Trends in Food Science & Technology 67 (2017) 207e219

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Trends in Food Science & Technology

journal homepage: http://www.journals.elsevier.com/trends-in-food-science-
and-technology

Review

Principles and applications of spectroscopic techniques for evaluating

food protein conformational changes: A review
Kaiqiang Wang a, b, c, Da-Wen Sun a, b, c, d, *, Hongbin Pu a, b, c, Qingyi Wei a, b, c
a
School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China
b
Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou 510006, China
c
Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process Control of Cold Chain Foods, Guangzhou Higher
Education Mega Center, Guangzhou 510006, China
d
Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of
Ireland, Beleld, Dublin 4, Ireland

a r t i c l e i n f o a b s t r a c t

Article history: Background: Proteins are essential nutrients required in various body functions and normal human life.
Received 16 February 2017 However, in the food industry, the application of proteins especially those of plant origin have been
Received in revised form limited due to their poor functionality. Although nowadays, diverse modication techniques are usually
12 June 2017
employed to improve their performance in food products, it is also important that effective methods for
Accepted 13 June 2017
monitoring the resultant conformational changes induced during protein modication are developed.
Available online 1 July 2017
Scope and approach: In this review, the relationship between protein conformation and functionality is
briey discussed. Thereafter, the underlying principles behind ve selected spectroscopic techniques i.e.
Keywords:
Protein conformation
Fourier transform infrared, Raman, circular dichroism, uorescence, and ultraviolet spectroscopies are
Fourier transform infrared spectroscopy introduced and their recent applications for monitoring conformational changes that occur during
Raman spectroscopy physical, chemical or enzymatic modication of proteins are addressed. In addition, the advantages and
Circular dichroism spectroscopy limitations of each spectroscopic technique are comparatively discussed and perspectives on the current
Fluorescence spectroscopy situation alongside future trends are highlighted.
Ultraviolet spectroscopy Key ndings and conclusions: Spectroscopic techniques present an attractive panacea for evaluation of
conformational changes during protein modication. Although certain challenges especially with com-
plex food materials require urgent attention thus, more robust spectroscopic solutions should be
exploited in the future.
2017 Elsevier Ltd. All rights reserved.

1. Introduction formulation (Buckow, Sikes, & Tume, 2013; Gharsallaoui, Saurel,

Proteins are considered principal components for body meta-
bolism and general human life. In recent years, there has been an
increasing interest in utilizing proteins from various sources as
functional ingredients in food products, primarily due to their high
nutritional value and unique functionality. The functional proper-
ties of protein in food including water- and fat-binding capacities,
gel forming and rheological behaviors, emulsifying capabilities,
foaming capabilities etc., providing desirable sensory characteris-
tics such as structure, texture, avor, and color during food product
* Corresponding author. School of Food Science and Engineering, South China
University of Technology, Guangzhou 510641, China.
E-mail address: dawen.sun@ucd.ie (D.-W. Sun).
URL: http://www.ucd.ie/refrig, http://www.ucd.ie/sun
Chambin, & Voilley, 2012; Hu et al., 2015b; Lam & Nickerson,
2013; Steen et al., 2016; Whitford, 2013). To cite an example, the
emulsifying properties of proteins permit the formation of emul-
sions owing to their amphiphilic nature and lm-forming capacity,
therefore protable in various food products, drugs and nutrient
delivery tactics (Lam & Nickerson, 2013). However, some native
proteins, especially those of plant origin exhibit poor functionalities
during food manufacturing. For instance, the poor digestibility of
red kidney bean protein isolates (Yin, Tang, Wen, Yang, & Li, 2008),
low solubility of soy protein isolates (Hu et al., 2013), poor gelation
property of rapeseed protein isolates (He, He, Chao, Ju, & Aluko,
2014), as well as poor solubility, foaming and emulsifying proper-
ties of wheat gluten proteins (Agyare, Addo, & Xiong, 2009). As a
result, certain modication treatments including physical (Zhang,
Yang, Zhou, Zhang, & Wang, 2017), chemical (Shilpashree, Arora,

http://dx.doi.org/10.1016/j.tifs.2017.06.015
0924-2244/ 2017 Elsevier Ltd. All rights reserved.
208 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219
Chawla, & Tomar, 2015), and enzymatic methods (Wang et al.,
2016b, 2016a) are required in order to obtain optimal nutritive
value such as high bioactive activities (Perreault, He
naux, Bazinet,
& Doyen, 2017), high digestibility (Yin et al., 2008) and low aller-
genicity (Li, Zhu, Zhou, & Peng, 2012; Tong et al., 2012). Such
treatments also ameliorate the functional properties of proteins
including gelling strength (He et al., 2014), emulsication (Raikos,
2010) and their ability to form foams (Morales, Martnez, Ruiz-
Henestrosa, & Pilosof, 2015).
Furthermore, it is well known that the modication of food
protein functionality is accompanied with conformational changes,
which inuence the quality of the end-product. For example,
Abaee, Madadlou, and Saboury (2017) found that the hardness of
the non-heat-treated whey protein cold-set hydrogels prepared at
pH 9.0 was signicantly higher than that of the samples obtained at
pH 8.0 and pH 7.0. Their results suggested that base-induced
denaturation and unfolding of b-lactoglobulin at pH 9.0 caused
the formation of more disulde bonds and hydrophobic in-
teractions, accounting for the increased a-helical structures. Li,
Kang, Zhao, Xu, and Zhou (2014) illustrated that high-intensity
ultrasound could induce conformational changes including a
decrease in the a-helical contents and increase in b-sheet, b-turns,
and unordered contents, contributing to protein aggregation and
gel formation in meat, leading to enhanced gel texture. On the other
hand, Rahaman, Vasiljevic, and Ramchandran (2016) reported that
different processing approaches could affect the conformational
changes related to digestibility and allergenicity of food proteins in
peculiar ways. Therefore, it is essential to develop effective detec-
tion methods for evaluating conformational changes in protein and
understanding the relationship between its structural and func-
tional characteristics during processing. Several chemical methods
for indirect reection of protein denaturation in meat are available
based on detection of protein solubility, free sulfhydryl content,
surface hydrophobicity, or myobrillar ATPase activity (Chen, Diao,
Li, Chen, & Kong, 2016). However, most of these above-mentioned
methods are time-consuming, environmentally unfriendly and do
not obtain conformational changes related to specic secondary
and tertiary structures.
In the past decades, nondestructive techniques such as com-
puter vision (Du & Sun, 2005; Jackman, Sun, & Allen, 2009, 2011;
Sun & Brosnan, 2003), spectroscopy, hyperspectral imaging
(Barbin, ElMasry, Sun, & Allen, 2013; Cheng & Sun, 2015; Cheng,
Sun, Pu, & Zhu, 2015; Cheng et al., 2016; Elmasry, Barbin, Sun, &
Allen, 2012; ElMasry, Sun, & Allen, 2013; Feng and Sun, 2013;
Feng et al., 2013; Kamruzzaman, ElMasry, Sun, & Allen, 2013; Liu,
Sun, & Zeng, 2014; Ma, Sun, & Pu, 2016; Pu, Kamruzzaman, &
Sun, 2015; Wu & Sun, 2013; Xiong et al., 2015), etc, have been
developed for rapid evaluation of food quality and properties, in
particular, spectroscopic techniques in the last few years have
signicantly improved, however, they are developed and used for
protein structure analysis for decades. These techniques include X-
ray diffraction (XRD) (Jenkins et al., 2013), nuclear magnetic reso-
nance (NMR) (Mao, Tejero, Baker, & Montelione, 2014), Fourier
transform infrared (FTIR) (Zhang, Waghmare, Chen, Xu, & Mitra,
2015), Raman (Li et al., 2014), circular dichroism (CD)
(Chandrapala, Zisu, Kentish, & Ashokkumar, 2012), uorescence
(Rufn, Schmit, Latte, Dollat, & Chambin, 2014) and ultraviolet
(UV) (Barrios-Peralta, Pe
rez-Won, Tabilo-Munizaga, & Briones-
Labarca, 2012) spectroscopies. These spectroscopic techniques are
particularly suitable for probing structural conversion such as
folding and unfolding. In particular, XRD and NMR spectroscopies
are used to obtain structural information of proteins in high reso-
lution. In fact, XRD is regarded as one of the best methods for
protein structure analysis and even minimal conformational
changes are detectable if only crystallization in two alternative
conformations is possible. However, XRD does not allow for real
time conformational transitions analysis. While NMR spectroscopy
is only able to detect low molecular weight proteins and has its
limitations for applying to proteins larger than a few hundred
residues (Demchenko, 2013).
Nonetheless, other spectroscopic techniques including FTIR,
Raman, CD, uorescence and UV are simple, rapid, convenient, and
have gained increase popularity for monitoring conformational
changes during protein modication. To the best of knowledge, a
review, which specically addresses their applications in this area,
is currently unavailable. Thus, the current review presents the
recent advances pertaining to their application in detection of
conformational changes in proteins alongside their advantages and
limitations. The underlying principles of aforementioned spectro-
scopic techniques are also summarized. In addition, certain areas
that could be further exploited and future research trends are
proposed.
2. Relationship between conformational and functional
properties of food protein
The conformational and functional properties of food protein
are closely related to various inherent (e.g. amino acids composi-
tion) and external (e.g., pH, temperature and ionic strength) factors.
The chemical nature of the amino acids side-chain groups decides
the shape and overall hydrophobicity of proteins. Generally, pro-
teins tend to assume an elongated rodlike shape when they contain
a large number of hydrophilic amino acids residues distributed
uniformly in its sequence; in contrast, they tend to assume a
globular shape when they contain a large number of hydrophobic
residues. In the native form of proteins, the hydrophobic segments
are mostly buried inside the core. The surface hydrophobicity and
hydrophilicity characteristics of protein surface can mostly affect
their solubility characteristics, which govern several functionalities
such as thickening, foaming, emulsication, and gelation properties
(Hettiarachchy, Sato, Marshall, & Kannan, 2012). The pH value af-
fects protein solubility in aqueous solutions. At the isoelectric pH,
the hydrophobic interaction between proteins reaches maximum,
inhibiting unfolding of the protein molecules and resulting in the
minimum solubility. When proteins are exposed to moderately pH
values above or below the isoelectric point, the electrostatic
repulsion and ionic hydration promote the solubilization of protein
and some functional properties are improved, which might be
related to unfolding of the protein and/or activation of buried sulf-
hydryl groups. The ionic strength of a solution also determines the
overall charge of the protein molecule. Ionic strength affects pro-
tein solubility (salting-in or salting-out) dependent on the
hydrophilicity-hydrophobicity characteristics of the protein sur-
face. Moreover, temperature induced protein denaturation could
also alter the surface hydrophobicity of proteins (Damodaran,
1997).
Functionalities of denatured food proteins are differ from their
native states. Therefore, physical, chemical or enzymatic modi-
cations often used to alter the conformation and functionalities of
food proteins. The exposure of hydrophobic regions, for example,
can lower the solubility, affect the surface activity as well as alter
the water and oil holding abilities of proteins (Shen & Tang, 2012).
The unfolding of protein structure can expose more amino acids
residues, facilitating the proteolysis and improve the digestibility of
proteins (Perreault et al., 2017). In addition, changes in the
conformation can inhibit the activity of anti-nutritional factors or
protein toxins and lower the allergenicity (Liu, Zhao, Sun, & Ren,
2013; Rahaman et al., 2016). More details about the relationship
between protein conformation and functionalities can be found
elsewhere (Hettiarachchy et al., 2012; Whitford, 2013). Apart from
 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219
the modication conditions, changes in the structural and func-
tional properties of food protein also depend on the state of protein
(e.g., brous or globular, wet or dry, in liquid or frozen state, and
presence of foreign substances or not) (Kuan, Bhat, Patras, & Karim,
2013). For instance, globular proteins that possess complex three-
dimensional shape depart considerably from brous proteins,
showing elongated structures that lack true tertiary structure. Due
to the cavities in the folded state of globular proteins, they tend to
be more susceptible to hydrostatic pressure than brous proteins
(Ustunol, 2014). The conformational changes of proteins can be
detected by spectroscopic techniques, and spectral characteristics
related to protein conformational properties are introduced in de-
tails in the current review.
3. Principles of spectroscopic techniques
3.1. Fourier transform infrared (FTIR) spectroscopy
Infrared (IR) spectrum in the range of 400e4000 cm
1 arises
from the absorption of energy by chemical bonds, primarily
stretching and bending motions, and has been recognized as a
powerful technique for the structural and chemical characteriza-
tion of proteins (Carbonaro & Nucara, 2010). Generally speaking,
the amide I band (1700e1600 cm
1) of an IR spectrum is primarily
attributed to CO stretching vibrations (approximately 80%) with
some in-plane N-H bending and C-H stretching modes. In partic-
ular, the CO stretching vibrations in proteins mainly depend on
their various secondary structures and inter- or intramolecular ef-
fects, including molecular geometry and hydrogen bonding pattern,
which makes the amide I band being the most sensitive IR spectral
region to predict the secondary structural components of proteins
(Kong & Yu, 2007; Zhao et al., 2013).
When the FTIR absorption of protein is measured in solutions,
the strong IR absorbance of H2O centered at 1640 cm
1 from O-H-O
bending mode may interfere the determination. In order to elimi-
nate the interference, the IR absorption spectrum of protein can be
acquired in solid state, e.g., for FTIR experiment, a protein sample of
2 mg is generally mixed with 198 mg of transparent media (usually
KBr) to form pellet (1e2 mm thick) for measurement. Besides, the
diffuse reectance and attenuated total reection (ATR) FTIR
spectrum have also been developed for evaluating food protein
secondary structure.
The observed amide I band is a complex of several overlapping
components that corresponding to specic secondary structures,
including a-helices, b-sheets, b-turns, and random coils. However,
these broad underlying components bands are instrumentally un-
resolvable (Carbonaro & Nucara, 2010). In order to enhance the
resolution of individual underlying component and quantitatively
estimate the relative contributions of various secondary structures,
Fourier self-deconvolution (FSD) tting and second derivative
analysis should be used to achieve maximum band narrowing,
degrade the signal-to-noise ratio and identify different types of
secondary structures present in proteins. For instance, Fig. 1 pre-
sents the handling process of IR spectra in amide I region of pulsed
electric eld (PEF) treated egg white protein (EWP) (Qian, Ma,
Wang, & Jiang, 2016). The peak location of the components does
not shift, whereas their areas change signicantly. The calculation
of the portion of the components indicates that a reduction of a-
helices is accompanied by an increase of b-sheets during PEF
treatment.
3.2. Raman spectroscopy
Raman spectrum has been proven to provide effective infor-
mation about protein secondary structures and the
209
microenvironment of protein side chains, which can be used as a
valid tool for evaluating protein denaturation. The spectrum can be
processed identically to the infrared spectroscopy data. In common
with IR spectra, among several distinct vibrational modes of the
-CO-NH- amide, the most available bands of Raman spectra for
determining protein secondary structures are the amide I and III
regions. The frequency position of these bands depends strongly on
the protein state, the environment and the intermolecular in-
teractions (Blanpain-Avet et al., 2012).
Fig. 2 shows the Raman spectra of b-conglycinin by high in-
tensity ultrasound (20 kHz at 400 W) treatment for 0, 5, 20 and
40 min (Hu, Cheung, Pan, & Li-Chan, 2015a). Apart from the amide I
and III regions, the major vibrational motions of the side chains,
including inter-chain disulde bands, tryptophan (Trp) bands,
tyrosine (Tyr) bands, and aliphatic hydrophobic residues in Raman
spectra can also be analyzed to provide some information about
protein tertiary structure. The Raman bands located at 760, 880,
1361 cm
1 are ascribed to Trp residues. A sharp line at 1361 cm
1 is
suggested as an indicator of buried Trp residues (Ferrer, Go
mez,
An~o

n, & Puppo, 2011). However, the Trp bands at 760 and
880 cm
1 have been proposed as an indicator of the strength of H-
bonding and hydrophobicity of the indole ring. An increase in the
intensity in these bands indicates that Trp residues are buried, and
in contrast a decrease is connected with the opposite phenomenon
(Go
mez, Ferrer, An~o

n, & Puppo, 2013). The Raman Tyr residues
vibrations are located at about 850 cm
1 and 830 cm
1, and the
ratio of Tyr doublet (I850/I830) is sensitive to the nature of hydrogen
bonding and ionization of phenolic hydroxyl groups. Tyr doublet is
considered as a good indicator for evaluating the degree of Tyr
residues exposed or buried (Go
mez et al., 2013).
In Raman spectra, the stretching vibration located in the range
of 500e550 cm
1 derive from disulde bridges that are formed by
two cysteines, and the peaks around 510, 525, and 545 cm
1 can be
assigned to disulde bonds in gauche-gauche-gauche (g-g-g),
gauche-gauche-trans (g-g-t), and trans-gauche-trans (t-g-t) con-
formations, respectively (Li, 2012). Disulde bonds belong to the
secondary bonds that could maintain the tertiary structure of
proteins, thereby changes in disulde bonds correlate with the
alteration of protein tertiary structure. In addition, the vibration of
aliphatic amino acid residues in Raman spectra near 2800-
3000 cm
1 (C-H stretching) and 1440-1465 cm
1 (C-H bending)
have also been investigated for monitoring protein conformational
changes. Although the indicators of changes in C-H bending vi-
bration are debatable, these changes can also provide information
on proteins hydrophobic interactions and conformational changes
due to processing (Sheng, Wang, Huang, Xu, & Ma, 2016).
3.3. Circular dichroism (CD) spectroscopy
CD spectroscopy is another well-established spectroscopic
technique for determining secondary structures, folding and
binding properties of proteins. In fact, when the plane polarized
light passes through a modulator that subjects to an alternating
50 kHz electric eld, it will be split into the rotating left-handed
(counter-clockwise) and the right-handed (clockwise) circularly
polarized components (Kelly, Jess, & Price, 2005). If the two circu-
larly polarized components have the same amplitude, the recom-
bination of the components can regenerate radiation polarized in
the original plane. Conversely, the resulting recombined compo-
nent radiation is then elliptically polarized. The principle of CD
spectroscopy is based on the unequal absorption of the two circu-
larly polarized components (DA AL-AR). In order to observe CD
signals, the sample should be optically active. As all amino acids
except glycine are asymmetric and hence optically active, protein
structures have been widely studied by CD spectroscopy.
210 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219

