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Advances in Understanding the Molecular Structures and Functionalities of Biodegradable Zein-based Materials Using Spectroscopic Techniques: A Review Hazal Turasan, and Jozef L. Kokini Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01455 • Publication Date (Web): 14 Dec 2016 Downloaded from http://pubs.acs.org on December 15, 2016
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Biomacromolecules
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Advances in Understanding the Molecular Structures
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and Functionalities of Biodegradable Zein-based
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Materials Using Spectroscopic Techniques: A
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Review
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Hazal Turasan, Jozef L. Kokini*
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Department of Food Science, Purdue University, 47907 West Lafayette Indiana
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Abstract
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Zein’s amphiphilic properties, film forming capability, and biodegradability make zein a highly
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demanded polymer for fabrication of packaging materials, production of drug carrier
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nanoparticles, scaffolds in tissue engineering, and formation of biodegradable platforms for
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biosensors including microfluidic devices. Zein properties can be improved by chemical
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modifications which are often analyzed with spectroscopic techniques. However, there is not a
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consensus on the structure of zein. For this reason, in this review the aim is to compile the recent
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studies conducted on zein-based products and compare them under five main spectroscopic
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techniques; Fourier Transform Infrared Spectroscopy (FTIR), Raman Spectroscopy, Circular
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Dichroism (CD), X-Ray Diffraction (XRD) and Atomic Force Microscopy (AFM). This review
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serves as a library of recent zein studies and helps readers to have a better perception of
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contradictions in the literature to take their studies one step further.
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Key words: Zein, biodegradable, 3D structure, spectroscopic properties, AFM, SPR
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1. Introduction
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Zein comprises about 50% of corn proteins and is the main storage protein found in corn 1. It is
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a prolamin which is insoluble in water unless water is a part of the diluting solvent in other
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alcoholic solutions2. The most commonly used solvent is 70% ethanol in water and other common
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solvents are aqueous methanol solutions, aqueous or glacial acetic acid solutions or aqueous
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propanol solutions. 2
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The amino acid sequence of zein has been studied by many researchers over the years and is
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well known1,3. However, there is no consensus on the tertiary structure of zein so far. Depending
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on their structural differences, zein proteins are categorized under four types as α-, β-, δ- and γ-
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zein with α-zeins being the most abundant. Just like its tertiary structure, the secondary structure
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of α-zein has also been extensively studied with various spectroscopic techniques. There are great
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variations between the secondary structure analysis results of studies. However, given that zein is
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a biological material that can be obtained from different varieties of corn and with different
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extraction methods, it would be highly optimistic to expect perfectly matching secondary structure
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content results. Also, as will be seen further in this paper, the sample preparation methods can
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cause variations in the results. Keeping these in mind, however, some of the inconsistencies in the
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literature comes from different interpretation ways of spectra. Clearly there is no consensus on
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how to analyze data to calculate secondary structure contents of zein. One of the aims of this paper
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is to show these differences by comparing studies.
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Due to its unique properties, zein has a very wide range of applications. Zein is in high demand
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for use in biodegradable packaging coating and packaging applications due to its film forming
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ability. Also, due to its high hydrophobicity, it is commonly used as moisture resistant layer in
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bilayer packaging production4. Zein is also an electrospinnable and extrudable protein, which
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allows it to be used as a drug carrier5,6. Other uses include adhesives, coatings, ceramic, cosmetics
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and textile applications and continue to grow1,7. For these applications, incorporation of different
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substances to zein matrix is needed. The verification of these complex formations are usually done
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with spectroscopic techniques in which chemical structures, secondary and tertiary structures and
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surface properties are tested. However, the wide variety of these studies necessitates a holistic
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understanding of how zein’s behavior changes under different conditions and how these
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interactions affect its functionality.
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The aim of this paper is to combine and critically compare the recent studies which play a
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significant role in understanding the conformational structure of pristine zein and its interactions
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with various components in complex formations. The studies were compared under five main
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analysis techniques for a better perception of the contradictions in the literature. Studies which
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include the secondary structure analyses and the chemical bond formations under different
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complex formations were reviewed under Fourier Transform Infrared Spectroscopy (FTIR),
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Raman Spectroscopy and Circular Dichroism (CD) subtitles separately. The comparison of surface
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properties of zein and other zein-based products were discussed in Atomic Force Microscopy
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(AFM) and the proposed models for the tertiary structure of zein were thoroughly compared in X-
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Ray Diffraction (XRD) parts. The organization of this review enables quick access to papers with
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specific techniques applied on zein. Also the discussion of the results of different techniques helps
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to understand how results are interpreted in the literature.
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The applications discussed in this paper will help researchers to view the successful
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modifications done on zein-based products in the literature and will help them produce superior
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materials with much better characteristics. With the proper combination of the treatments
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discussed in this paper, zein-based materials with either very elastic or very stiff structures, super
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hydrophilic or super hydrophobic surfaces, much smoother surfaces, high transparency or other
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demanded properties can be achieved.
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2. FTIR studies of conformational transitions resulting from different modification procedures of zein and zein-based products
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Fourier Transform Infrared Spectroscopy (FTIR) is one of the first choices of experimentalists
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to have a better understanding of chemical structure changes including bond formations occurring
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during various processes that include extrusion, encapsulation, electrospinning, plasma treatment,
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nanoparticulation and others which result in crosslink formation, changes in the distribution of
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hydrophilic and hydrophobic bonds and inevitably impact secondary structure. FTIR data becomes
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very valuable when it is used with other complementary spectroscopic techniques to detect
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chemical changes. FTIR spectroscopy has become very popular in understanding the
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characteristics of zein itself and the interactions of zein with other molecules in zein based
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materials.
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2.1 Effects of different zein solvents
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For almost all the applications of it, zein has to be dissolved in solvents. Therefore understanding
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the conformational changes of zein in solution is crucial. The effects of different solvents on the
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chemical organization of zein films has been studied with FTIR8. The effects of different alcohol
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solutions on chemical changes and their impact on mechanical properties, water permeability and
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surface hydrophilicity were evaluated using different concentrations of ethanol or isopropanol as
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solvent. Dissolving zein in both ethanol and isopropanol reduced the percentage of random coil
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structures observed at 1640-1650 cm-1 to zero and increased ordered α-helix conformations
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observed at 1650-1660 cm-1 and β-sheets observed at 1610-1640 cm-1. Isopropanol caused a higher
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β-turn content observed at 1660-1700 cm-1 than ethanol. It has been proposed that the disordered
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structures like β-turns expose the hydrophobic sites more than ordered structures and increase the 5
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hydrophobicity. However, films prepared from isopropanol solutions were more hydrophilic than
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the films with ethanol contradicting their hypothesis. Also, there are many studies proving that α-
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helix structures are linked through glutamine β-turns which are hydrophillic9,10. Also increasing
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exposure of β-turns has proven to increase the surface hydrophillicity of the films11,12. Their results
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are compatable with the latter hypothesis. The addition of glycerol as plasticizer decreased the β-
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turn fraction significantly which lowered the number of hydrogen bonds between proteins,
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resulting in more free volume and more random coil structure formation8. Increasing concentration
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of glycerol increased the ability of zein films to elongate before rupture from 20% to 40% and
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increased the tensile strength of the films from 10 MPa to almost 50 Mpa up to a concentration of
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20% w/w beyond which tensile strength and the ability to elongate started decreasing. This
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changing trend in mechanical properties around 20% glycerol concentration may be explained by
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the critical plasticization point. As Athamneh et al. explained in their study, proteins have critical
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plasticization points at which the secondary structure contents start changing course13. For zein,
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the critical glycerol plasticization point was found at around 16% w/w. However, the findings of
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Athamneh et al. show that, β-sheet content increase until critical plasticization point and then start
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decreasing as glycerol concentration increase. These findings are inconsistent with findings of
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Chen et al.8.
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These results are in agreement with the FTIR results of plasticized films because increasing free
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volume is expected to introduce more mobility to the polymer enabling the polymer molecules to
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elongate more under the same stress8. Also increasing glycerol concentration increases the
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accumulation of the glycerol clusters around the binding sites of glycerol and zein which
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strengthens the association of glycerol with zein. As a result, the tensile strength of the films
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increased. Increasing alcohol concentration in water during the preparation of the zein films also 6
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increased the tensile strength of the films up to 90% (v/v) alcohol concentration which was
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assumed to be due to increasing electrostatic interaction between the protein molecules8. In the
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case of alcohol concentrations above 90% (v/v), the reduced the tensile strength of films from for
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both ethanol and isopropanol solutions was attributed to increasing electrostatic interactions
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between zein molecules, causing repulsion. However, it has to be noted that, in a later study, zeta
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potential of zein nanoparticles in 50% (v/v) ethanol solution was found to be -46.0 mV, indicating
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repulsion between particles, which contradicts with the findings of this study14. The water
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permeability and the water absorption values of films prepared with either ethanol or isopropanol
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solutions decreased with increasing alcohol concentration up to 90% and then started increasing
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above 90% alcohol concentration, consistent with the free volume arguments leading to an
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explanation of how the mechanical properties change as a function of ethanol concentration.
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Isopropanol resulted in higher values of water permeability and water absorption.
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FTIR, as explained above, is a very useful tool to understand how secondary structures of zein
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vary in different solvents. The findings help not only understanding why zein behaves differently
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in solutions but also improving the final zein product.
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2.2 Investigating the interaction between zein and other molecules
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FTIR spectroscopy is also commonly used to understand bond formations occurring between
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zein and other molecules. For example Wheelright et al. studied the esterification of zein using
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methanol in a one-step reaction15. In this esterification process, specifically the amide groups of
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glutamine and asparagine in zein structure were methylated since these amide groups are in high
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concentrations in zein and are good candidates to create bonds with methyl groups. The results of
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FTIR experiments showed that the secondary amides of zein were not affected by the esterification
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process. However, the changes in the intensity of the relevant peaks confirmed the replacement of 7
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the primary amide groups of zein with methoxy groups indicating that the esterification process
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was successful.
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Zhang et al. also used FTIR spectroscopy to validate the successful blending of silver and zein
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molecules16. These blends show antimicrobial properties due to silver particles. FTIR spectra of
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zein-silver composites were compared to zein spectra. The disappearance of the peaks at 2987-
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2900 cm-1 in the composites, which show C-H stretching, and the appearance of new peaks at
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1297-1303 cm-1, which show Ag+-N bonding, confirmed the interaction between silver molecules
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and zein molecules.
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In another study, the formation of microspheres from soy/zein protein blend was investigated
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and the characteristics of blend microspheres were compared to pure soy protein or pure zein
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microspheres using FTIR17. The results showed that when microspheres were formed from zein
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only, the α-helix content increased while β-sheet content decreased, α-helix being the dominating
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structure. On the other hand, in soy protein microspheres, β-sheet content was dominating the
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secondary structures. The blend microspheres had higher α-helix content than β-sheet content,
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which changed however with the concentrations of zein and soy protein. With increasing zein
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concentration in the blend, α-helix content increased, and with increasing soy protein concentration
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β-sheet content increased. XRD experiments supported FTIR findings and also revealed that in
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blend microspheres, zein showed more crystallinity than soy proteins and zein was miscible with
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the amorphous regions of soy protein molecules.
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The studies prove that FTIR is a very quick and easy method to explore the bond formations in
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zein-based complexes. This way, the reaction mechanisms of chemical interactions can be clearly
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understood.
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2.3 Characterization of chemical changes during plasticization
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The films produced from pristine zein show high brittleness unless plasticizers are added. The
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chemistry of molecular interactions between zein and plasticizer molecules has been explored by
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many researchers using FTIR spectroscopy. In a study of the interactions of different plasticizers
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with zein using FTIR, it was found that water and glycerol hydrogen bonded with the amide groups
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of zein when they are used as plasticizers18. On the other hand, 2-Mercaptoethanol was bound to
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zein through the breakage of disulfide bonds which caused almost no detectable changes in the
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FTIR spectra. Similarly, building on the pioneering studies of Gioia and Guilbert, and Lai and
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Padua on the effect of glycerol and oleic acid on plasticization of zein, Xu et al. showed the
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plasticizing effects of oleic acid and glycerol on the mechanical properties of zein films both
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separately and in combination19–21. The contribution of this interesting study was to combine the
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molecular plasticization of oleic acid and the structural plasticization of glycerol to observe the
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differences in tensile strength, elongation and water permeability of the zein films. The chemical
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basis of changes occurring in these properties were explored with FTIR spectroscopy. Changing
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plasticizer ratios did not affect the distribution of secondary structures of zein. Increasing
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elongation of proteins are usually explained by increasing β-sheet content at the expense of α-helix
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content, which in this case does not seem to be valid for zein. Therefore, the authors concluded
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that plasticization of oleic acid and glycerol were achieved through modification of the
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supramolecular structures of zein.
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The choice of the right plasticizer and the explanation of plasticization mechanisms have always
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been a puzzle for researchers. The complete mechanisms have not been fully understood yet.
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However, these studies show that FTIR spectroscopy plays a key role for beginning to crack this
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puzzle and offer theories for mechanisms. 9
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2.4 Structural changes of zein due to electrospinning method and resulting chemical transformations
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Fabrication of zein fibers by electrospinning has also been studied using FTIR. For example,
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Torres-Giner et al. studied the characterization of electrospun zein nanostructures by examining
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the effects the acidity of the solvent (for which aqueous ethanol solutions were either acidified
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with acetic acid or alkalinized with sodium hydroxide) the polymer concentration, the flow rate of
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the solvent and the voltage applied during the procedure22. Electrospinning was compared with
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casting using FTIR and it was shown that, the evaporation of the solvent is more successful during
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electrospinning than solvent casting, demonstrated by the absence of the peak appearing around
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1241 cm-1 generated by the solvent. Also, amide I bands of the spectra showed that electrospun
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zein fibers obtained from acidified ethanol solutions have lower β-sheet content compared to either
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pure zein powder or solvent cast zein films. This study shows that due to the difference in solvent
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evaporation, electrospinning has an effect on the secondary structures of zein.
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2.5 Characterization of chemical changes during crosslinking
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Crosslinking is a common technique used to improve mechanical properties of protein films.
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Many studies have been done on crosslinking of zein films with many different crosslinking agents
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in the literature. For example Sessa et al. conducted a detailed study on the structural changes
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occurring during crosslinking of zein23. Crosslinking of zein was achieved with different
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concentrations of glutaraldehyde in acetic acid solutions. FTIR analyses on the films showed that
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the crosslinked zein films had new peaks at 951 cm-1, 858 cm-1 and 895 cm-1, all of which were
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attributed to the introduction of glutaraldehyde to the film matrix. Also it has been shown that
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glutaraldehyde caused a slight denaturation, which resulted in a modification of amide III band. 10
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Based on the interpretation of FTIR data, the authors proposed a crosslinking reaction series which
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consist of dihydroxy-piperidine zein complex binding covalently to self-oligomerized
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glutaraldehyde cyclic hemiacetals. Addition of glutaraldehyde also improved the mechanical
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properties of zein films such that tensile strength, elongation and young’s modulus of the films
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increased as crosslinker ratio of the films increased.
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When succinic anhydride, eugenol and citric acid were added as crosslinkers, opposite results
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were observed, such that the addition of any of the crosslinkers did not cause any major changes
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in the spectra24. The addition of eugenol was observed at 1955.68 cm-1 due to –C=C– stretching.
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Also peaks for –COOH groups were observed at 2910-29170 cm-1 and at 3033-3097 cm-1 which
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were considered to be due to bonding between the croslinker molecules and zein molecules. The
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differences were considered as “small” and therefore the addition of eugenol, citric acid or succinic
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anhydride were assumed not to cause significant changes in the chemistry and no pathway for the
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crosslinking reaction was proposed24.
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These studies show that the type of the crosslinking agent and its concentration have a significant
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effect on the chemistry of the final zein product as well as its mechanical properties. Therefore the
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choice of the crosslinker has to be made carefully for zein applications.
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2.6 Characterization of zein-dough complexes
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FTIR has also been used to examine zein-dough products. Sly et al. prepared zein dough products
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in which zein was used as a substitute for gluten25. They studied tensile properties and quality
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parameters of the zein-dough samples. The dependence of the quality of dough samples on the
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acidity parameters were also examined. FTIR analyses were conducted to better understand the
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molecular origins of changing tensile properties of dough with the addition of zein. The authors
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also investigated the effect of acidic conditions on the conformational changes in dough samples 11
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with FTIR. An increase in α-helix content was observed as the concentrations of the organic acids
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were increased, however, in general it was found that zein has less α-helix content when it is in
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dough form. The authors attributed this to the deamidation occurring during dough formation. Also
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the extensibility of the zein-dough samples were also attributed to β-sheet content of zein in dough
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form, which shows consistency with the findings of Xu et al., who attributed the elongation of zein
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films to their β-sheet content19. Mejia et al. also studied the rheology of zein dough and observed
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increased β-sheet content with increasing viscoelasticity with hydration26. These FTIR studies
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agree that while zein forms a dough, the β-sheet content increases significantly which also gives
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the dough its elongation.
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2.7 Studies examining decolorization of zein
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Recently, zein films have been considered as good candidates for microfluidic device platforms
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or platforms for cell cultures. Therefore, transparency of the films became an important issue. Han
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et al. studied the transparency and the swelling of zein films. Zein films were treated with heat and
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moisture (121⁰C and 100% RH) to see the differences between the transparency of the films27. The
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changes in the secondary structures of the films were characterized with FTIR and the results of
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the FTIR analyses were also confirmed by XRD analysis. FTIR results showed that heat and
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moisture treatment caused an increase in the β-sheet content and a reduction of α-helix content.
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The difference in secondary structures were attributed to the deamination of glutamine content of
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zein which plays an important role in the formation of antiparallel helices. Decolorization of zein
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was also investigated by using FTIR to assess changes in zein during its purification through
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column filtration28. Column filtration caused an increase in the α-helix content of zein which was
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observed as a shift from 1643 cm-1 to 1649 cm-1. The CD analyses also supported the findings of
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FTIR, indicating that purification step increased α-helix content by 5%. 12
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In brief, this study shows that heat and moisture treatment causes changes in zein’s secondary
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structure. As expected, heat treatment disrupts the bonds in highly ordered helical structures and
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therefore decrease the α-helix content. The increase in β-sheet content with the introduction of
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water to the system is consistent with dough formation results as explained above.
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2.8 Chemical changes occurring in zein during extrusion
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Due to its high stability in high temperatures, its film forming ability and mouldable
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characteristics, zein usage in extrusion processes has also increased significantly in recent years.
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The effects of extrusion processes have been investigated by many over the years. Selling
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investigated the effects of extrusion on the structural properties of zein using different
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spectroscopic techniques to provide accurate information29. FTIR was specifically employed to
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observe the cleavage of amino acids and the loss of primary structure of zein after extrusion.
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Changes in the secondary structures were also observed by FTIR analyses. The results indicated
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that, extrusion caused a loss of both the side chains of amino acid. It was also observed that
272
especially at temperatures higher than 220⁰C, extrusion caused a rapid narrowing in the bands of
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α-helix and β-sheet structures which indicates that both structures were decreasing in content
274
(Figure 1). The results of CD analysis confirmed the findings of FTIR analysis.
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Figure 1. IR spectra of zein + 5% water extrudates in 90% EtOH/water after extrusion at
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temperatures (⁰C) indicated. Reprinted from ref 29, Copyright (2010), with permission from
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Elsevier.
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On the other hand, it was suggested that hot melt extrusion had a slight reducing effect on α-
280
helix content of zein while it caused an increase in β-sheet content30. As can be seen from the FTIR
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results of dried zein powder and extruded dry zein powder in Figure 2, the general content of
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secondary structures do not show significant changes. These conclusions support the idea of using
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zein as a drug carrier since it is very stable at high temperatures of extrusion.
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Figure 2. ATR-FTIR spectra for dried zein powder (DZP) and dried ground zein extrudates
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(DGZE). Solid lines are the baseline corrected spectra of DZP (dark) and DGZE (grey). Dashed
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lines are the secondary derivatives of DZP (dark) and DGZE (grey). Detailed assignments of
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different forms of secondary structures of zein protein are shown in the secondary derivative
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spectra. Reprinted from ref 30. