Principles and applications of spectroscopic techniques for evaluating food protein conformational changes: A review

Accepted Manuscript Principles and applications of spectroscopic techniques for evaluating food protein conformational changes: A review Kaiqiang Wang, Da-Wen Sun, Hongbin Pu, Qingyi Wei PII:

S0924-2244(17)30090-0

DOI:

10.1016/j.tifs.2017.06.015

Reference:

TIFS 2035

To appear in:

Trends in Food Science & Technology

Received Date: 16 February 2017 Revised Date:

12 June 2017

Accepted Date: 13 June 2017

Please cite this article as: Wang, K., Sun, D.-W., Pu, H., Wei, Q., Principles and applications of spectroscopic techniques for evaluating food protein conformational changes: A review, Trends in Food Science & Technology (2017), doi: 10.1016/j.tifs.2017.06.015. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Principles and Applications of Spectroscopic Techniques for Evaluating

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Food Protein Conformational Changes: A Review

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Kaiqiang Wang1,2,3, Da-Wen Sun1,2,3,4∗, Hongbin Pu1,2,3, Qingyi Wei1,2,3

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School of Food Science and Engineering, South China University of Technology, Guangzhou 510641, China 2

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Academy of Contemporary Food Engineering, South China University of Technology, Guangzhou Higher Education Mega Center, Guangzhou 510006, China

Engineering and Technological Research Centre of Guangdong Province on Intelligent Sensing and Process

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Control of Cold Chain Foods, Guangzhou Higher Education Mega Center, Guangzhou 510006, China

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Food Refrigeration and Computerized Food Technology (FRCFT), Agriculture and Food Science Centre, University College Dublin, National University of Ireland, Belfield, Dublin 4, Ireland

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Abstract

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Background: Proteins are essential nutrients required in various body functions and normal human

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life. However, in the food industry, the application of proteins especially those of plant origin have

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been limited due to their poor functionality. Although nowadays, diverse modification techniques are

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usually employed to improve their performance in food products, it is also important that effective

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methods for monitoring the resultant conformational changes induced during protein modification

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are developed.

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Scope and approach: In this review, the relationship between protein conformation and functionality

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is briefly discussed. Thereafter, the underlying principles behind five selected spectroscopic

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Corresponding author. Tel: +353-1-7167342; Fax: +353-1-7167493.

E-mail address: [email protected]. Website: www.ucd.ie/refrig; www.ucd.ie/sun. 1

ACCEPTED MANUSCRIPT techniques i.e. Fourier transform infrared, Raman, circular dichroism, fluorescence, and ultraviolet

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spectroscopies is introduced and their recent applications for monitoring conformational changes that

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occur during physical, chemical or enzymatic modification of proteins are addressed. In addition, the

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advantages and limitations of each spectroscopic technique are comparatively discussed and

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perspectives on the current situation alongside future trends are highlighted.

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Key findings and conclusions: Spectroscopic techniques present an attractive panacea for evaluation

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of conformational changes during protein modification. Although certain challenges especially with

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complex food materials require urgent attention thus, more robust spectroscopic solutions should be

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exploited in the future.

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Keywords: Protein conformation, Fourier transform infrared spectroscopy, Raman spectroscopy,

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circular dichroism spectroscopy, fluorescence spectroscopy, ultraviolet spectroscopy

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1. Introduction

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Proteins are considered principal components for body metabolism and general human life. In recent

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years, there has been an increasing interest in utilizing proteins from various sources as functional

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ingredients in food products, primarily due to their high nutritional value and unique functionality.

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The functional properties of protein in food including water- and fat-binding capacities, gel forming

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and rheological behaviors, emulsifying capabilities, foaming capabilities etc., providing desirable

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sensory characteristics such as structure, texture, flavor, and color during food product formulation

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(Buckow et al., 2013; Hu et al., 2015b; Lam & Nickerson, 2013; Whitford, 2013). To cite an

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example, the emulsifying properties of proteins permit the formation of emulsions owing to their

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amphiphilic nature and film-forming capacity, therefore profitable in various food products, drugs

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and nutrient delivery tactics (Lam & Nickerson, 2013). However, some native proteins, especially

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those of plant origin exhibit poor functionalities during food manufacturing. For instance, the poor

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digestibility of red kidney bean protein isolates (Yin et al., 2008), low solubility of soy protein

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as poor solubility, foaming and emulsifying properties of wheat gluten proteins (Agyare et al., 2009).

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As a result, certain modification treatments including physical (Zhang et al., 2017), chemical

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(Shilpashree et al., 2015), and enzymatic methods (Wang et al., 2016a; 2016b) are required in order

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to obtain optimal nutritive value such as high bioactive activities (Perreault et al., 2017), high

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digestibility (Yin et al., 2008) and low allergenicity (Li et al., 2012; Tong et al., 2012). Such

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treatments also ameliorate the functional properties of proteins including gelling strength (He et al.,

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2014), emulsification (Raikos, 2010) and their ability to form foams (Morales et al., 2015).

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Furthermore, it is well known that the modification of food protein functionality is accompanied with

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conformational changes, which influence the quality of the end-product. For example, Abaee et al.

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(2017) found that the hardness of the non-heat-treated whey protein cold-set hydrogels prepared at

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pH 9.0 was significantly higher than that of the samples obtained at pH 8.0 and pH 7.0. Their results

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suggested that base-induced denaturation and unfolding of β-lactoglobulin at pH 9.0 caused the

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formation of more disulfide bonds and hydrophobic interactions, accounting for the increased

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α-helical structures. Li et al. (2014) illustrated that high-intensity ultrasound could induce

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conformational changes including a decrease in the α-helical contents and increase in β-sheet, β-turns,

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and unordered contents, contributing to protein aggregation and gel formation in meat, leading to

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enhanced gel texture. On the other hand, Rahaman et al. (2016) reported that different processing

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approaches could affect the conformational changes related to digestibility and allergenicity of food

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proteins in peculiar ways. Therefore, it is essential to develop effective detection methods for

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evaluating conformational changes in protein and understanding the relationship between its

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structural and functional characteristics during processing. Several chemical methods for indirect

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reflection of protein denaturation in meat are available based on detection of protein solubility, free

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sulfhydryl content, surface hydrophobicity, or myofibrillar ATPase activity (Chen et al., 2016).

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However, most of these above-mentioned methods are time-consuming, environmentally unfriendly

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Undoubtedly, spectroscopic techniques in the last few years have significantly improved, however,

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they are developed and used for protein structure analysis for decades. These techniques include

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X-ray diffraction (XRD) (Jenkins et al., 2013), nuclear magnetic resonance (NMR) (Mao et al.,

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2014), Fourier transform infrared (FTIR) (Zhang et al., 2015), Raman (Li et al., 2014), circular

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dichroism (CD) (Chandrapala et al., 2012), fluorescence (Ruffin et al., 2014) and ultraviolet (UV)

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(Barrios-Peralta et al., 2012) spectroscopies. These spectroscopic techniques are particularly suitable

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for probing structural conversion such as folding and unfolding. In particular, XRD and NMR

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spectroscopies are used to obtain structural information of proteins in high resolution. In fact, XRD

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is regarded as one of the best methods for protein structure analysis and even minimal

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conformational changes are detectable if only crystallization in two alternative conformations is

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possible. However, XRD does not allow for real time conformational transitions analysis. While

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NMR spectroscopy is only able to detect low molecular weight proteins and has its limitations for

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applying to proteins larger than a few hundred residues (Demchenko, 2013).

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Nonetheless, other spectroscopic techniques including FTIR, Raman, CD, fluorescence and UV are

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simple, rapid, convenient, and have gained increase popularity for monitoring conformational

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changes during protein modification. To the best of knowledge, a review, which specifically

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addresses their applications in this area, is currently unavailable. Thus, the current review presents

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the recent advances pertaining to their application in detection of conformational changes in proteins

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alongside their advantages and limitations. The underlying principles of aforementioned

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spectroscopic techniques are also summarized. In addition, certain areas that could be further

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exploited and future research trends are proposed.

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2. Relationship between conformational and functional properties of food protein

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The conformational and functional properties of food protein are closely related to various inherent 4

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chemical nature of the amino acids side-chain groups decides the shape and overall hydrophobicity

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of proteins. Generally, proteins tend to assume an elongated rodlike shape when they contain a large

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number of hydrophilic amino acids residues distributed uniformly in its sequence; in contrast, they

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tend to assume a globular shape when they contain a large number of hydrophobic residues. In the

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native form of proteins, the hydrophobic segments are mostly buried inside the core. The surface

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hydrophobicity and hydrophilicity characteristics of protein surface can mostly affect their solubility

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characteristics, which govern several functionalities such as thickening, foaming, emulsification, and

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gelation properties (Hettiarachchy, 2012). The pH value affects protein solubility in aqueous

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solutions. At the isoelectric pH, the hydrophobic interaction between proteins reaches maximum,

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inhibiting unfolding of the protein molecules and resulting in the minimum solubility. When proteins

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are exposed to moderately pH values above or below the isoelectric point, the electrostatic repulsion

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and ionic hydration promote the solubilization of protein and some functional properties are

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improved, which might be related to unfolding of the protein and/or activation of buried sulf-hydryl

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groups. The ionic strength of a solution also determines the overall charge of the protein molecule.

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Ionic

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hydrophilicity-hydrophobicity characteristics of the protein surface. Moreover, temperature induced

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protein denaturation could also alter the surface hydrophobicity of proteins (Damodaran, 1997).

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Functionalities of denatured food proteins are differ from their native states. Therefore, physical,

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chemical or enzymatic modifications often used to alter the conformation and functionalities of food

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proteins. The exposure of hydrophobic regions, for example, can lower the solubility, affect the

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surface activity as well as alter the water and oil holding abilities of proteins (Shen & Tang, 2012).

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The unfolding of protein structure can expose more amino acids residues, facilitating the proteolysis

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and improve the digestibility of proteins (Perreault et al., 2017). In addition, changes in the

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conformation can inhibit the activity of anti-nutritional factors or protein toxins and lower the

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ACCEPTED MANUSCRIPT allergenicity (Liu et al., 2013; Rahaman et al., 2016). More details about the relationship between

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protein conformation and functionalities can be found elsewhere (Hettiarachchy, 2012; Whitford,

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2013). Apart from the modification conditions, changes in the structural and functional properties of

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food protein also depend on the state of protein (e.g., fibrous or globular, wet or dry, in liquid or

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frozen state, and presence of foreign substances or not) (Kuan et al., 2013). For instance, globular

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proteins that possess complex three-dimensional shape depart considerably from fibrous proteins,

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showing elongated structures that lack true tertiary structure. Due to the cavities in the folded state of

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globular proteins, they tend to be more susceptible to hydrostatic pressure than fibrous proteins

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(Ustunol, 2014). The conformational changes of proteins can be detected by spectroscopic

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techniques, and spectral characteristics related to protein conformational properties are introduced in

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details in the current review.

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3. Principles of spectroscopic techniques

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3.1 Fourier transform infrared (FTIR) spectroscopy

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Infrared (IR) spectrum in the range of 400 to 4000 cm-1 arises from the absorption of energy by

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chemical bonds, primarily stretching and bending motions, and has been recognized as a powerful

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technique for the structural and chemical characterization of proteins (Carbonaro & Nucara, 2010).

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Generally speaking, the amide I band (1700-1600 cm-1) of an IR spectrum is primarily attributed to

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C=O stretching vibrations (approximately 80%) with some in-plane N-H bending and C-H stretching

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modes. In particular, the C=O stretching vibrations in proteins mainly depend on their various

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secondary structures and inter- or intramolecular effects, including molecular geometry and

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hydrogen bonding pattern, which makes the amide I band being the most sensitive IR spectral region

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to predict the secondary structural components of proteins (Kong & Yu, 2007).

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When the FTIR absorption of protein is measured in solutions, the strong IR absorbance of H2O

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centered at 1640 cm-1 from O-H-O bending mode may interfere the determination. In order to

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for FTIR experiment, a protein sample of 2 mg is generally mixed with 198 mg of transparent media

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(usually KBr) to form pellet (1-2 mm thick) for measurement. Besides, the diffuse reflectance and

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attenuated total reflection (ATR) FTIR spectrum have also been developed for evaluating food

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protein secondary structure.

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The observed amide I band is a complex of several overlapping components that corresponding to

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specific secondary structures, including α-helices, β-sheets, β-turns, and random coils. However,

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these broad underlying components bands are instrumentally unresolvable (Carbonaro & Nucara,

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2010). In order to enhance the resolution of individual underlying component and quantitatively

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estimate the relative contributions of various secondary structures, Fourier self-deconvolution (FSD)

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fitting and second derivative analysis should be used to achieve maximum band narrowing, degrade

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the signal-to-noise ratio and identify different types of secondary structures present in proteins. For

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instance, Figure 1 presents the handling process of IR spectra in amide I region of pulsed electric

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field (PEF) treated egg white protein (EWP) (Qian et al., 2016). The peak location of the

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components does not shift, whereas their areas change significantly. The calculation of the portion of

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the components indicates that a reduction of α-helices is accompanied by an increase of β-sheets

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during PEF treatment.

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3.2 Raman spectroscopy

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Raman spectrum has been proven to provide effective information about protein secondary structures

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and the microenvironment of protein side chains, which can be used as a valid tool for evaluating

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protein denaturation. The spectrum can be processed identically to the infrared spectroscopy data. In

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common with IR spectra, among several distinct vibrational modes of the -CO-NH- amide, the most

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available bands of Raman spectra for determining protein secondary structures are the amide I and

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III regions. The frequency position of these bands depends strongly on the protein state, the 7

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Figure 2 shows the Raman spectra of β-conglycinin by high intensity ultrasound (20 kHz at 400 W)

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treatment for 0, 5, 20 and 40 min (Hu et al., 2015a). Apart from the amide I and III regions, the major

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vibrational motions of the side chains, including inter-chain disulfide bands, tryptophan (Trp) bands,

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tyrosine (Tyr) bands, and aliphatic hydrophobic residues in Raman spectra can also be analyzed to

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provide some information about protein tertiary structure. The Raman bands located at 760, 880,

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1361 cm-1 are ascribed to Trp residues. A sharp line at 1361 cm-1 is suggested as an indicator of

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buried Trp residues (Ferrer et al., 2011). However, the Trp bands at 760 and 880 cm-1 have been

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proposed as an indicator of the strength of H-bonding and hydrophobicity of the indole ring. An

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increase in the intensity in these bands indicates that Trp residues are buried, and in contrast a

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decrease is connected with the opposite phenomenon (Gómez et al., 2013). The Raman Tyr residues

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vibrations are located at about 850 cm-1 and 830 cm-1, and the ratio of Tyr doublet (I850/I830) is

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sensitive to the nature of hydrogen bonding and ionization of phenolic hydroxyl groups. Tyr doublet

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is considered as a good indicator for evaluating the degree of Tyr residues exposed or buried (Gómez

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et al., 2013).

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In Raman spectra, the stretching vibration located in the range of 500-550 cm-1 derive from disulfide

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bridges that are formed by two cysteines, and the peaks around 510, 525, and 545 cm-1 can be

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assigned to disulfide bonds in gauche-gauche-gauche (g-g-g), gauche-gauche-trans (g-g-t), and

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trans-gauche-trans (t-g-t) conformations, respectively (Li, 2012). Disulfide bonds belong to the

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secondary bonds that could maintain the tertiary structure of proteins, thereby changes in disulfide

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bonds correlate with the alteration of protein tertiary structure. In addition, the vibration of aliphatic

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amino acid residues in Raman spectra near 2800-3000 cm-1 (C-H stretching) and 1440-1465 cm-1

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(C-H bending) have also been investigated for monitoring protein conformational changes. Although

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the indicators of changes in C-H bending vibration are debatable, these changes can also provide

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information on proteins hydrophobic interactions and conformational changes due to processing

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(Sheng et al., 2016).

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3.3 Circular dichroism (CD) spectroscopy

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CD spectroscopy is another well-established spectroscopic technique for determining secondary

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structures, folding and binding properties of proteins. In fact, when the plane polarized light passes

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through a modulator that subjects to an alternating 50 kHz electric field, it will be split into the

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rotating left-handed (counter-clockwise) and the right-handed (clockwise) circularly polarized

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components (Kelly et al., 2005). If the two circularly polarized components have the same amplitude,

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the recombination of the components can regenerate radiation polarized in the original plane.

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Conversely, the resulting recombined component radiation is then elliptically polarized. The

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principle of CD spectroscopy is based on the unequal absorption of the two circularly polarized

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components (∆A=AL-AR). In order to observe CD signals, the sample should be optically active. As

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all amino acids except glycine are asymmetric and hence optically active, protein structures have

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been widely studied by CD spectroscopy.

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Normally, CD spectra are collected in high-transparent rectangular or cylindrical quartz cells. Buffers

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for dissolving protein samples should be transparent and not contain any materials that are optically

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active. In addition, protein solutions for CD measurement should be at least 95% pure with

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concentrations between 0.005 and 5 mg/mL depending on the pathlength of the cell. When CD

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spectrum is acquired, software such as CDPro, CONTIN, SELCON3, DICROPROT, and CDSSTR

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and computational methods such as singular value decomposition, optimization algorithms,

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regression, and neural networks are used for analyzing the CD spectrum. A detailed introduction of

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the acquirement and analysis of the CD spectrum can be found in a previous review (Martin &

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Schilstra, 2008).

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The CD spectrum of a protein in the far UV region is dominated by a weak but broad n→π*

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transition at about 220 nm and an intense π→π* transition centered around 200 nm of amide groups,

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ACCEPTED MANUSCRIPT and are influenced by the geometries of the polypeptide backbones, making it one of the most

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commonly used techniques to determine the protein secondary structure content (Whitmore &

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Wallace, 2008). Generally, α-helices show a strong positive band at 191-193 nm and a typical double

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negative bands at 208-210 and 222 nm, β-sheets produce an intense positive band at about 195-200

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nm and a negative band at about 216-218 nm, whereas random coils have a strong negative bands at

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195-200 nm and a much weaker band (either positive or negative) between 215 and 230 nm. On the

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other hand, the CD spectrum in near UV region (250-320 nm) can provide useful information related

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to aromatic chromophores (Phe, Tyr, and Trp residues) of proteins in asymmetric environment, which

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has been widely used to assess the tertiary and occasionally quaternary structures of proteins during

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processing. Generally speaking, Phe residues have sharp fine structure between 255 and 270 nm with

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peaks observed near 262 and 268 nm, whereas the bands arising from Tyr and Trp residues are

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located at 275-282 nm and 290-305 nm in near-UV CD spectrum, respectively (Kelly et al. 2005;

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Martin & Schilstra, 2008). An increase in the band magnitudes and intensities is an indication of

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structural changes, which are related to the loss of native-like structure and increasing interactions of

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the aromatic amino acid residues during processing (He et al., 2014). Figure 3 shows the CD spectra

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of myoglobin in solution, absorbed at tricaprin oil/water interface and at hexadecane oil/water

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interface (Day et al., 2014). As shown in Figure 3a, myoglobin is a high helical protein with strong

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positive ellipticity at 193 nm and two distinctive negative minima at 208 and 222 nm respectively.

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Upon adsorption to oil/water interfaces, the reduction in the intensity of peaks at 193, 208 and 222

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nm suggests the loss of helical structure. Figure 3b shows the near-UV CD spectra, providing the

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evidence of the change in the Trp residues environment.

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In recent years, with the improvements in instrumentation for conventional CD and advent of

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synchrotron radiation circular dichroism (SRCD), lower wavelength bands (as low as 140 nm) and

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more information about proteins conformation of the spectra are obtainable. SRCD spectroscopy can

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provide important static and dynamic structural information on proteins in solution. Furthermore, the

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high flux of a synchrotron source increases the signal-to-noise levels of the CD spectrum, allowing

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measurement with turbid samples (e.g., in the presence of lipids, salts, and detergents) (Wallace &

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Janes, 2010).

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3.4 Fluorescence spectroscopy

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Fluorescence is the emission of photons due to the absorption of UV or visible light of chromophores

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that can emit photons. The principles of fluorescence generation can be elucidated by a Jablonski

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diagram (Karoui & Blecker, 2011). In general, a spectrofluorimeter system comprises of six

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components: a light source, which is typically a mercury or xenon lamp for emitting UV or Vis light,

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a sample holder, two monochromator and/or filter(s) with one for selecting the excitation

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wavelengths and the other for selecting the emission wavelengths, a detector for converting the

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emitted light to the electronic signal, and a data acquisition unit (Karoui & Blecker, 2011).

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Fluorescence spectroscopy as one of the oldest and powerful analytical methods has been extensively

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studied for the analysis of molecular structure and function, as well as protein conformations. In

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terms of proteins, Trp, Tyr, and Phe residues are natural chromophores and are responsible for

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fluorescence. The intrinsic Trp fluorescence spectrum is generally used to study protein unfolding

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and dynamics (Albani, 2008). Figure 4 shows the intrinsic Trp fluorescence emission spectra of black

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bean protein dispersion with different ultrasonic treatments (Jiang et al., 2014). When Trp residues

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are fully or partially buried in the hydrophobic core of protein interiors (before ultrasonic treatment,

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in the environment with a low polarity), Trp fluorescence emission maximum wavelength (λmax) is <

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330 nm, whereas λmax shifts to a longer wavelength (bathochronic shift) in the presence of a polar

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environment and the loss of the protein tertiary or quaternary structure (after ultrasonic treatment).

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For Tyr residues, their λmax locates at about 305 nm. Tyr residues are more fluorescent than Trp

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residues in solutions, but the fluorescence quantum yield significantly decreases when Tyr is present

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in proteins. This phenomenon may be interpreted by the fact that the protein tertiary or quaternary

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proteins, inducing a certain extent of quenching the Tyr fluorescence. Consequently, Tyr fluorescence

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is lack of sensitivity to the polarity of the environment and is only used as an intrinsic fluorescent

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probe in studying Trp-lacking proteins (Munishkina & Fink, 2007). On the other hand, Phe with a

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fluorescence λmax at near 280 nm is rarely used as fluorescent probe because of its relatively low

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quantum yield.

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Moreover, extrinsic fluorescent dyes such as 1- anilinonaphthalene-8-sulfonic acid (ANS),

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4,4’-bis-1-anilinonaphthalene-8-sulfonate (Bis-ANS), and nile red can attach to proteins via covalent

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interactions and/or non-covalent interactions, and therefore providing additional possibility for

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studying proteins conformational changes (Hawe et al., 2008). However, it should be noted that the

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extrinsic fluorescent dye may also change the protein properties.

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3.5 UV spectroscopy

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The near-UV absorption spectra of aromatic amino acid residues in proteins contain abundant

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information related to protein conformations. UV spectroscopy uses the algorithms for molecular

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surface topography and for the accessibility of certain groups of atoms to the solvent. The algorithms

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also enable analysis of the three-dimensional arrangement of atoms and groups within the

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environment of chromophore groups (Demchenko, 2013). The spectral peaks in the range of 250-265

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nm correspond to Phe residues, and those in the region 265-280 nm attribute to Tyr-Trp electronic

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interactions, whereas peaks above 285 nm are identified exclusively as Trp contributions (Randall et

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al., 2016). Both changes in protein conformation and dissociation, as well as protein denaturation

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may lead to the change in the microenvironment of one or more aromatic amino acid residues.

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However, the larger bandwidth of UV absorption spectrum masks most useful information. Other

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components such as cysteine and histidine can also contribute to the UV absorbance of proteins

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resulting in rather structure less spectra, thus making it difficult to detect small changes that occur

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ACCEPTED MANUSCRIPT due to varying local environment of the chromophores. Figure 5a displays the normal UV spectra of

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lysozyme before and after PEF treatment. The poor separation of the UV absorption bands causes

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non-informative for detailed analysis of protein tertiary structure (Zhao & Yang, 2008).

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Spectrophotometry is an analytical method that using mathematical transformation of normal

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spectral curve into a derivative, which can extract qualitative and quantitative information from

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overlapping bands of the analytes. In the derivative near-UV spectrum, the ability to detect and to

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measure minor spectral features is considerably enhanced (Chang et al., 2017). As a result, the

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numerical derivative spectra are often calculated to achieve a higher resolution with increased

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sensitivity in near-UV regions of proteins. As shown in Figure 5b, the UV absorption peaks related to

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Phe, Tyr and Trp can be easily differentiated from the second-derivative UV spectra of lysozyme.

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The blue shift of the second-derivative peaks after PEF treatment indicates an unfolding of the

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tertiary structure. Changes in amplitude can be best described by calculating the ratio (r = a/b) of the

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two peak to trough values between differences in second-derivative absorbance peaks. The value of

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‘r’ is greatly relevant to solvent polarity for Tyr, while it is rarely dependent on solvent polarity for

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Trp.

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4. Applications in evaluating proteins conformational changes

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Protein modification commonly refers to purposive alteration in protein conformation by physical,

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chemical or enzymatic treatments. In such a case, even small alteration in protein conformation are

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capable of causing significant changes in their physicochemical and functional properties, thus

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enhancing their utilization as ingredients in the food industry. Therefore, evaluating the

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conformational changes of the modified protein can be helpful for further understanding of the

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relationship between the structural and functional properties, and for gaining proteins with desired

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functional properties suitable for many food formulations. Table 1 summarizes applications of

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spectroscopic techniques for monitoring proteins conformational changes due to different treatments.

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4.1 Physical treatments

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The modification of protein by physical treatment is generally safe with no chemical reagent addition.

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Particularly, heat treatment is commonly used. Gelation capacity is an important property of food

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proteins during heat treatment, and heat-induced denaturation process is accompanied by protein

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unfolding and an exposure of reactive groups (such as sulfhydryl groups and hydrophobic groups),

328

which is believed to be crucial for protein gel formation (Raikos, 2010). In one study, Liu et al.

329

(2011)

330

intra-/intermolecular disulfide exchanges between pork and fish protein during heating triggered a

331

distinguished gelation process. They found that heating induced unfolding of α-helices and formation

332

of β-sheets was associated with the exposure of reactive groups that were beneficial for

333

protein-protein interaction and gelling. Recently, Wang et al. (2017b) employed UV spectroscopy,

334

intrinsic fluorescence spectroscopy, and FTIR spectroscopy to interpret the mechanism of

335

heat-induced wheat gluten gel formation and observed a pronounced transition towards β-sheet-like

336

structures.