Fig. 1. Handling process of effect of pulsed electric eld on IR spectra in amide I region of egg white protein powder; (a)e(f) represent peak-tting of the secondary derived curves
from IR spectra for samples treated at 0, 5, 10, 15, 20, and 25 kV, respectively. Six peaks are observed for four components, which are attributed to a-helices (1657 cm
1), b-sheets
(1611 and 1626 cm
1), b-turns (1673 and 1688 cm
1) and random coils (1642 cm
1) (Qian et al., 2016).
 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219
Fig. 2. Raman spectra of freeze-dried high intensity ultrasound (20 kHz at 400 W)
treated b-conglycinin at 0, 5, 20 and 40 min (Hu et al., 2015a). 500e550 cm
1: S-S
stretching vibrational bands; 760, 880 and 1361 cm
1: Trp vibrational bands; 830 and
850 cm
1: Tyr vibrational bands; 1200e1340 cm
1: amide III bands; 1600e1700 cm
1:
amide I band; 1440e1465 cm
1: C-H bending; 2800e3000 cm
1: C-H stretching.
Normally, CD spectra are collected in high-transparent rectan-
gular or cylindrical quartz cells. Buffers for dissolving protein
samples should be transparent and not contain any materials that
are optically active. In addition, protein solutions for CD measure-
ment should be at least 95% pure with concentrations between
0.005 and 5 mg/mL depending on the pathlength of the cell. When
CD spectrum is acquired, software such as CDPro, CONTIN, SEL-
CON3, DICROPROT, and CDSSTR and computational methods such
as singular value decomposition, optimization algorithms, regres-
sion, and neural networks are used for analyzing the CD spectrum.
A detailed introduction of the acquirement and analysis of the CD
spectrum can be found in a previous review (Martin & Schilstra,
2008).
The CD spectrum of a protein in the far UV region is dominated
by a weak but broad n/p* transition at about 220 nm and an
Fig. 3. (a) Far-UV CD spectra and (b) near-UV CD spectra of myoglobin in solution at pH 7, absorbed at tricaprin oil/water interface and at hexadecane oil/water interface (Day et al.,
2014).
211
intense p/p* transition centered around 200 nm of amide groups,
and are inuenced by the geometries of the polypeptide backbones,
making it one of the most commonly used techniques to determine
the protein secondary structure content (Whitmore & Wallace,
2008). Generally, a-helices show a strong positive band at
191e193 nm and a typical double negative bands at 208e210 and
222 nm, b-sheets produce an intense positive band at about
195e200 nm and a negative band at about 216e218 nm, whereas
random coils have a strong negative bands at 195e200 nm and a
much weaker band (either positive or negative) between 215 and
230 nm. On the other hand, the CD spectrum in near UV region
(250e320 nm) can provide useful information related to aromatic
chromophores (Phe, Tyr, and Trp residues) of proteins in asym-
metric environment, which has been widely used to assess the
tertiary and occasionally quaternary structures of proteins during
processing. Generally speaking, Phe residues have sharp ne
structure between 255 and 270 nm with peaks observed near 262
and 268 nm, whereas the bands arising from Tyr and Trp residues
are located at 275e282 nm and 290e305 nm in near-UV CD
spectrum, respectively (Kelly et al. 2005; Martin & Schilstra, 2008).
An increase in the band magnitudes and intensities is an indication
of structural changes, which are related to the loss of native-like
structure and increasing interactions of the aromatic amino acid
residues during processing (He et al., 2014). Fig. 3 shows the CD
spectra of myoglobin in solution, absorbed at tricaprin oil/water
interface and at hexadecane oil/water interface (Day et al., 2014). As
shown in Fig. 3a, myoglobin is a high helical protein with strong
positive ellipticity at 193 nm and two distinctive negative minima
at 208 and 222 nm respectively. Upon adsorption to oil/water in-
terfaces, the reduction in the intensity of peaks at 193, 208 and
222 nm suggests the loss of helical structure. Fig. 3b shows the
near-UV CD spectra, providing the evidence of the change in the Trp
residues environment.
In recent years, with the improvements in instrumentation for
conventional CD and advent of synchrotron radiation circular di-
chroism (SRCD), lower wavelength bands (as low as 140 nm) and
more information about proteins conformation of the spectra are
obtainable. SRCD spectroscopy can provide important static and
dynamic structural information on proteins in solution. Further-
more, the high ux of a synchrotron source increases the signal-to-
noise levels of the CD spectrum, allowing measurement with turbid
samples (e.g., in the presence of lipids, salts, and detergents)
(Wallace & Janes, 2010).
212 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219

3.4. Fluorescence spectroscopy providing additional possibility for studying proteins conforma-
tional changes (Hawe, Sutter, & Jiskoot, 2008). However, it should
Fluorescence is the emission of photons due to the absorption of be noted that the extrinsic uorescent dye may also change the
UV or visible light of chromophores that can emit photons. The protein properties.
principles of uorescence generation can be elucidated by a
Jablonski diagram (Karoui & Blecker, 2011). In general, a spectro-
3.5. UV spectroscopy
uorimeter system comprises of six components: a light source,
which is typically a mercury or xenon lamp for emitting UV or Vis
The near-UV absorption spectra of aromatic amino acid residues
light, a sample holder, two monochromator and/or lter(s) with
in proteins contain abundant information related to protein con-
one for selecting the excitation wavelengths and the other for
formations. UV spectroscopy uses the algorithms for molecular
selecting the emission wavelengths, a detector for converting the
surface topography and for the accessibility of certain groups of
emitted light to the electronic signal, and a data acquisition unit
atoms to the solvent. The algorithms also enable analysis of the
(Karoui & Blecker, 2011).
three-dimensional arrangement of atoms and groups within the
Fluorescence spectroscopy as one of the oldest and powerful
environment of chromophore groups (Demchenko, 2013). The
analytical methods has been extensively studied for the analysis of
spectral peaks in the range of 250e265 nm correspond to Phe
molecular structure and function, as well as protein conformations.
residues, and those in the region 265e280 nm attribute to Tyr-Trp
In terms of proteins, Trp, Tyr, and Phe residues are natural chro-
electronic interactions, whereas peaks above 285 nm are identied
mophores and are responsible for uorescence. The intrinsic Trp
exclusively as Trp contributions (Randall et al., 2016). Both changes
uorescence spectrum is generally used to study protein unfolding
in protein conformation and dissociation, as well as protein dena-
and dynamics (Albani, 2008). Fig. 4 shows the intrinsic Trp uo-
turation may lead to the change in the microenvironment of one or
rescence emission spectra of black bean protein dispersion with
more aromatic amino acid residues. However, the larger bandwidth
different ultrasonic treatments (Jiang et al., 2014). When Trp resi-
of UV absorption spectrum masks most useful information. Other
dues are fully or partially buried in the hydrophobic core of protein
components such as cysteine and histidine can also contribute to
interiors (before ultrasonic treatment, in the environment with a
the UV absorbance of proteins resulting in rather structure less
low polarity), Trp uorescence emission maximum wavelength
spectra, thus making it difcult to detect small changes that occur
(lmax) is < 330 nm, whereas lmax shifts to a longer wavelength
due to varying local environment of the chromophores. Fig. 5a
(bathochronic shift) in the presence of a polar environment and the
displays the normal UV spectra of lysozyme before and after PEF
loss of the protein tertiary or quaternary structure (after ultrasonic
treatment. The poor separation of the UV absorption bands causes
treatment). For Tyr residues, their lmax locates at about 305 nm. Tyr
non-informative for detailed analysis of protein tertiary structure
residues are more uorescent than Trp residues in solutions, but
(Zhao & Yang, 2008).
the uorescence quantum yield signicantly decreases when Tyr is
Spectrophotometry is an analytical method that using mathe-
present in proteins. This phenomenon may be interpreted by the
matical transformation of normal spectral curve into a derivative,
fact that the protein tertiary or quaternary structure suppresses Tyr
which can extract qualitative and quantitative information from
uorescence. Besides, energy transfer from Tyr to Trp residues oc-
overlapping bands of the analytes. In the derivative near-UV
curs in proteins, inducing a certain extent of quenching the Tyr
spectrum, the ability to detect and to measure minor spectral fea-
uorescence. Consequently, Tyr uorescence is lack of sensitivity to
tures is considerably enhanced (Chang et al., 2017). As a result, the
the polarity of the environment and is only used as an intrinsic
numerical derivative spectra are often calculated to achieve a
uorescent probe in studying Trp-lacking proteins (Munishkina &
higher resolution with increased sensitivity in near-UV regions of
Fink, 2007). On the other hand, Phe with a uorescence lmax at
proteins. As shown in Fig. 5b, the UV absorption peaks related to
near 280 nm is rarely used as uorescent probe because of its
Phe, Tyr and Trp can be easily differentiated from the second-
relatively low quantum yield.
derivative UV spectra of lysozyme. The blue shift of the second-
Moreover, extrinsic uorescent dyes such as 1- anilinonaph-
derivative peaks after PEF treatment indicates an unfolding of the
thalene-8-sulfonic acid (ANS), 4,4-bis-1-anilinonaphthalene-8-
tertiary structure. Changes in amplitude can be best described by
sulfonate (Bis-ANS), and nile red can attach to proteins via cova-
calculating the ratio (r a/b) of the two peak to trough values
lent interactions and/or non-covalent interactions, and therefore
between differences in second-derivative absorbance peaks. The
value of r is greatly relevant to solvent polarity for Tyr, while it is
rarely dependent on solvent polarity for Trp.

4. Applications in evaluating proteins conformational

changes

Protein modication commonly refers to purposive alteration in

Fig. 4. Intrinsic uorescence emission spectra for 0.15 mg/mL black bean protein
dispersion of different ultrasonic treatment (Jiang et al., 2014).
protein conformation by physical, chemical or enzymatic treat-
ments. In such a case, even small alteration in protein conformation
are capable of causing signicant changes in their physicochemical
and functional properties, thus enhancing their utilization as in-
gredients in the food industry. Therefore, evaluating the confor-
mational changes of the modied protein can be helpful for further
understanding of the relationship between the structural and
functional properties, and for gaining proteins with desired func-
tional properties suitable for many food formulations (Guo et al.,
2017). Table 1 summarizes applications of spectroscopic tech-
niques for monitoring proteins conformational changes due to
different treatments.
 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219 213

Fig. 5. (a) The zero-order and (b) second-derivative UV spectra of lysozyme before and after pulsed electric eld (PEF) treated at 35 kV/cm for 1200 ms (Zhao & Yang, 2008).

Table 1
Recent advances in application of spectroscopy techniques for monitoring proteins conformational changes during modication.

Samples Processing methods Spectroscopic techniques References

a-Lactalbumin and b-Lactoglobulin High intensity ultrasound CD Chandrapala et al. (2012)

b-conglycinin Ultrasound Maillard reaction CD Zhang, Chi, and Li (2014)
Barley proteins Deamidation FTIR Zhang et al. (2015)
Beer protein Z Mashing, boiling CD, FTIR Han et al. (2015)
Black bean protein isolates Ultrasound CD, uorescence Jiang et al. (2014)
Bovine b-Lactoglobulin High pressure microuidization Fluorescence, UV, CD Zhong et al. (2012)
Bovine haemoglobin Heat UV, CD Coleman and Mitchell (2016)
Bovine lactoferrin Heat Fluorescence Stanciuc et al. (2013)
Buckwheat protein Maillard reaction UV, uorescence Guo and Xiong (2013)
Chicken breast meat High intensity ultrasound Raman Li et al. (2014)
Egg allergen ovotransferrin Heat Fluorescence, CD, UV Tong et al. (2012)
Egg white protein Pulsed electric eld UV, FTIR Qian et al. (2016)
Flaxseed protein isolate High hydrostatic pressure Fluorescence Perreault et al. (2017)
Gelatin Phosphorylation FTIR Kaewruang et al. (2014)
Immunoglobulins High hydrostatic pressure FTIR George et al. (2013)
Milk protein concentrate Acylation Fluorescence Shilpashree et al. (2015)
Myobrillar protein High pressure UV Barrios-Peralta et al. (2012)
Myobrillar protein High pressure Fluorescence, UV, Raman, CD Qiu, Xia, and Jiang (2014)
Myobrillar protein High pressure Raman Zhang et al. (2017)
Peanut protein isolate Maillard reaction Fluorescence, CD Liu, Zhao, Zhao, Ren, and Yang (2012)
Phytohemagglutinin High hydrostatic pressure FTIR Liu et al. (2013)
Rapeseed protein isolate High pressure, heat CD He et al. (2014)
Sauvignon blanc wine proteins High hydrostatic pressure FTIR Tabilo-Munizaga et al. (2014)
Silver carp actomyosin Acidication CD, UV, uorescence Xu, Xia, and Jiang (2012)
Skeletal muscle myoglobin High pressure carbon dioxide UV, CD, uorescence Yan, Xu, Jia, Dai, and Li (2016)
Soy glycinin High pressure FTIR Savadkoohi et al. (2016)
Soybean b-conglycinin and glycinin High intensity ultrasound FTIR, CD, Raman Hu et al. (2015a)
Soybean glycinin High intensity ultrasound CD, uorescence Zhou et al. (2016)
Soybean protein Glycosylation, enzymatic cross-linking CD Song and Zhao (2014)
Soybean protein isolate High hydrostatic pressure Fluorescence, CD Li et al. (2012)
Soy protein Ultrasound enzymatic cross-linking Raman Hu et al. (2015a)
Soy protein isolate Microuidization Fluorescence Shen and Tang (2012)
Soy protein isolate Low-frequency ultrasonication CD Hu et al. (2013)
Soy protein isolate Maillard reaction Fluorescence, CD Zhuo et al. (2013)
Wheat gluten Deamidation FTIR, uorescence Liao et al. (2016)
Wheat gluten Extrusion FTIR Wang et al. (2017a)
Wheat gluten Heat UV, uorescence, FTIR Wang et al. (2017b)
Whey protein Heat Raman Blanpain-Avet et al. (2012)
Whey protein Heat Fluorescence Rufn et al. (2014)
Whey protein Heat Fluorescence, CD, FTIR Zhang and Zhong (2012)
Whey protein Maillard reaction Fluorescence Spotti et al. (2014)
Whey protein isolate Maillard reaction FTIR, CD Wang, Bao, and Chen (2013)
Whey protein isolate Enzymatic deamidation CD, uorescence Miwa et al. (2013)
Yak casein micelles Acylation Fluorescence, FTIR Yang et al. (2015)
214 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219
4.1. Physical treatments
The modication of protein by physical treatment is generally
safe with no chemical reagent addition. Particularly, heat treatment
is commonly used. Gelation capacity is an important property of
food proteins during heat treatment, and heat-induced denatur-
ation process is accompanied by protein unfolding and an exposure
of reactive groups (such as sulfhydryl groups and hydrophobic
groups), which is believed to be crucial for protein gel formation
(Raikos, 2010). In one study, Liu, Zhao, Xie, and Xiong (2011) used
Raman spectroscopy demonstrated that the difference Tyr ratios
and intra-/intermolecular disulde exchanges between pork and
sh protein during heating triggered a distinguished gelation pro-
cess. They found that heating induced unfolding of a-helices and
formation of b-sheets was associated with the exposure of reactive
groups that were benecial for protein-protein interaction and
gelling. Recently, Wang et al. (2017b) employed UV spectroscopy,
intrinsic uorescence spectroscopy, and FTIR spectroscopy to
interpret the mechanism of heat-induced wheat gluten gel for-
mation and observed a pronounced transition towards b-sheet-like
structures.
Apart from heat treatment, high-intensity ultrasound (HIU),
high pressure (HP), dynamic high-pressure microuidization
(DHPM), pulsed electric eld (PEF) have also been extensively
studied as physical treatment methods for food protein modica-
tion. Li et al. (2014) explored HIU (20 kHz, 450 W, and 6 min) for
modifying the functional properties of pale, soft and exudative
(PSE) chicken breast meat. Raman spectroscopy indicated that HIU
treatment appeared to induce changes in the spatial structure of
myosin, rendering the unfolding of a-helices, followed by a signif-
icant increase in b-sheets, b-turns, and unordered contents. The
conformational changes contributed to protein aggregates and gel
formation in the meat system, which in turn explained the
improvement in gel texture and water retention of the treated PSE-
like meat gels. Jia et al. (2010) attempted to utilize ultrasound
treatment to accelerate the enzymatic hydrolysis of wheat germ
protein. An increase of ANS uorescence intensity of wheat germ
protein with the increase of ultrasonic power was observed, indi-
cating the exposure of more hydrophobic groups and regions inside
the protein and the unfolded protein structure being more bene-
cial to alcalase hydrolysis. Furthermore, the angiotensin convert-
ing enzyme (ACE) inhibitory activity of wheat germ protein
hydrolysate treated with ultrasound was higher than that without
ultrasound treatment.
HP processing is a novel nonthermal method that can be used as
a physical treatment for protein modication. Unlike the heating-
denatured counterparts, which disrupt protein structure by trans-
ferring nonpolar hydrocarbons from the hydrophobic core toward
the water, the pressure treatment allows penetration of water into
the hydrophobic region interior of the protein matrix. Conse-
quently, the hydration patterns of the protein side chains would
signicantly affect the structural dynamic properties under high-
pressure conditions, and the structure stability is primary inu-
enced by its conformational exibility to compensate for losses of
noncovalent bonds due to relocation of water molecules (Buckow
et al., 2013). HP effect potentially accounts for the exchange of S-
S/SH and alteration of non-covalent bonds (ionic, hydrophobic, and
hydrogen bridges) of proteins. Although the primary structure re-
mains unchanged, the secondary, tertiary, and quaternary struc-
tures of protein would unfold and disassociate during HP
processing (Tabilo-Munizaga et al., 2014). Li et al. (2012) provided
direct evidence that high hydrostatic pressure (HPP) treatment
could reduce the allergenic property of soy protein isolates (SPI)
and improve the security of SPI for cow milk allergic babies.
Meanwhile, the extrinsic emission uorescence spectroscopy
suggested an approximate 11.5-fold increase of uorescence in-
tensity and a blue shift of lmax from 516 to 466 nm. Their CD
spectral analysis also indicated that there was a signicant increase
in helices and turns contents while reduction occurred in strands
and unordered contents. Li et al. (2012) speculated that the epi-
topes of SPI allergens could be closely related to its secondary
structures. In addition, effects of HHP on the modication of the
conformational and functional properties of proteins in condensed
systems have been extensively studied in recent years. Based on
FTIR spectral analysis, Savadkoohi, Bannikova, Mantri, and Kasapis
(2016) investigated the conformational behavior of soy glycinin
after HPP treatment and indicated that the pressurization induced
the loosing of b-sheet and a-helical structures and the concomitant
increase of random coils in soy glycinin samples with 10, 30 and
60% (w/w) solid contents, while the twelve disulphide linkages
assisted in retaining secondary structure in concentrated systems
(>70%, w/w).
DHPM technology uses the combined forces of high velocity
impact, high-frequency vibration, instantaneous pressure drop,
intense shear, cavitation and ultra-high pressures of up to 200 MPa,
with a short treatment time (less than 5 s) and continuous opera-
tion. Zhong et al. (2012) evaluated the relationship between the
antigenicity and conformation of b-lactoglobulin (b-LG) subjected
to DHMP treatment. UV, CD and uorescence spectra characterized
that the conformational unfolding and aggregation of b-LG under
DHMP were dramatically related to its antigenicity. At low level of
DHMP treatment (0.1e80 MPa), the disaggregation and unfolding
of b-LG were accompanied by an increase in the antigenicity, and
the aggregation of b-LG at pressures greater than 80 MPa contrib-
uted to a decrease of antigenicity. In another study, Hu et al. (2011)
analyzed CD spectra and demonstrated some a-helices and b-turns
converted to b-sheets in peanut allergen Ara h2 samples after
60 MPa DHMP treatment, which corresponded with an obvious
reduction of antigenicity and presumably was caused by either
burying or damaging of the conformational IgG-binding epitopes.
On the other hand, PEF is widely used in the food industry for
inactivation of microorganisms, which can also be used to preserve
nutrients and modify the structure and function of proteins in order
to achieve specic and/or desired functional properties. Qian et al.
(2016) discussed the effect of PEF on structural properties of egg
white protein (EWP) in solid state. UV spectral analysis suggested
more Trp exposure, while FTIR spectral analysis revealed quanti-
tative change of the relative portions of molecular secondary
structure of EWP. The decrease of a-helices and increase of b-sheets
would cause the alteration of the functional properties of proteins.
4.2. Chemical treatments
Chemical treatments mainly aim to modify -NH2, -OH, -SH, or
-COOH in protein side chains, and are effective approaches to
improve further the physicochemical and functional properties of
food proteins. Chemical modication can be achieved by phos-
phorylation, Maillard reaction, deamidation, acylation, oxidative
and so on.
Phosphorylation modication of proteins is very important
either in biological systems or in vitro for improving the physico-
chemical and functional properties of proteins. The major effect of
phosphorylation is to increase the net negative charge on protein
surface and alter protein conformations. Kaewruang, Benjakul, and
Prodpran (2014) studied phosphorylation gelatin from the skin of
unicorn leatherjacket in solutions (pH 7.0, 65  C, 1 and 3 h), and
their FTIR spectral analysis showed that the phosphate incorpo-
rated might affect the helical structure of gelatin mainly via the
increased repulsion between charged residues in gelatin chains.
Enomoto et al. (2010) employed dry-heating (pH 4.0, 85  C, 1 day)
 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219
to phosphorylate ovalbumin (OVA) derived from egg white (N-
OVA) and recombinant OVA (Re-OVA), and their CD spectra of N-
OVA and Re-OVA showed double minima at 208 and 222 nm,
whereas these minima were slightly decreased through phos-
phorylation by dry-heating. These phenomena revealed that the
secondary structure of OVA was scarcely affected by phosphoryla-
tion. However, the tertiary structure was signicantly altered as
suggested by uorescence spectra.
Maillard reaction generally occurs spontaneously during long-
time storage or heat treatment, which can effectively improve
some functionalities without the addition of extraneous chemicals.
Spotti et al. (2014) reported that the molecular weight of dextrans
(DX) affected the structural and rheological characteristics of WPI/
DX conjugates obtained by Maillard reaction. According to uo-
rescence spectra, the uorescence intensity of WPI decreased
compared with native WPI, and the effect accentuated more in the
case of lower molecular weights of DX. In addition, Zhang et al.
(2012) used near-UV CD spectroscopic technique to analyze the
tertiary conformations of soy b-conglycinin/DX conjugate prepared
by Maillard reaction in a crowded liquid system, and showed that
the decrease of the near-UV CD spectral intensity of b-conglycinin/
DX conjugate was due to the loss of b-conglycinin tertiary structure
caused by an exposure of aromatic side chains.
Deamidation can convert the amide groups of protein side
chains into acid groups and thus increase the number of negative
charges, which is attractive for modifying plant proteins that
contain a number of glutamine and asparagine residues. In one
work, a stronger absorption located at 1641 cm
1 or 1640 cm
1 was
observed in all deamidated barley proteins, indicating the forma-
tion of more exible or extended structures. An increase of the
absorption at 1608 cm
1 in deamidated barley proteins was
ascribed to the exposure of hidden parts in native protein mole-
cules during deamidation. These conformational changes might
arise from the increase of electronic repulsion and loss of hydrogen
bonding, which would facilitate emulsion formation (Zhang et al.,
2015). More recently, Liao et al. (2016) investigated the intrinsic
uorescence emission spectra of citric-acid-deamidated wheat
gluten (CDWG) and found an increase of a deamidated degree from
25% to 55% and a lmax red shift of CDWG, indicating the expansion
of the wheat gluten structure and exposure of hydrophobic groups,
which resulted in a signicant change in the tertiary structure.
In addition, it has been reported that acylation of proteins would
lead to an increase in the electrostatic repulsion forces in the pro-
tein, hence resulting in a transformation of the conformational and
functional characteristics. Acetic anhydride and succinic anhydride
are the two most commonly used acylation agents for modifying
proteins structural and functional properties. Yin, Tang, Wen, Yang,
and Yuan (2010) reported that there was a good relationship be-
tween physicochemical properties and conformational features of
acetylated and succinylated kidney bean protein isolate (KPI). In
their study, intrinsic uorescence and CD spectroscopic techniques
were carried out to investigate tertiary and secondary conforma-
tional changes of KPI during acylation. Acetylation and succinyla-
tion caused signicant and gradual decreases in uorescence
intensity with increasing anhydride-to-protein ratio to 1.0, sug-
gesting protein unfolding during acylation. The increase in negative
ellipticity showed the conformational transformation to a-helices
or random coils under acylation. Shilpashree et al. (2015) studied
succinylation on milk protein concentrate (MPC) and found that
succinylation could be used for ameliorating the functional prop-
erties of MPC and its application could be extended at a succiny-
lation degree of 90.43%. Simultaneously, the red shift from 347 to
359 nm of the maximum uorescence wavelength demonstrated
that the succinylation led to denaturation of MPC.
It should also be noted that proteins are vulnerable to oxidative
215
damage because of their abundance in foods and high oxidation
reaction rates. Protein oxidation is a covalent interaction, which
could induce protein fragmentation, cross-linking, and conforma-
tional changes, leading to decrease of its nutritional value and
functional characteristics. Spectrouorometric methods were
demonstrated to be the feasible techniques in evaluating proteins
oxide. Wu, Zhang, Kong, and Hua (2009) investigated intrinsic Trp
uorescence spectra to trace 2, 2-azobis (2-amidinopropane)
dihydrochloride (AAPH) mediated soy protein oxidation, and found
AAPH resulted in a decrease in Trp uorescence intensity and a blue
shift of lmax from 330 to 319 nm. In addition, their CD spectra
suggested that oxidation led to a gradual loss of a-helix and b-sheet
structures. Furthermore, it was found that these conformational
changes decreased the water holding capacity and gel strengthen of
soy protein gel.
4.3. Enzymatic treatments
Compared with physical and chemical treatment, enzymatic
modication is more acceptable for improving the functional
properties of proteins due to milder process conditions required,
easier control of the reaction, high efciency of modication and
less formation of by-products. Among the enzymatic modication,
the enzymatic proteolysis, cross-linking and deamidation are
commonly used. Proteolysis can result in hydrolysis of peptide
bonds and affect proteins primary structure. Therefore, secondary
and tertiary structural changes result from proteolysis are not
discussed here.
Unlike the proteolysis process, transglutaminase (TGase) could
catalyze intra- and intermolecular isopeptide bonds cross-linking
by an acyl transfer reaction between glutamine (acyl donors) and
lysine residues (acyl acceptors) of the proteins. Currently, a number
of researches reported that TGase could be utilized to improve the
textural properties of various proteins and spectroscopic tech-
niques are thus used to monitor protein conformational changes.
For example, Herrero, Cambero, Ordonez, De la Hoz, and Carmona
(2008) used Raman spectra of amide I and amide III bands to
analyze structural conversion of cross-linked meat proteins with
different amounts of TGase. A signicant decrease in a-helices
accompanied by an increase in b-sheets and turns percentages
upon addition of TGase was observed. Herrero et al. (2008) also
observed positive correlation of springiness with b-sheet structure
and negative correlation with a-helices content, positive correla-
tion of adhesiveness with a-helices and turns and negative corre-
lation with b-sheets, and positive correlation of hardness and
springiness with turns. Furthermore, studies on Trp band at
759 cm
1, Tyr doublet ratio (I850/I830), Raman bands at 1450 cm
1
and 2935 cm
1 suggested an alteration of tertiary structure of meat
proteins. In another study, CD spectra were analyzed to assess the
TGase-induced structural alteration of soybean proteins. The
ellipticity around 195, 208 and 216 nm decreased in TGase cross-
linked soybean protein, showing that TGase modication induced
a more random secondary structure of soybean protein (Song &
Zhao, 2014).
In recent years, protein-glutaminase (PG) deamidation has
gradually gained more and more attention because it is more
desirable than chemical deamidation. Miwa, Yokoyama, Nio, and
Sonomoto (2013) reported that PG-deamidated WPI tended to
form a soft texture gel with a higher water-binding capacity, which
to some extent was attributed to the structural changes and would
be meaningful for practical uses. Structural analysis of WPI by using
CD spectroscopy and uorescence spectroscopy revealed that the
partial disruption of the tertiary structures of WPI proteins with
respect to Trp residues shift to a more-polar environment by the
electrical repulsion of the negative charge derived from carboxyl
216 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219

groups under PG deamidation.
5. Advantages and limitations
Spectroscopic techniques including FTIR, Raman, CD, uores-
cence, and UV spectroscopy have been successfully applied for
evaluating conformational changes in proteins. In contrast to their
traditional counterparts, known to reect food protein denatur-
ation indirectly, spectroscopic techniques present considerable
advantages. For example, FTIR spectroscopy provides high-quality
spectra with spectrum in the region of 1600e1700 cm
1 highly
sensitive to alterations in secondary structure. Raman spectroscopy
can obtain information about molecular vibrations related to sec-
ondary structures, side chains of proteins and interference resulting
from H2O is rare in Raman spectra. In addition, it is feasible to
analyze samples in many cases with fewer sample preparation
procedures, thereby providing the potential for direct, non-
destructive, and faster detection of conformational changes in
situ. CD spectroscopy is uniquely sensitive to the detection of pro-
tein conformational changes at a low concentration. Besides, CD
spectra in the far-UV and near-UV region can provide some useful
information related to protein secondary structures and tertiary
structures, respectively. On the other hand, UV spectroscopy and
uorescence are inexpensive and easy to operate, and can effec-
tively monitor changes in the tertiary structure of proteins. In
particular, data acquisition in uorescence spectroscopy occurs
within nanoseconds, thereby making it possible to investigate in-
depth thermodynamics involving multiple experiments under
different conditions. Table 2 provides a summary of the advantages
of these spectroscopic techniques. It should be noted that other
methods, including differential scanning calorimetry (Kazemi,
Ngadi, & Garie
py, 2011), sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (Perreault et al., 2017), scanning tunneling mi-
croscopy (Rinke et al., 2014), hydrogen/deuterium exchange mass
spectrometry (Li, Williams, & Topp, 2008), small-angle X-ray scat-
tering and multi-angle laser light scattering in conjunction with a
size exclusion chromatography (Zhao, Li, Liu, Liu, & Li, 2012) could
be used with spectroscopies complementarily, which offer further
advantages.
However, certain limitations are associated with some of these
spectroscopic techniques. Notably, reliable evaluation of confor-
mational changes in protein is only possible with high sample
purity and strictly dened environmental conditions. Most food
systems comprise of complex matrices, containing not only pro-
teins but also some other components such as lipids, starches,
pigments, etc. which interfere during analysis and overlap the in-
formation of protein spectra, posing difculties in monitoring
protein conformation in real food systems. Even for proteins with
high purity, the FTIR spectrum of protein in aqueous conditions
may experience interference by H2O. Although this interference can
be eliminated by using D2O solution, the H-D substitution may
change the protein structural characteristics somewhat in com-
parison with its native state. In some cases, Raman spectroscopy is
greatly restricted by inherent weaker Raman scattering and
stronger disturbance of biological uorescence, which could
hamper the gaining of high-resolution Raman spectra. Besides,
lengthy laser radiation can generate heat that may alter the
conformation of samples, thus affecting the measurement accuracy.
In the case of CD, UV and uorescence spectroscopies, only diluted
protein samples can be analyzed. However, diluted samples are not
the usual concentrations of protein in food products. Therefore, the
sample preparation procedure will be time-consuming. In addition,
CD spectra can only provide low-resolution secondary structural
information because the accuracy of this technique highly depends
on the reference databases veried by other techniques. On the
other hand, another drawback of FTIR, Raman and CD spectrome-
ters is their high cost in the instruments.
6. Conclusions and future trends
In this review, the principles and recent applications of ve
spectroscopic techniques including FTIR, Raman, CD, UV and uo-
rescence spectroscopies for protein conformation evaluation are
described. Although these techniques cover several advantages for
measuring the secondary or tertiary structures, some challenges
still exist to realize protein conformation detection in real food-
stuffs due to their complex components. Therefore, spectroscopic
solutions for protein conformational monitoring of complex food

Table 2
Comparison of different spectroscopic techniques for detecting proteins conformational changes.

Spectroscopies Protein structure Protein Advantages Limitations

state

FTIR Secondary structure Liquid or Fast and Convenient High cost

solid Sensitive to conformational changes Strong IR absorbance of H2O
under various conditions H-D substitution affect protein properties
Lack of dependence on the physical state
of samples
Raman Secondary and Liquid or Non-destructive High cost
tertiary structures solid Convenient Inherently weaker Raman scattering
On-line and in situ Stronger biological uorescence interference
Weaker H2O interference Thermal effect generated by the laser
CD Secondary and Liquid Fast Time-consuming sample preparation procedures
tertiary structures Low protein concentration Samples should be highly clear
Accurate sample concentrations and reference databases being essential for
determining secondary structure content
Not suitable for direct measurements of solid-state and high concentration
samples
Fluorescence Tertiary structure Liquid Economic and simple Time-consuming sample preparation procedures
The data acquisition is quite fast Not suitable for direct measurements of samples in solid-state
Useful for in-depth thermodynamic Unable to determine secondary structures
studies
UV Tertiary structure Liquid Fast Time-consuming sample preparation procedures
Economic and simple Not suitable for direct measurements of samples in solid-state
Unable to determine secondary structures
Overlapping Tyr, Trp, and Phe spectra
 K. Wang et al. / Trends in Food Science & Technology 67 (2017) 207e219
systems should be further developed in the future.
Noteworthy, there are increasing studies of Raman spectroscopy
to evaluate protein conformational changes in meat products. This
broadens the perspective to detect proteins in food products with
few preparation procedures, and provides the potential for on-line
and in-situ monitoring protein conformational changes in the
course of different types of treatments, which has practical signif-
icance for food protein processing. On the other hand, due to the
inherent weak Raman scattering effect and strong biological uo-
rescence interference of Raman spectroscopy, development of new
techniques such as surface-enhanced Raman spectroscopy might a
promising way to gain a spectrum with strong signals. Considering
the use of far-UV CD for protein secondary structure analyses
greatly depends on the reference databases, consequently,
amelioration of the CD spectrum processing software and compu-
tational approaches, as well as establishment of more enormous
and accurate reference databases should be considered in the
future work. Fluorescence and UV spectrophotometers have rela-
tively low cost with simple operations and future research interests
can be focused on software improvements and overlapping spectral
processing.
It should be pointed out that any spectroscopic technique
should not be used alone. In terms of UV and uorescence spec-
troscopies, their limitations on the detection of peptide backbone
structures should be compensated for other complementary ap-
proaches, and combined spectroscopic techniques are promising
for increasing prediction accuracy of protein conformations trans-
formation. Apart from the aforementioned techniques, develop-
ment of other emerging spectroscopies such as using
electromagnetic spectra in terahertz (THz) frequency ranges
(0.1e10 THz) for protein conformation measurement is attractive in
future work. Although fundamental research and applications of
THz spectroscopy for protein conformational monitoring are still in
its infancy, with the development of THz sources and detector, this
technique could be a novel and powerful nondestructive technique
with great potential for denatured proteins detection. It is hoped
that future studies could develop robust solutions for proteins
conformational monitoring for the food industry.
Acknowledgments
The authors are grateful to the International S&T Cooperation
Program of China (2015DFA71150) for its support. This research was
also supported by the Collaborative Innovation Major Special Pro-
jects of Guangzhou City (201508020097, 201604020007,
201604020057), the Guangdong Provincial Science and Technology
Plan Projects (2015A020209016, 2016A040403040), the Key Pro-
jects of Administration of Ocean and Fisheries of Guangdong
Province (A201401C04), the National Key Technologies R&D Pro-
gram (2015BAD19B03), the International and Hong Kong - Macau -
Taiwan Collaborative Innovation Platform of Guangdong Province
on Intelligent Food Quality Control and Process Technology &
Equipment (2015KGJHZ001), the Guangdong Provincial R & D
Centre for the Modern Agricultural Industry on Non-destructive
Detection and Intensive Processing of Agricultural Products, the
Common Technical Innovation Team of Guangdong Province on
Preservation and Logistics of Agricultural Products (2016LM2154),
the China Postdoctoral Science Foundation (2017M612672) and the
Fundamental Research Funds for the Central Universities
(2017MS067).
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