.60 3.2 11.05
WPI7 3.04 2.8 10.43
3.28 2.7 10.39
3.57 3
2.59 3.0 7.25
WPI 8 2.87 3.1 8.6
3.08 3.1 6.8
2.47 3.8 8.67
2.81 3.2 8.73
WPI9 3.11 3.7 8.61
3.46 3.2 8.39
3.80 3 12.03
Table 3.1. Sensory astringency scores perceived from whey protein beverage samples at
various pH values ranging from . to 3.9.
64
Protein Soures pH Levels1 Protein Types R (%)
P(regression)
WPI 9 . .
WPC 6 6 .3 . 1
WPH 9 . .
Table 3.2. The multiple linear regression using pH levels and protein types as predictor
and astringency as response.
1
For each protein types from different protein sources, 5 different pH levels ranging from
2.2 to 3.9 were used as explanatory variable.
3.4.2 Comparing spectra information from whey protein powder and beverage
We developed a novel protocol to analyze whey protein beverages by IR icro-
spectroscopy by using a SpectRI (Tienta Sciences) slide, which provided a high
infrared reflectance surface and the ability to concentrate the samples up to 1 times due
to the presence of a hydrophobic coating. This technique permitted collection of high-
quality and reproducible spectral data from small amounts of samples (~ . - μL)
through formation of thin homogeneous films after vacuum-drying. This technique was
selected because it provided high-throughput capabilities allowing data collection in <1
min.
The infrared spectrum of whey protein isolate powder and beverage (Figure 3.1)
in the information-rich region of 18 -7 cm-1 showed characteristic bands arising from
functional group vibrations. The large broad bands in the raw spectrum at 169 ad 1637
cm-1 in the raw spectrum were associated with amide I (α-helix and β-sheet structures of
proteins) while the signal at and 1 cm-1 corresponded to amide II (combined δ(N-H)
and ν(C-N) contributions of the peptide bond) group vibrations of proteins. The band at
65
approximately 1 cm-1 resulted from the deformation bending of C-H in the >CH
groups and the band at 139 cm-1 corresponded to the stretching of C= in the C -
groups (Figure 3.1). A distinct feature in the spectrum was associated with the broad pea
band ~ 1 cm-1 corresponding to the combined signal from the amide III groups and
-
P= stretching in phosphodiesters (>P ). The bands between 1 and 9 cm-1
resulted from the vibration of C- -C and C- were attributed to sugars, acids and/or
polysaccharide components of the samples. This latter region was prevalent in the
beverage samples (Figure 3.1) as they contained added sugars and acids in the matrix.
Whey Protein powder

Whey Protein beverage uC-O-C of
Amide II
polysaccharides
of acyl chain of triacylglycerols
CH groups of lipids
Amide I
CH and carbonyl stretching
O-H groups
water
Absorbance
3600 3200 2800 2400 2000 1600 1200 800
Wavenumber (cm-1)
Figure 3.1. Attenuated Total Reflectance (ATR) id-Infrared spectra of Whey protein
powder and beverage.
66
The spectral complexity required the application of mathematical filter functions
(SNV and second derivative) and selection and combination of certain spectral regions to
enhance spectral resolution. athematical (second derivative) transformations of the
spectra highlighted distinct features between the samples (Figure 3. ) and reduced
spectral noise helping to minimize the variability between replicates.
Powder
Absorbance
Beverage
3600 3200 2800 2400 2000 1600 1200 800

Wavenumber
Figure 3. . Second derivative spectral transformation collected using an Attenuated Total

Reflectance Infrared (ATR-IR) accessory equipped either with a ZnSe crystal plate
(bench-top) or Germanium crystal ( icro-spectroscopy).
3.4.3 SIMCA classification of whey protein beverages with fixed pH
Evaluation of whey protein beverages with fixed pH (3. ) value was examined
using FTIR-ATR icro-spectroscopy and SI CA classification plot (Figure 3.3) was
67
generated to visualize well-separated clustering among the beverage samples. Evaluation
of the SI CA classification plots showed that most samples formed tight clusters, and
were far away from others with interclass distance values greater than 1 for
discrimination among WPI, WPC and WPH samples, evidencing the ability of infrared
spectroscopy combined with chemometrics to achieve reliable resolution of unique
spectral mar ers. Usually interclass distances greater than 3 between clusters were
considered significant for identification of data points as members of a group (Kvalheim
and Kratang, 199 ).
Figure 3.3. Soft independent modeling of class analogy (SI CA) classification plot and
discriminating power based on the infrared spectra of whey protein beverage samples.
The SI CA discriminating power algorithm showed that the 16 1 cm-1 band was
most important for separating the different classes, corresponding to disordered β-sheet
!
68
bands. In peptides, the frequency of the C-amide I band is a sensitive local probe of
secondary structure and the alignment of residues within a β-sheet. As the peptides align,
the C-amide I band at 16 1 cm-1 (due to the misaligned form) decreases in intensity and
the band at 1 91 cm-1 (due to the aligned form) increases (Petty et al., ). These
irreversible changes in protein structure are possibly due to heating or allowing the
sample to stand at room temperature for extended periods of time (Petty et al., ). The
band at 16 1 cm-1 has also been assigned to the characteristic N-H bending vibrations of
Asp. Although the original Asp band can be observed at 1 97 cm-1, with increasing
concentration the band shifts toward 16 1 cm-1.
3.4.4 PLSR calibration models of whey protein beverages with fixed pH
The PLSR plots and loadings for whey protein beverages with fixed pH value
(WPI, WPC, and WPH beverages) were generated separately in Figure 3. . Among all
three types of whey protein products, WPH generated the best calibration models with the
highest R ( .98) and lowest standard error of cross validation (SECV= .11) shown in
Table 3.3. WPI and WPC beverages also performed strong correlations between IR
spectra and sensory astringency score (R> .81, SECV< . ).
69
3.8 A. WPI B. WPI
0.2
3.4
-0.0
1582
3.0 -0.2
1601
2.6 -0.4
IR Predicted Value
2.6 3.0 3.4 1543 1620 1697 1775

C. WPC D. WPC
3.4 0.2
1535
Loadings
3.0 -0.0
1504
-0.2 1582
2.6
1647
-0.4
2.2
2.4 2.8 3.2 3.6 1466 1543 1620 1697
E. WPH F. WPH
4.0 0.2
3.5 -0.0
1582
3.0 -0.2
1574 1690
-0.4
2.5
-0.6
2.0 2.5 3.0 3.5 4.0 1543 1620 1697
Wavenumber (cm-1)
Sensory Astringency Score
(3.5) using FTIR-ATR germanium Micro-spectroscopy.
70
WPI WPC WPH
1
Sample Number 16 13
-1
Wavenumber Range (cm ) 1 -18 1 -1697 1 97-1736
Factor 6
Cumulative 1 . 99.99 1 .
SECV3 .17 . .11
R .89 .81 .98
SEC .16 . 3 .1
R Cal6 .91 .8 .99
7
RPD .18 1.7 .

whey protein beverages with fixed pH (3.5) using FTIR-ATR Micro-spectroscopy
spectra. 1Sample Number: Whey protein beverage samples used in individual model.
Cumulative: Cumulative variances explained by the calibration model. 3SECV: Standard
error of cross-validated calibration model. R: Coefficient of cross-validated calibration
model. SEC: Standard error of calibration model. 6R Cal: Coefficient of calibration
model. 7RPD: relative percent deviation
The model producing the minimum SECV, standard error of prediction
(magnitude of error expected when independent samples are predicted using the model),
was selected as the best model for the spectral data set. Examination of the loading
indicated bands explaining the regression between infrared spectra and astringency scores
in all three models was 1 8 cm-1, which was associated with anti-symmetric stretching
−
of Asp C groups ( izuguchi et al., 1997).
3.4.5 PLSR calibration models of whey protein beverages with various pH
The effect of various pH values of whey protein beverages were modeled for
predicting the astringency scores by trained sensory panel. The performance of models
generated from spectra collected by using FTIR icro-spectroscopy (ATR and
71
reflectance modes) and Portable FTIR-ATR spectrometer were evaluated for predicting
whey protein astringency. As illustrated in both Figure 3. and Table 3. , good regression
models were obtained for all three whey protein products using FTIR-ATR icro-
spectroscopy, with the smallest R equal to .8 . The most important bands for Factor 1
(solid line) in the loading plot for WPI, WPC, and WPH beverages were centered at 1
and 16 7 cm-1 that were related to the structural information on proteins. The large broad
bands in the raw spectrum at 16 7 cm-1 were associated with amide I (α-helix structures
of proteins) while the signal at and 1 cm-1 corresponds to amide II (combined δ(N-H)
and ν(C-N) contributions of the peptide bond) group vibrations of proteins (Kong and Yu,
7). While Factor 1 explained a large percentage of the model variance, Factor
(dashed line) also played an important role in revealing functional groups in protein
contributing to the establishment of astringency model. The bands 1 8 cm-1 observed in

−
Factor of WPC beverage models indicated the importance of C groups ( izuguchi
et al., 1997).
72
3.5 A. WPI B. WPI
0.2
3.0 1539
-0.0
1562
2.5
-0.2
1647 1690
2.5 3.0 3.5 1543 1620 1697 1775
C. WPC D. WPC
0.2
IR Predicted Value
0.1
3.5
Loadings
-0.0 1431
1585
1539
3.0 -0.1
1443
-0.2
1686
1647
2.5
2.5 3.0 3.5 1466 1659
E. WPH 0.3
F. WPH
3.6
3.2
0.1
2.8
1543
2.4 -0.1 1458
1427
1620
2.0
1647
-0.3
2.2 2.6 3.0 3.4 3.8 1466 1659
Sensory Astringency Score Wavenumber (cm-1)
(ranging from 2.2 to 3.9) using FTIR-ATR germanium Micro-spectroscopy.
PLSR calibration models of WPI, WPC, and WPH based on FTIR-Reflectance icro-
spectroscopy and Portable FTIR-ATR spectrometer also formed good relationship
between infrared spectra and sensory astringency scores, as illustrated in Table 3. . With
all three applications of IR techniques, very robust calibration models (R> .93) were
73
constructed based on WPH beverage samples. However WPI beverages showed the
lowest performance possibly due to the complexity of the matrix that increases the signal
interference and decreases the ability to resolve the target signal.
FTIR-ATR icro- FTIR-Reflectance Portable FTIR-ATR

spectroscopy icro-spectroscopy Spectrometer
WPI WPC WPH WPI WPC WPH WPI WPC WPH
Sample number 3 36 8 9 7 1
Wavenumber 1 1 1 1 1 37 13 1 1 1 1
range (cm-1) -1798 -1798 -1798 -1699 -18 -18 -17 8 -176 -1698
Factor 6 6 6 6 6 6 6
Cumulative 99.99 99.98 99.9 99.89 99.98 99.7 99.9 99.99 99.9
SECV .16 .1 . .19 .1 . .18 .17 .1
R .8 .9 .93 .77 .91 .9 .7 .8 .93
SEC .16 .1 . .18 .13 .18 .1 .16 .1
R Cal .86 .96 .9 .8 .93 .96 .83 .88 .96
RPD 1.8 .98 .67 1. 7 .39 .91 1. 8 1.91 .8
whey protein beverages with various pH using FTIR-ATR Micro-spectroscopy, FTIR-
Reflectance Micro-spectroscopy, or Portable FTIR-ATR Spectrometer spectra.
Comparing the loading of both FTIR-Reflectance icro-spectroscopy (Figure
3.6) and Portable FTIR-ATR spectrometer (Figure 3.7) with FTIR-ATR icro-
spectroscopy (Figure 3. ), similar bands arising from Factor 1 in every loading plot were
confirmed to be related to amide I (centered at 16 cm-1) and amide II (centered at 1
cm-1) regions, which indicated that when pH was involved as a variable in product
manufacturing, protein secondary structure played a very important role in regulating
astringency generated from whey protein beverages. eanwhile it should not be
74
negligible that the loading plot disclosed important bands including 1333, 1 ,1 9 ,
1 96, and 1 99 associated with functional groups rather than protein secondary structure,
which were meaningful in understanding the regression models. IR bands at 1 99 and
1 9 cm-1 exhibited in WPI and WPC calibration models by FTIR-Reflectance icro-
spectroscopy can be assigned to the characteristic N-H bending vibrations of Asp
( izuguchi et al., 1997). Calibration model based on the WPH spectra by FTIR-
Reflectance icro-spectroscopy also gave an important band at 1333 cm-1, which most
li ely can be attributed to the carboxylate vibrations of Asp groups due to protonation
change (Gerwert et al., 1989). Examining the loadings of WPI, WPC, and WPH beverage
calibration models constructed by Portable FTIR-ATR spectrometer, IR bands at 1 96
cm-1 arising from WPI model, while IR bands at 1 cm-1 existed in all three models.
The absorbance bands at 1 cm-1 was possibly related to the δ(CH ) group frequencies
of aliphatic side chains of amino acid ( arth, ).
75
A. WPI 0.2 B. WPI
3.4
0.1
3.0
-0.0
1329
2.6 1256 1352
-0.1 1541 1599
1454
1649
2.2 -0.2
2.5 3.0 3.5 1275 1468 1661
C. WPC D. WPC
3.8
0.2
IR Predicted Value
3.4 0.1
Loadings
-0.0
3.0
-0.1 1595
1541
2.6 -0.2 1647 1715
2.5 3.0 3.5 1564 1661 1757
E. WPH 0.2 F. WPH

3.5
0.1
3.0
-0.0
2.5
1333 1362
2.0 -0.1 1541

1456
1653
1639
2.2 2.6 3.0 3.4 3.8 1468 1661
(ranging from 2.2 to 3.9) using FTIR-reflectance Micro-spectroscopy.
76
3.5 A. WPI 0.2 B. WPI
0.1
3.0
-0.0
-0.1 1542 1454

2.5 1596
-0.2 1655 1639
2.5 3.0 3.5 1672 1579 1486
C. WPC 0.2 D. WPC

3.8
IR Predicted Value
0.1
Loadings
3.4 -0.0
-0.1 1542 1454

3.0 1491
-0.2 1639
3.0 3.4 3.8 1765 1672 1579 1486
E. WPH F. WPH
0.2
3.4
0.1
-0.0
3.0
1534 1454
-0.1 1620
1486 1430
2.6 1640
2.5 3.0 3.5 1672 1579 1486

(ranging from 2.2 to 3.9) using portable FTIR-ATR diamond equipment.
Interestingly, three models (WPI, WPC and WPH) generated with the spectra
collected for beverage samples with fixed and various pH showed good performance
statistics (Table 3. ) and correlation with astringency sensory scores (Figure 3.8) by a
descriptive sensory analysis. The PLSR models based on FTIT-ATR spectra collected
77
from whey protein-containing beverages (R> .79, and SECV< . 7) gave satisfactory
performance statistics.
A. WPI B. WPI
3.5
0.2
3.0 -0.0
1539
1562
-0.2
2.5 1686
1647
2.5 3.0 3.5 1543 1620 1697 1775

4.0 C. WPC D. WPC
0.2
IR Predicted Value
3.6
Loadings
3.2 -0.0
1539 1593
2.8 1686
-0.2 1431
1647
2.6 3.0 3.4 3.8 1466 1659
4.0
E. WPH F. WPH
0.2
3.5
3.0
-0.0
2.5 1543
1431
2.0 -0.2 1620
1647
2.0 2.5 3.0 3.5 4.0 1466 1543 1620 1697

(3.5) and various pH (ranging from 2.2 to 3.9) using FTIR-ATR germanium Micro-
spectroscopy
78
WPI WPC WPH
Sample Number 39 31
-1
Wavenumber Range (cm ) 1 93-1798 1 -1798 1 -17 8
Factor 6
Cumulative 99.9 99.99 99.67
SECV .18 . . 7
R .8 .79 .88
SEC .17 . 3 .
R Cal .83 .83 .9
RPD 1.77 1.6 .13

whey protein beverages with fixed and various pH using FTIR-ATR Micro-spectroscopy
spectra.
The regions that gave the lowest SECV were chosen for developing cross-
validated PLSR calibration models given in Table 3.3, 3. , and 3. , which were different
for each individual model. The factor selected for each model should be able to represent
enough information to build the calibration model, and at the same time, should not be
too large to include the structural information (irrelevant variables). The factors selected
in all models were ranging from to 6, explaining larger than 99% variables for all of our
calibration models. Ta ing into consideration that the standard deviation among panelist
for evaluating astringency scores ranged from . - . units with a %RSD ranging from
. to .1 (Table 3. ), the predictive ability of our infrared models with SECV < . 7
units showed good reliability, since the %RSD will also be a source of variability for the
regression models. y maximizing the variance from whey protein sources, pH values,
processing techniques, and sensory evaluation, robust calibration models to predict
79
astringency generated from acidic whey protein beverages based on different whey
protein types were constructed by multivariate regression combined with infrared spectra.
During the investigation of loadings, infrared bands responsible in constructing the PLSR
astringency models were also revealed, which could help us to understand the reasons
behind. It pointed out that whey protein itself, and pH were possible reasons to address
astringency from acidic whey protein beverages.
3.4.6 PLSR prediction of a separate set of whey protein beverages
Table 3.6 showed the astringency prediction value of a separate set of beverages
made from specific whey protein powders and ad usted to various pH levels. Astringency
score predictions were done by collecting sample infrared spectra and using the
previously cross-validated WPI, WPC and WPH models by FTIR-reflectance icro-
spectroscopy. The predicted scores showed good precision associated with low standard
deviation calculated from replicates. verall, as the pH level of whey protein beverage
increased (from to 7), the astringency associated with each sample showed a decreasing
tendency.
The impact of modifying the pH on the predicted astringency scores on beverage
samples was higher when formulated with WPC and WPH powders, while the
astringency scores of beverage samples formulated from WPI powders showed a slight
declined in astringency scores as pH levels were raised. The astringency scores of GPH1
showed the smallest changes among all whey protein powders, probably because the
ma or protein in GPH1 was actually gelatin. Total solids measurement indicated similar
solid concentrations among all samples. ur results showed that the pH values had
80
apparent impact on the induced astringency of whey protein beverages, while the pH
ranges were evaluated from acidic conditions to neutral conditions. This result agreed
with previous wor done by eecher and others ( 8), who also emphasized the ma or
effect of pH on astringency from whey protein solutions.
Assigned ID Actua Prediction SD °Brix Assigned ID Actual Prediction SD °Brix

l pH Score pH Score
WPC2_01 6.516 1.90 0.06 10.8 WPC6_0 6.572 1.00 0.17 10.8
WPC2_2 2.066 3.73 0.01 11 WPC6_2 2.079 3.40 0.13 11
WPC2_3 3.108 3.04 0.03 10.6 WPC6_3 3.085 2.78 0.03 10.6
WPC2_4 4.046 2.45 0.17 10.4 WPC6_4 4.102 2.16 0.11 10.2
WPC2_5 4.993 2.19 0.04 10.4 WPC6_5 4.988 1.48 0.04 10.5
WPC2_6 5.907 1.95 0.01 10.8 WPC6_6 6.183 0.96 0.03 10.5
WPC2_7 7.349 1.65 0.00 11.2 WPC6_7 6.911 0.79 0.01 10.4
GPH1_0 5.511 1.72 0.02 10.2 WPH3_0 6.654 2.71 0.15 11

GPH1_2 2.099 2.00 0.04 10.4 WPH3_2 2.08 4.08 0.08 11.4
GPH1_3 3.066 1.77 0.01 10.2 WPH3_3 3.079 3.23 0.02 11
GPH1_4 4.011 1.79 0.03 10.2 WPH3_4 3.962 2.99 0.01 10.6
GPH1_5 4.756 1.73 0.02 10 WPH3_5 5.077 2.89 0.02 10.6
GPH1_6 6.022 1.71 0.02 10 WPH3_6 6.072 2.81 0.17 10.8
GPH1_7 6.959 1.63 0.01 10 WPH3_7 7.008 2.76 0.05 10.8
WPI2_0 3.671 2.57 0.02 10.2

WPI2_2 2.036 2.96 0.01 10.4 WPI1_2 2.086 2.91 0.02 10.4
WPI2_3 2.959 2.72 0.00 10.2 WPI1_3 3.094 2.73 0.01 10.2
WPI2_4 4 2.55 0.02 10.1 WPI1_4 4 2.61 0.00 10.2
WPI2_5 5.276 2.35 0.01 10 WPI1_5 5.051 2.47 0.02 10
WPI2_6 5.992 2.29 0.01 10.2 WPI1_6 5.64 2.41 0.00 10.2
WPI2_7 7 2.18 0.01 10.2 WPI1_72 7.027 2.24 0.01 10
Table 3.6. The predicted values of astringency in formulated whey protein beverages
using IR spectra and calibration models by FTIR-reflectance Micro-spectroscopy.
1
Sample codes including indicate that they are beverages before pH ad ustment and the
actual pH levels are natural pH levels when whey protein beverages were formulated
from powder
The pH level of WPI1 beverage was 7. 7 before any pH ad ustment hence WPI1_7
represents both WPI1_ and WPI1_7
81
3.5 Conclusion
FTIR spectroscopy has allowed for the rapid (~ min analysis time) and accurate
analysis of WPI, WPC, and WPH beverages and contributed to the development of
simple and rapid protocols for predicting astringency and the final quality of the
beverages, which finally could enhance the flavor of whey protein products and
increasing consumer li ing of the whey protein food products.
82
Chapter 4 Understanding Factors Influencing Development of Astringency in Whey
Protein Products
4.1 Abstract
Whey proteins beverages are often formulated to be acidic in order to improve
their clarity and microbial stability but as a result develop an unpleasant and astringent
flavor. ur ob ective was to evaluate the factors regulating astringency in whey protein
products by the statistical and instrumental methods. Whey protein isolate (WPI), whey
protein concentrate (WPC) and whey protein hydrolyzate (WPH) from different
manufacturers were used to formulate beverages at acidic pH ranges. Powders were
pressed onto a three-reflection ATR-IR diamond crystal with a high-pressure clamp for
spectra. everages were measured by FTIR Reflectance icro-Spectroscopy with the aid
of vacuum drying. Trained panelists from Sensory Service Center at North Carolina State
University using descriptive analysis were involved to determine astringency scores.
Whey protein powder samples were acid hydrolyzed to free amino acids and analyzed by
Gas Chromatography method to quantify the concentrations of acidic amino acids.
ultiple Linear Regression ( LR), Partial Least Square Regression (PLSR) and Pearson
Correlation were used to evaluate the factors regulating astringency in whey protein.
LR using pH and protein types as explanatory variables explained high levels of
variance ( . % - 9 . %), illustrating their importance in regulating astringency in whey
protein beverages. Good PLSR models based on whey protein powders were generated
83
for astringency determination (SECV< .3 and correlation coefficient (r> .7 ). a or
characteristic absorption bands detected for astringency, in the range of 1396 to 17
cm1, were also associated with the C -

groups and amide I region. Understanding the
factors affecting astringency of whey proteins beverages manufactured at low pH can
help to develop strategies to improve consumer acceptance of these products.
4.2 Introduction
Astringency is the combination of complex sensations due to the perception of
shrin ing, drawing or puc ering of oral mucosa and muscles around the mouth (Lee and
Lawless, 1991 AST , ). Among foods that are astringent, Tannins (polyphenol
compounds) were found responsible in causing astringency most of the time in wine,
grape seeds, teas, fruits and vegetables. Tannins were able to interact and bind proline-
rich proteins and histidine-rich proteins in salivary proteins, followed by the aggregation
and precipitation of protein-tannin complex, and finally wea en the salivary’s ability
(Luc et al, 199 axter et al., 1997 Dinnella et al., 9).
Whey protein, a complex mixture of different proteins including α-lactalbumin, β-
lactoglobulin, bovine serum albumin, immunoglobulins, lactoferrin, lactoperoxidase,
glycomacropeptides and others (Laetitia . onnaillie and Peggy . Tomasula, 8),
was also recognized as an astringent agent but could not simply share the same
mechanism as tannins. ne comparable suggestion stated that the interactions between
the positively charged whey proteins (pH < pI) and negatively charged saliva proteins
could contribute to the astringency in acidic whey protein solutions ( eecher et al., 8
84
Vardhanabhuti et al., 1 ). Although efforts to explore the reasons behind astringency
triggered by acidic whey protein solutions have been made, the mechanism remains
unclear. ur ob ective was to evaluate the factors regulating astringency in whey protein
products by the statistical and instrumental methods.
Two batches of beverage samples were prepared separately. ne batch included
different types of whey protein powder, including whey protein isolate (WPI, 3 different
samples), whey protein concentrate (WPC, 18) and whey protein hydrolysate (WPH, 1 )
were obtained from a leading beverage industry. After formulated from these powder
samples, they were controlled under uniform pH value (pH=3. ). The other batch
included 9 WPI powder, 6 WPC powder and WPH powder samples from different
manufacturers. After formulated from these powder samples, the beverages from every
powder sample were further ad usted to pH values ranging from . to 3.9. Each sample
was coded with 3 digit number and vacuum pac ed in glass bottle. everage samples
were maintained at ºC until spectra collection using FT-IR.
4.3.2 Astringency prediction
Quantitative sensory test on relevant whey protein beverage samples was
conducted at the Sensory Service Center in North Carolina State University, which is an
internationally recognized program in Dairy products. Sensory analysis was conducted in
compliance with institutional review board human sub ect regulations.
85
4.3.3 Fourier-Transform Infrared Spectroscopy (FT-IR)
Whey protein power samples were measured using an IR spectrometer (Excalibur
FTS 3 GX Varian, Palo Alto, CA) coupled with an attenuated total reflectance (ATR)
accessory. The optical bench included a dynamically aligned ichelson interferometer,
potassium bromide beam splitter, and a deuterated tri-glycine sulfate (DTGS) detector. IR
spectra were observed using commercial software (Win-IR Pro, Version . Varian Inc.)
from to 7 cm-1 with resolution of 8 cm-1, co-adding 6 scans to increase signal to
noise ratio. easurements were performed on a diamond crystal, with refractive index of
o
. and an incidence angle of yielding three internal reflections. The instrument was
calibrated before measurements using the crystal alone as bac ground. The powder
samples were pressed onto the ATR-IR crystal using a high-pressure clamp (PIKE Tech,
adison, WI), and replicate spectra were obtained.
Whey protein beverage samples were measured using FTIR-reflectance micro-
spectroscopy, the methodology was the same as mentioned in Chapter 3.
4.3.4 Statistical analysis
ultiple Linear Regression ( LR) attempts to model the relationship between
two or more explanatory variables (pH and whey protein types) and a response variable
(astringency) by fitting a linear equation to observed data. This line describes how the
mean response changes with the explanatory variables.
The astringency scores of whey protein powder samples were used to test the
ability to predict the sensory attributes based on their IR spectra in multivariate model.
ultivariate regression analysis (Partial least squares regression, PLSR) was performed
86
to build calibration models to simultaneously correlate the reference sensory score data to
the infrared spectral information. Partial Least Squares Regression (PLSR) was cross-
validated (leave-one-out approach) to generate calibration models. PLSR models were
evaluated in terms of loading vectors, standard error of calibration (SEC), standard error
of cross validation (SECV), coefficient of determination (r-value) and outlier diagnostics.
Pearson correlation was used to discover the correlation coefficient between the
concentrations of specific amino acids in whey protein powder and the astringency score
at fitted pH value of corresponding whey protein powder.
4.3.5 Gas Chromatography (GC) analysis
Whey protein powder was firstly acid hydrolyzed to yield free amino acids. mg
of each powder was mixed with 1ml of HCl (6N) in a sealed glass tube and placed in
oven at 11 ºC overnight to fully hydrolyze the protein. The resulting solution was blow-
dried with nitrogen gas and dissolved in 1ml of water (HPLC grade). This water solution
contained free amino acids yielding from whey protein powder and was analyzed by
following GC method with hours.
Amino acids hydrolyzed from whey protein powder samples were analyzed by
EZ:Faast™ amino acid analysis it (Phenomenex, Torrance, CA), which provided rapid
clean-up, derivatization and GC analysis of amino acids from complex mixtures. The
method was based on solid phase extraction (SPE), followed by a rapid derivatization
reaction and GC-Flame ionization detector (FID) analysis. The clean-up step using SPE
cartridges could save time on prior removal of lengthy proteins and interfering
substances, the next steps involved production of chloroformate derivatives of both the
87
amino and carboxylic acid groups, and the final step was GC-FID analysis. An Agilent
689 series (Santa Clara, CA) gas chromatograph equipped with an FID was used for
sample analysis. The chromatographic column with the dimensions 1 m* . mm i.d. of
a proprietary phase was provided with the it. The temperature programme of oven
started at 11 ºC, followed by a linear ramp of 3 ºC/min up to 3 ºC, and a hold time of
1 min. The carrier gas was helium at a rate of 1. ml/min at 1 ºC. The in ection using an
auto sampler was . ul and in ector temperature was ºC, while the detector was ept
at 3 ºC.
The results of LR analysis used powder type and pH values as explanatory
variables to predict the corresponding astringency score was shown in table .1. While
simple linear regression using pH of whey protein beverages, as single explanatory
variable cannot sufficiently explain variance of beverage astringency (R ranging from
1 . % to 6. %), the incorporation of both variables by LR largely enhanced the
variance explained (R ranging from 71.8% to 9 . %). As indicated in table .1, the p
values associated with pH variable were all smaller than . . The statistical analysis
indicated that both pH and protein types played important roles in the development of
whey protein astringency. While protein types were under consideration, it was possibly
implying that astringency generated from acidic whey protein beverages could be
attributed to the protein secondary structure, amino acids compositions and all other
factors related to the whey protein types. oth figures in Figure .1 showed the normal
88
distribution of data set (WPI was used as an example), examining it was appropriate to
use LR to analyze the data.
(%)
Protein Types R P(regression) P (pH)
WPI 9 7 .3 . . 3
WPC 6 71.8 . 6 . 3
WPH 9 . . .
Table 4.1. Multiple Linear Regression using pH and whey protein types as predictor and
astringency scores as response for WPI, WPC, and WPH.
Figure 4.1. The examination of validity using MLR to analyze WPI samples. Residuals vs
Fits for Astringency (A) and Normal plot of Residuals for Astringency (B).
89
The relationship between pH values and astringency in acidic whey protein
beverages was further proved by PLSR analysis based on the IR spectra. As shown in
Figure . , strong correlation between pH values and IR spectra of acidic whey protein
beverages with various pH levels was observed in WPI, WPC and WPH models, which
could be confirmed by high R (> .97) and low standard errors (< .11) shown in Table
. . The wavenumber ranges used in all three models were from 1 to 18 cm-1, which
basically covered the ma or bands involved in protein structure. The most outstanding
bands in all loadings plots were amide I (~1 cm-1) and amide II (~16 cm-1) bands,
which indicated that pH was closely related to the alteration of protein’s secondary
structure, hence implied the relationship between pH and astringency.
PLSR model
WPI WPC WPH
Wavenumber range (cm-1) 1 1 -1796 1 -18 1 -18
Factor 6
Cumulative1 99.83 99.8 99.76
SECV .11 . 8 .1
R3 .97 .99 .98
SEC .1 . 8 . 9
Table 4.2. Calibration and cross-validation results of PLSR models developed by pH of
whey protein beverages using FTIR-Reflectance Micro-spectroscopy spectra.
1
Cumulative: Cumulative variances explained by the calibration model.
SECV: Standard error of cross-validated calibration model.
3
R: Coefficient of cross-validated calibration model.
SEC: Standard error of calibration model.
90
3.8 A. WPI B. WPI
0.2
3.4 0.1
3.0 -0.0
1454 1522
-0.1 1541 1578
2.6
1684
1649
-0.2
2.2
2.2 2.6 3.0 3.4 3.8 1468 1661
4.0
C. WPC D. WPC
0.2
3.6 0.1
IR Predicted pH
Loadings
3.2 -0.0
-0.1 1454
2.8 1541
1520
1647
-0.2 1626
2.5 3.0 3.5 1468 1661
4.0 E. WPH 0.2 F. WPH
0.1
3.5
-0.0
3.0
1499
-0.1 1539
1456 1518
2.5 1603 1657
-0.2 1638
2.6 3.0 3.4 3.8 1468 1661
Measured pH Wavenumber (cm-1)
Figure 4.2. PLSR plots and loadings based on the infrared spectra of whey protein WPI,
WPC and WPH beverage samples and measured pH using FTIR-Reflectance Micro-
spectroscopy.
To explore the effect of whey protein type on the development of astringency, a
regression analysis of each individual whey protein powder samples, correlating their
infrared spectra using FTIR-ATR with sensory astringency scores of corresponding
beverages at fitted pH (3. ) was done for WPI, WPC, and WPH separately. y
91
controlling one factor (pH) at a certain level, it might be possible to unmas other
mechanisms behind astringency. The regression plots and loadings were showing in
Figure .3, and the statistics performance of these data were also generated and showed
in Table .3.
Figure 4.3. PLSR plots and loadings based on the infrared spectra of whey protein
powder samples (WPI, WPC, and WPH).
92
PLSR model
WPI WPC WPH
Factor 6 7 8
Cumulative 99.3 93. 99.
SECV .19 .3 . 8
R .87 .7 .99
SEC .17 . .
Table 4.3. Calibration and cross-validation statistic performance of PLSR models
developed by using whey protein powder samples.
The regions that gave the lowest SECV were chosen for developing cross-
validated PLSR calibration models illustrated in Figure .3 and Table .3, which included
the frequency ranges of 18 -9 cm-1 and provided the best predictive ability. SECV is
an estimate of the standard error of prediction (magnitude of error expected when
independent samples are predicted using the model) and the model producing the
minimum SECV is selected as the best model for the spectral data set. Within this
experiment, WPH had the lowest SECV value, whereas WPC showed the highest error
for prediction.
The cumulative variance gives the proportion of variability of the property that is
described by the model. ost of the variance were explained in calibration models
(>93. %), while reasonable factor numbers were associated (<8). The numbers of factors
should not be too high, in that way too much structural noise will be incorporated in
building the model, and generated unreliable data.
Among all of the whey protein output, WPH generated the best calibration
models, while WPC did not show results as good as others, it had relatively low
cumulative variance (93. ), high SECV ( .3 ), and also a low R value ( .7 ). The reason
93
why this happened was probably that WPC may contain various protein levels, with all
other component such as high amount of lactose not removed. The complexity of the
matrix were possibly bringing signal interference and decreasing the ability to resolve the
target signal. Nevertheless, the ability of predicting astringency by direct analysis of the
whey protein powders provided unique opportunity for rapid assessment of the quality of
the incoming material as related to their impact on astringency.
Figure .3 also showed the PLSR loadings for WPI, WPC and WPH powder
samples. The most important bands explaining the astringency scores were in the 1396,
1 66, 1 97 and 16 -17 cm-1 region. 1396 cm-1 was associated the stretching of C=
in the C - groups (Coates, ), 1 97 cm-1 was assigned to the vibrations of C -
+ -
bound with Ca and the C antisymmetric stretching vibrations of the glutamyl side-
chain give rise to a band at 1 67 cm-1 ( izuguchi et al., 1997). The broad bands between
16 -17 cm-1 were associated with amide I region (Kong and Yu, 7). These signals
were related to modifications occurring in the secondary structure of the native protein
possibly attributed to calcium binding with acidic amino acids in the β-sheet
conformation.
Since carbonyl groups in amino acids side chains were suggested to be important
in the construction of correlation between whey protein powder’s IR spectra and their
corresponding astringency, a GC analysis to quantify the amino acids with carbonyl side
chains in whey protein powders and Pearson correlation was used to exam the connection
between the amino acids concentration with carbonyl side chains in whey protein
powders and the corresponding astringency of beverages at pH 3. . The results of
94
Pearson correlation were shown in Table . .
Pearson correlation P
GLU .8 . 3
ASP .6 8 . 9
ASP+GLU .78 . 13
Table 4.4. Pearson correlation between concentrations of glutamic acid (GLU) and
aspartic acid (ASP) in whey protein powders and the corresponding astringency of
beverages at pH 3.5.
In GC analysis, the selected whey protein powders, soy protein powder, and
gelatin powder were first acid hydrolyzed to yield free amino acids. Soy protein and
gelatin were added to improve the sampling in Pearson correlation, because soy protein
was nown to be very astringent and gelatin was recognized as low astringent. After
digestion, both glutamine and glutamic acid in protein were determined together because
glutamine was deaminated to form glutamic acid similarly asparagine and aspartic acid
were determined together. The resulting concentrations of GLU and ASP were analyzed
by Pearson correlation. The highest correlation coefficient was generated from GLU
concentration and astringency ( .8 ), and the lowest one was from ASP and astringency
( .6 8). However all three coefficients suggested the possibility of lin age between
amino acids with carbonyl side chains in whey protein powders and their corresponding
astringency. The GC analysis and Pearson correlation supported that whey protein itself
was responsible for the astringency and amino acids with carbonyl side chains were
possibly contributing to the development of astringency.

95
4.5 Conclusion
verall, both pH and protein types played important roles in the development of
whey protein astringency. FT-IR was able to monitor protein properties as related to
astringency development, acidic amino acids (Glu and Asp) and the β–sheet protein
conformation were pointed out as important explanation of astringency prediction.
Pearson correlation and PLSR analysis illustrated that carbonyl groups and secondary
conformation of denatured whey proteins were associated with the astringency, which
was match the results from our LR analysis.
4.6 Significance and future work
y the combination of infrared spectroscopy with pattern recognition technique,
we found the unique bands from commercial whey protein powder to authenticate whey
protein, not only based on the protein levels but also the possible processing techniques.
The authentication of raw protein ingredient can ensure desired functionalities in various
food applications and finally benefit food industry with a better quality.
With the comprehensive information obtained from food samples infrared spectra,
the PLSR analysis showed relationship between the reference value and the predicted
value, indicating the high possibility to predict the perceived astringency induced by
whey protein compounds in acidic beverages based on the detection of specific functional
groups.
ased on 6 whey protein beverage samples with fixed pH, our results showed
that the protein types had obvious impact on the perceived astringency. Then statistical
96
analysis ( LR and Pearson correlation) further indicated that both pH and protein types
were important explanatory variables to predict astringency in acidic whey protein
beverages (variance explained was larger than 71.8%), which matched the results of
important bands resolved from PLSR models of beverage samples with various pH.
However introducing other food ingredients such as citric acid into acidic whey
protein beverages might interfere with the bands associated to astringency. And the
complex matrix of whey protein, including minerals, lactose, lactic acid, and others might
also influence the signal perceived as infrared spectra. Future wor can be done to
explore the mechanism of astringency triggered by whey protein. The change of protein’s
secondary conformation and the location of amino acids with carbonyl side chains in
whey protein can be possible factors related to astringency. We can also evaluate the
effect of mas ing agents to decrease the perceived astringency in acidic whey protein
beverages. Possible mas ing agents can be tested are hydrocolloid, sugar, salt and the
combination of them.
97
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Dissertation For Graduate School - Ting Wang 2nd Edition

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The Authentication of Whey Protein Powder Ingredients and Understanding

Factors Regulating Astringency in Acidic Whey Protein Beverages to Estimate

Graduate Program in Food Science and Technology

The Ohio State University

Dr. Luis E. Rodriguez-Saona, Advisor

Dr. Monica Giusti

Dr. Michael E Mangino

Dr. Lynn Knipe

functionalities such as water binding, gelation, emulsification, and foaming. The

become to an essential ingredient in food industry. The advanced techniques involving

transformed from waste to a value-added ingredient, it still confronts problems: The

formulated under acidic pH.

The overall objectives of this dissertation were to provide an accurate and

convenient instrumental method, Infrared Spectroscopy (IR), to authenticate the whey

application of whey protein as a key ingredient in food products.

reveal the factors modulating whey protein astringency.

significant factors in the development of astringency in whey protein products.

In conclusion, with the comprehensive information obtained from food samples

be extremely attractive to both scientific research and food industry practice.

my life. He taught me how to become a confident people; he guided me how to choose

the career; he supported me to gain experience from food industry; He encouraged me to

best decision I have ever made to choose him as my advisor.

I also would like to thank my dissertation committee members, Dr. Monica

September 14, 1982 ....................................... Yang Quan, China

2001 – 2005 ................................................... B.S. Bioengineering, South China

2008 - 2011 .................................................. M.S. Food Science and Technology, The

Ohio State University

2011 - present ............................................... Ph.D. Food Science and Technology, The

Ohio State University

Major Field: Food Science and Technology

List of Figures ................................................................................................................. xiii

Chapter 1: Literature Review ............................................................................................ 1

1.1 A Review of whey protein processing and functionality .......................................... 1

1.1.1 Whey and whey protein fractions ...................................................................... 1

1.1.2 Whey protein processing and production ........................................................... 5

1.1.3 Whey protein functionality .............................................................................. 14

1.1.4 Whey protein application ................................................................................. 21

1.1.5 Whey protein analytical methods ..................................................................... 22

1.2 Astringency and mechanisms in various food ........................................................ 26

1.2.1 Terminology of astringency ............................................................................. 26

1.2.2 Mechanisms of astringency in four main groups of astringent compounds ..... 27

1.2.3 Exploration of mechanisms of astringency caused by whey protein ............... 33

Chapter 2: Authentication of Whey Protein Powders by Combining Infrared

Spectroscopy and Pattern Recognition Analysis .............................................................. 40

2.2 Introduction ............................................................................................................ 41

2.3 Materials and methods ............................................................................................ 43

2.3.1 Whey protein sample ....................................................................................... 43

2.3.2 Fourier-Transform Infrared Spectroscopy (FT-IR) .......................................... 43

2.3.3 Multivariate classification analysis .................................................................. 44

2.4 Results and discussion ............................................................................................ 46

2.5 Conclusion .............................................................................................................. 55

Chapter 3: A Novel Application of FT-IR Technique Combined with Chemometrics in

3.2 Introduction ............................................................................................................ 57

3.3 Materials and methods ............................................................................................ 59

3.3.1 Whey protein sample ....................................................................................... 59

3.3.2 Astringency prediction ..................................................................................... 60

3.3.4 Portable FTIR Spectroscopy ............................................................................ 61

3.3.5 Multivariate regression analysis ....................................................................... 61

3.4 Results and discussion ............................................................................................ 63

3.4.1 Perceived astringency scores for whey protein beverages ............................... 63

3.4.3 SIMCA classification of whey protein beverages with fixed pH ..................... 67

3.4.6 PLSR prediction of a separate set of whey protein beverages ......................... 80

3.5 Conclusion .............................................................................................................. 82