337

Apart from heat treatment, high-intensity ultrasound (HIU), high pressure (HP), dynamic

338

high-pressure microfluidization (DHPM), pulsed electric field (PEF) have also been extensively

339

studied as physical treatment methods for food protein modification. Li et al. (2014) explored HIU

340

(20 kHz, 450 W, and 6 min) for modifying the functional properties of pale, soft and exudative (PSE)

341

chicken breast meat. Raman spectroscopy indicated that HIU treatment appeared to induce changes

342

in the spatial structure of myosin, rendering the unfolding of α-helices, followed by a significant

343

increase in β-sheets, β-turns, and unordered contents. The conformational changes contributed to

344

protein aggregates and gel formation in the meat system, which in turn explained the improvement in

345

gel texture and water retention of the treated PSE-like meat gels. Jia et al. (2010) attempted to utilize

346

ultrasound treatment to accelerate the enzymatic hydrolysis of wheat germ protein. An increase of

Raman

spectroscopy

demonstrated

that

the

difference

Tyr

ratios

and

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ACCEPTED MANUSCRIPT ANS fluorescence intensity of wheat germ protein with the increase of ultrasonic power was

348

observed, indicating the exposure of more hydrophobic groups and regions inside the protein and the

349

unfolded protein structure being more beneficial to alcalase hydrolysis. Furthermore, the angiotensin

350

converting enzyme (ACE) inhibitory activity of wheat germ protein hydrolysate treated with

351

ultrasound was higher than that without ultrasound treatment.

352

HP processing is a novel nonthermal method that can be used as a physical treatment for protein

353

modification. Unlike the heating-denatured counterparts, which disrupt protein structure by

354

transferring nonpolar hydrocarbons from the hydrophobic core toward the water, the pressure

355

treatment allows penetration of water into the hydrophobic region interior of the protein matrix.

356

Consequently, the hydration patterns of the protein side chains would significantly affect the

357

structural dynamic properties under high-pressure conditions, and the structure stability is primary

358

influenced by its conformational flexibility to compensate for losses of noncovalent bonds due to

359

relocation of water molecules (Buckow et al., 2013). HP effect potentially accounts for the exchange

360

of S-S/SH and alteration of non-covalent bonds (ionic, hydrophobic, and hydrogen bridges) of

361

proteins. Although the primary structure remains unchanged, the secondary, tertiary, and quaternary

362

structures of protein would unfold and disassociate during HP processing (Tabilo-Munizaga et al.,

363

2014). Li et al. (2012) provided direct evidence that high hydrostatic pressure (HPP) treatment could

364

reduce the allergenic property of soy protein isolates (SPI) and improve the security of SPI for cow

365

milk allergic babies. Meanwhile, the extrinsic emission fluorescence spectroscopy suggested an

366

approximate 11.5-fold increase of fluorescence intensity and a blue shift of λmax from 516 to 466 nm.

367

Their CD spectral analysis also indicated that there was a significant increase in helices and turns

368

contents while reduction occurred in strands and unordered contents. Li et al. (2012) speculated that

369

the epitopes of SPI allergens could be closely related to its secondary structures. In addition, effects

370

of HHP on the modification of the conformational and functional properties of proteins in condensed

371

systems have been extensively studied in recent years. Based on FTIR spectral analysis, Savadkoohi

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ACCEPTED MANUSCRIPT et al. (2016) showed the conformational behavior of soy glycinin after HPP treatment and indicated

373

that the pressurization induced the loosing of β-sheet and α-helical structures and the concomitant

374

increase of random coils in soy glycinin samples with 10, 30 and 60% (w/w) solid contents, while the

375

twelve disulphide linkages assisted in retaining secondary structure in concentrated systems (> 70%,

376

w/w).

377

DHPM technology uses the combined forces of high velocity impact, high-frequency vibration,

378

instantaneous pressure drop, intense shear, cavitation and ultra-high pressures of up to 200 MPa, with

379

a short treatment time (less than 5 s) and continuous operation. Zhong et al. (2012) evaluated the

380

relationship between the antigenicity and conformation of β-lactoglobulin (β-LG) subjected to

381

DHMP treatment. UV, CD and fluorescence spectra characterized that the conformational unfolding

382

and aggregation of β-LG under DHMP were dramatically related to its antigenicity. At low level of

383

DHMP treatment (0.1 - 80 MPa), the disaggregation and unfolding of β-LG were accompanied by an

384

increase in the antigenicity, and the aggregation of β-LG at pressures greater than 80 MPa

385

contributed to a decrease of antigenicity. In another study, Hu et al. (2011) analyzed CD spectra and

386

demonstrated some α-helices and β-turns converted to β-sheets in peanut allergen Ara h2 samples

387

after 60 MPa DHMP treatment, which corresponded with an obvious reduction of antigenicity and

388

presumably was caused by either burying or damaging of the conformational IgG-binding epitopes.

389

On the other hand, PEF is widely used in the food industry for inactivation of microorganisms, which

390

can also be used to preserve nutrients and modify the structure and function of proteins in order to

391

achieve specific and/or desired functional properties. Qian et al. (2016) discussed the effect of PEF

392

on structural properties of egg white protein (EWP) in solid state. UV spectral analysis suggested

393

more Trp exposure, while FTIR spectral analysis revealed quantitative change of the relative portions

394

of molecular secondary structure of EWP. The decrease of α-helices and increase of β-sheets would

395

cause the alteration of the functional properties of proteins.

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ACCEPTED MANUSCRIPT 4.2 Chemical treatments

398

Chemical treatments mainly aim to modify -NH2, -OH, -SH, or -COOH in protein side chains, and is

399

an effective approach to improve further the physicochemical and functional properties of food

400

proteins. Chemical modification can be achieved by phosphorylation, Maillard reaction, deamidation,

401

acylation, oxidative and so on.

402

Phosphorylation modification of proteins is very important either in biological systems or in vitro for

403

improving the physicochemical and functional properties of proteins. The major effect of

404

phosphorylation is to increase the net negative charge on protein surface and alter protein

405

conformations. Kaewruang et al. (2014) studied phosphorylation gelatin from the skin of unicorn

406

leatherjacket in solutions (pH 7.0, 65 oC, 1 and 3 h), and their FTIR spectral analysis showed that the

407

phosphate incorporated might affect the helical structure of gelatin mainly via the increased repulsion

408

between charged residues in gelatin chains. Enomoto et al. (2010) employed dry-heating (pH 4.0, 85

409

o

410

OVA (Re-OVA), and their CD spectra of N-OVA and Re-OVA showed double minima at 208 and

411

222 nm, whereas these minima were slightly decreased through phosphorylation by dry-heating.

412

These phenomena revealed that the secondary structure of OVA was scarcely affected by

413

phosphorylation. However, the tertiary structure was significantly altered as suggested by

414

fluorescence spectra.

415

Maillard reaction generally occurs spontaneously during long-time storage or heat treatment, which

416

can effectively improve some functionalities without the addition of extraneous chemicals. Spotti et

417

al. (2014) reported that the molecular weight of dextrans (DX) affected the structural and rheological

418

characteristics of WPI/DX conjugates obtained by Maillard reaction. According to fluorescence

419

spectra, the fluorescence intensity of WPI decreased compared with native WPI, and the effect

420

accentuated more in the case of lower molecular weights of DX. In addition, Zhang et al. (2012)

421

used near-UV CD spectroscopic technique to analyze the tertiary conformations of soy

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C, 1 day) to phosphorylate ovalbumin (OVA) derived from egg white (N-OVA) and recombinant

17

ACCEPTED MANUSCRIPT β-conglycinin/DX conjugate prepared by Maillard reaction in a crowded liquid system, and showed

423

that the decrease of the near-UV CD spectral intensity of β-conglycinin/DX conjugate was due to the

424

loss of β-conglycinin tertiary structure caused by an exposure of aromatic side chains.

425

Deamidation can convert the amide groups of protein side chains into acid groups and thus increases

426

the number of negative charges, which is attractive for modifying plant proteins that contain a

427

number of glutamine and asparagine residues. In one work, a stronger absorption located at 1641

428

cm-1 or 1640 cm-1 was observed in all deamidated barley proteins, indicating the formation of more

429

flexible or extended structures. An increase of the absorption at 1608 cm-1 in deamidated barley

430

proteins was ascribed to the exposure of hidden parts in native protein molecules during deamidation.

431

These conformational changes might arise from the increase of electronic repulsion and loss of

432

hydrogen bonding, which would facilitate emulsion formation (Zhang et al., 2015). More recently,

433

Liao et al. (2016) investigated the intrinsic fluorescence emission spectra of citric-acid-deamidated

434

wheat gluten (CDWG) and found an increase of a deamidated degree from 25% to 55% and a λmax

435

red shift of CDWG, indicating the expansion of the wheat gluten structure and exposure of

436

hydrophobic groups, which resulted in a significant change in the tertiary structure.

437

In addition, it has been reported that acylation of proteins would lead to an increase in the

438

electrostatic repulsion forces in the protein, hence resulting in a transformation of the conformational

439

and functional characteristics. Acetic anhydride and succinic anhydride are the two most commonly

440

used acylation agents for modifying proteins structural and functional properties. Yin et al. (2010)

441

reported that there was a good relationship between physicochemical properties and conformational

442

features of acetylated and succinylated kidney bean protein isolate (KPI). In their study, intrinsic

443

fluorescence and CD spectroscopic techniques were carried out to investigate tertiary and secondary

444

conformational changes of KPI during acylation. Acetylation and succinylation caused significant

445

and gradual decreases in fluorescence intensity with increasing anhydride-to-protein ratio to 1.0,

446

suggesting protein unfolding during acylation. The increase in negative ellipticity showed the

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18

ACCEPTED MANUSCRIPT conformational transformation to α-helices or random coils under acylation. Shilpashree et al. (2015)

448

studied succinylation on milk protein concentrate (MPC) and found that succinylation could be used

449

for ameliorating the functional properties of MPC and its application could be extended at a

450

succinylation degree of 90.43%. Simultaneously, the red shift from 347 to 359 nm of the maximum

451

fluorescence wavelength demonstrated that the succinylation led to denaturation of MPC.

452

It should also be noted that proteins are vulnerable to oxidative damage because of their abundance

453

in foods and high oxidation reaction rates. Protein oxidation is a covalent interaction, which could

454

induce protein fragmentation, cross-linking, and conformational changes, leading to decrease of its

455

nutritional value and functional characteristics. Spectrofluorometric methods were demonstrated to

456

be the feasible techniques in evaluating proteins oxide. Wu et al. (2009) investigated intrinsic Trp

457

fluorescence spectra to trace 2, 2’-azobis (2-amidinopropane) dihydrochloride (AAPH) mediated soy

458

protein oxidation, and found AAPH resulted in a decrease in Trp fluorescence intensity and a blue

459

shift of λmax from 330 to 319 nm. In addition, their CD spectra suggested that oxidation led to a

460

gradual loss of α-helix and β-sheet structures. Furthermore, it was found that these conformational

461

changes decreased the water holding capacity and gel strengthen of soy protein gel.

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4.3 Enzymatic treatments

464

Compared with physical and chemical treatment, enzymatic modification is more acceptable for

465

improving the functional properties of proteins due to milder process conditions required, easier

466

control of the reaction, high efficiency of modification and less formation of by-products. Among the

467

enzymatic modification, the enzymatic proteolysis, cross-linking and deamidation are commonly

468

used. Proteolysis can result in hydrolysis of peptide bonds and affect proteins primary structure.

469

Therefore, secondary and tertiary structural changes result from proteolysis are not discussed here.

470

Unlike the proteolysis process, transglutaminase (TGase) could catalyze intra- and intermolecular

471

isopeptide bonds cross-linking by an acyl transfer reaction between glutamine (acyl donors) and

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19

ACCEPTED MANUSCRIPT lysine residues (acyl acceptors) of the proteins. Currently, a number of researches reported that

473

TGase could be utilized to improve the textural properties of various proteins and spectroscopic

474

techniques are thus used to monitor protein conformational changes. For example, Herrero et al.

475

(2008) used Raman spectra of amide I and amide III bands to analyze structural conversion of

476

cross-linked meat proteins with different amounts of TGase. A significant decrease in α-helices

477

accompanied by an increase in β-sheets and turns percentages upon addition of TGase was observed.

478

Herrero et al. (2008) also observed positive correlation of springiness with β-sheet structure and

479

negative correlation with α-helices content, positive correlation of adhesiveness with α-helices and

480

turns and negative correlation with β-sheets, and positive correlation of hardness and springiness

481

with turns. Furthermore, studies on Trp band at 759 cm-1, Tyr doublet ratio (I850/I830), Raman bands at

482

1450 cm-1 and 2935 cm-1 suggested an alteration of tertiary structure of meat proteins. In another

483

study, CD spectra were analyzed to assess the TGase-induced structural alteration of soybean

484

proteins. The ellipticity around 195, 208 and 216 nm decreased in TGase cross-linked soybean

485

protein, showing that TGase modification induced a more random secondary structure of soybean

486

protein (Song & Zhao, 2014).

487

In recent years, protein-glutaminase (PG) deamidation has gradually gained more and more attention

488

because it is more desirable than chemical deamidation. Miwa et al. (2013) reported that

489

PG-deamidated WPI tended to form a soft texture gel with a higher water-binding capacity, which to

490

some extent was attributed to the structural changes and would be meaningful for practical uses.

491

Structural analysis of WPI by using CD spectroscopy and fluorescence spectroscopy revealed that

492

the partial disruption of the tertiary structures of WPI proteins with respect to Trp residues shift to a

493

more-polar environment by the electrical repulsion of the negative charge derived from carboxyl

494

groups under PG deamidation.

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495

496

5. Advantages and Limitations 20

ACCEPTED MANUSCRIPT Spectroscopic techniques including FTIR, Raman, CD, fluorescence, and UV spectroscopy have

498

been successfully applied for evaluating conformational changes in proteins. In contrast to their

499

traditional counterparts, known to reflect food protein denaturation indirectly, spectroscopic

500

techniques present considerable advantages. For example, FTIR spectroscopy provides high-quality

501

spectra with spectrum in the region of 1600-1700 cm-1 highly sensitive to alterations in secondary

502

structure. Raman spectroscopy can obtain information about molecular vibrations related to

503

secondary structures, side chains of proteins and interference resulting from H2O is rare in Raman

504

spectra. In addition, it is feasible to analyze samples in many cases with fewer sample preparation

505

procedures, thereby providing the potential for direct, non-destructive, and faster detection of

506

conformational changes in situ. CD spectroscopy is uniquely sensitive to the detection of protein

507

conformational changes at a low concentration. Besides, CD spectra in the far-UV and near-UV

508

region can provide some useful information related to protein secondary structures and tertiary

509

structures, respectively. On the other hand, UV spectroscopy and fluorescence are inexpensive and

510

easy to operate, and can effectively monitor changes in the tertiary structure of proteins. In particular,

511

data acquisition in fluorescence spectroscopy occurs within nanoseconds, thereby making it possible

512

to investigate in-depth thermodynamics involving multiple experiments under different conditions.

513

Table 2 provides a summary of the advantages of these spectroscopic techniques. It should be noted

514

that other methods, including differential scanning calorimetry (Kazemi et al., 2011), sodium dodecyl

515

sulfate-polyacrylamide gel electrophoresis (Perreault et al., 2017), scanning tunneling microscopy

516

(Rinke et al., 2014), hydrogen/deuterium exchange mass spectrometry (Li et al., 2008), small-angle

517

X-ray scattering and multi-angle laser light scattering in conjunction with a size exclusion

518

chromatography (Zhao et al., 2012) could be used with spectroscopies complementarily, which offers

519

further advantages.

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520

However, certain limitations are associated with some of these spectroscopic techniques. Notably,

521

reliable evaluation of conformational changes in protein is only possible with high sample purity and 21

ACCEPTED MANUSCRIPT strictly defined environmental conditions. Most food systems comprise of complex matrices,

523

containing not only proteins but also some other components such as lipids, starches, pigments, etc.

524

which interfere during analysis and overlap the information of protein spectra, posing difficulties in

525

monitoring protein conformation in real food systems. Even for proteins with high purity, the FTIR

526

spectrum of protein in aqueous conditions may experience interference by H2O. Although this

527

interference can be eliminated by using D2O solution, the H-D substitution may change the protein

528

structural characteristics somewhat in comparison with its native state. In some cases, Raman

529

spectroscopy is greatly restricted by inherent weaker Raman scattering and stronger disturbance of

530

biological fluorescence, which could hamper the gaining of high-resolution Raman spectra. Besides,

531

lengthy laser radiation can generate heat that may alter the conformation of samples, thus affecting

532

the measurement accuracy. In the case of CD, UV and fluorescence spectroscopies, only diluted

533

protein samples can be analyzed. However, diluted samples are not the usual concentrations of

534

protein in food products. Therefore, the sample preparation procedure will be time-consuming. In

535

addition, CD spectra can only provide low-resolution secondary structural information because the

536

accuracy of this technique highly depends on the reference databases verified by other techniques.

537

On the other hand, another drawback of FTIR, Raman and CD spectrometers is their high cost in the

538

instruments.

539

6. Conclusions and future trends

540

In this review, the principles and recent applications of five spectroscopic techniques including FTIR,

541

Raman, CD, UV and fluorescence spectroscopies for protein conformation evaluation are described.

542

Although these techniques cover several advantages for measuring the secondary or tertiary

543

structures, some challenges still exist to realize protein conformation detection in real foodstuffs due

544

to their complex components. Therefore, spectroscopic solutions for protein conformational

545

monitoring of complex food systems should be further developed in the future.

546

Noteworthy, there are increasing studies of Raman spectroscopy to evaluate protein conformational

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522

22

ACCEPTED MANUSCRIPT changes in meat products. This broadens the perspective to detect proteins in food products with few

548

preparation procedures, and provides the potential for on-line and in-situ monitoring protein

549

conformational changes in the course of different types of treatments, which has practical

550

significance for food protein processing. On the other hand, due to the inherent weak Raman

551

scattering effect and strong biological fluorescence interference of Raman spectroscopy,

552

development of new techniques such as surface-enhanced Raman spectroscopy might a promising

553

way to gain a spectrum with strong signals. Considering the use of far-UV CD for protein secondary

554

structure analyses greatly depends on the reference databases, consequently, amelioration of the CD

555

spectrum processing software and computational approaches, as well as establishment of more

556

enormous and accurate reference databases should be considered in the future work. Fluorescence

557

and UV spectrophotometers have relatively low cost with simple operations and future research

558

interests can be focused on software improvements and overlapping spectral processing.

559

It should be pointed out that any spectroscopic technique should not be used alone. In terms of UV

560

and fluorescence spectroscopies, their limitations on the detection of peptide backbone structures

561

should be compensated for other complementary approaches, and combined spectroscopic

562

techniques are promising for increasing prediction accuracy of protein conformations transformation.

563

Apart from the aforementioned techniques, development of other emerging spectroscopies such as

564

using electromagnetic spectra in terahertz (THz) frequency ranges (0.1 to 10 THz) for protein

565

conformation measurement is attractive in future work. Although fundamental research and

566

applications of THz spectroscopy for protein conformational monitoring are still in its infancy, with

567

the development of THz sources and detector, this technique could be a novel and powerful

568

nondestructive technique with great potential for denatured proteins detection. It is hoped that future

569

studies could develop robust solutions for proteins conformational monitoring for the food industry.

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570 571

Acknowledgments 23

ACCEPTED MANUSCRIPT The authors are grateful to the International S&T Cooperation Program of China (2015DFA71150)

573

for its support. This research was also supported by the Collaborative Innovation Major Special

574

Projects of Guangzhou City (201508020097, 201604020007, 201604020057), the Guangdong

575

Provincial Science and Technology Plan Projects (2015A020209016, 2016A040403040), the Key

576

Projects of Administration of Ocean and Fisheries of Guangdong Province (A201401C04), the

577

National Key Technologies R&D Program (2015BAD19B03), the International and Hong Kong -

578

Macau - Taiwan Collaborative Innovation Platform of Guangdong Province on Intelligent Food

579

Quality Control and Process Technology & Equipment (2015KGJHZ001), the Guangdong Provincial

580

R & D Centre for the Modern Agricultural Industry on Non-destructive Detection and Intensive

581

Processing of Agricultural Products and the Common Technical Innovation Team of Guangdong

582

Province on Preservation and Logistics of Agricultural Products (2016LM2154).

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

Spectroscopic techniques

References

α-Lactalbumin and β-Lactoglobulin

High intensity ultrasound

CD

Chandrapala et al. (2012)

β‑conglycinin

Ultrasound + Maillard reaction

CD

Zhang et al. (2014)

Barley proteins

Deamidation

FTIR

Beer protein Z

Mashing, boiling

CD, FTIR

Black bean protein isolates

Ultrasound

CD, fluorescence

Bovine β-Lactoglobulin

High pressure microfluidization

Fluorescence, UV, CD

Bovine haemoglobin

Heat

UV, CD

Bovine lactoferrin

Heat

Fluorescence

Buckwheat protein

Maillard reaction

Chicken breast meat

High intensity ultrasound

Egg allergen ovotransferrin

Heat

Egg white protein

Pulsed electric field

Flaxseed protein isolate

High hydrostatic pressure

Gelatin

Phosphorylation

Immunoglobulins

High hydrostatic pressure

Milk protein concentrate

Acylation

Myofibrillar protein

High pressure

Myofibrillar protein

High pressure

Myofibrillar protein

High pressure

Peanut protein isolate

Maillard reaction

Phytohemagglutinin

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Zhang et al. (2015) Han et al. (2015) Jiang et al. (2014) Zhong et al. (2012) Coleman & Mitchell (2016) Stănciuc et al. (2013) Guo & Xiong (2013)

Raman

Li et al. (2014)

Fluorescence, CD, UV

Tong et al. (2012)

UV, FTIR

Qian et al. (2016)

Fluorescence

Perreault et al. (2017)

FTIR

Kaewruang et al. (2014)

FTIR

George et al. (2013)

Fluorescence

Shilpashree et al. (2015)

UV

Barrios-Peralta et al. (2012)

Fluorescence, UV, Raman, CD

Qiu et al. (2014)

Raman

Zhang et al. (2017)

Fluorescence, CD

Liu et al. (2012)

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

Acidification

CD, UV, fluorescence

Xu et al. (2012)

Skeletal muscle myoglobin

High pressure carbon dioxide

UV, CD, fluorescence

Yan et al. (2016)

Soy glycinin

High pressure

FTIR

Savadkoohi et al. (2016)

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UV, fluorescence

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FTIR, CD, Raman

Hu et al. (2015a)

Soybean glycinin

High intensity ultrasound

CD, fluorescence

Zhou et al. (2016)

Soybean protein

Glycosylation, enzymatic cross-linking

CD

Song & Zhao (2014)

Soybean protein isolate

High hydrostatic pressure

Fluorescence, CD

Li et al. (2012)

Soy protein

Ultrasound + enzymatic cross-linking

Raman

Soy protein isolate

Microfluidization

Fluorescence

Soy protein isolate

Low-frequency ultrasonication

CD

Soy protein isolate

Maillard reaction

Fluorescence, CD

Wheat gluten

Deamidation

FTIR, fluorescence

Liao et al. (2016)

Wheat gluten

Extrusion

FTIR

Wang et al. (2017a)

Wheat gluten

Heat

UV, fluorescence, FTIR

Wang et al. (2017b)

Whey protein

Heat

Raman

Blanpain-Avet et al. (2012)

Whey protein

Heat

Fluorescence

Ruffin 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 et al. (2013)

Whey protein isolate

Enzymatic deamidation

CD, fluorescence

Miwa et al. (2013)

Yak casein micelles

Acylation

Fluorescence, FTIR

Yang et al. (2015)

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High intensity ultrasound

Hu et al. (2015a)

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Hu et al. (2013) Zhuo et al. (2013)

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Shen & Tang (2012)

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Soybean β-conglycinin and glycinin

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

Protein state

Advantages

Limitations

FTIR

Secondary structure

Liquid or solid

Fast and Convenient Sensitive to conformational changes under various conditions Lack of dependence on the physical state of samples

High cost Strong IR absorbance of H2O H-D substitution affect protein properties

Raman

Secondary and tertiary structures

Liquid or solid

Non-destructive Convenient On-line and in situ Weaker H2O interference

High cost Inherently weaker Raman scattering Stronger biological fluorescence interference Thermal effect generated by the laser

CD

Secondary and tertiary structures

Liquid

Fast Low protein concentration

Fluorescence

Tertiary structure

Liquid

Economic and simple The data acquisition is quite fast Useful for in-depth thermodynamic studies

UV

Tertiary structure

Liquid

Fast Economic and simple

Time-consuming sample preparation procedures 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 Time-consuming sample preparation procedures Not suitable for direct measurements of samples in solid-state Unable to determine secondary structures Time-consuming sample preparation procedures Not suitable for direct measurements of samples in solid-state Unable to determine secondary structures Overlapping Tyr, Trp, and Phe spectra

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Figure captions Figure 1. Handling process of effect of pulsed electric field on IR spectra in amide I region of egg white protein powder; (a)-(f) represent peak-fitting of the secondary derived curves from IR

810

spectra for samples treated at 0, 5, 10, 15, 20, and 25 kV, respectively. Six peaks are observed

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for four components, which are attributed to α-helices (1657 cm-1), β-sheets (1611 and 1626

812

cm-1), β-turns (1673 and 1688 cm-1) and random coils (1642 cm-1) (Qian et al., 2016).

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Figure 2. Raman spectra of freeze-dried high intensity ultrasound (20 kHz at 400 W) treated

814

β-conglycinin at 0, 5, 20 and 40 min (Hu et al., 2015a). 500-550 cm-1: S-S stretching

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vibrational bands; 760, 880 and 1361 cm-1: Trp vibrational bands; 830 and 850 cm-1: Tyr

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vibrational bands; 1200-1340 cm-1: amide III bands; 1600-1700 cm-1: amide I band; 1440-1465

817

cm-1: C-H bending; 2800-3000 cm-1: C-H stretching.

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Figure 3. (a) Far-UV CD spectra and (b) near-UV CD spectra of myoglobin in solution at pH 7,

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absorbed at tricaprin oil/water interface and at hexadecane oil/water interface (Day et al.,

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2014).

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different ultrasonic treatment (Jiang et al., 2014). Figure 5. (a) the zero-order and (b) second-derivative UV spectra of lysozyme before and after

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Figure 4. Intrinsic fluorescence emission spectra for 0.15 mg/mL black bean protein dispersion of

pulsed electric field (PEF) treated at 35 kV/cm for 1200µs (Zhao & Yang, 2008).

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Protein modification is essential for obtaining optimal functionalities. Protein functionalities are closely related to its conformational characteristics.

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Spectroscopies for evaluating protein conformational changes are reviewed. Advantages and limitations of each spectroscopic technique are discussed

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Spectroscopic techniques should be used complementary.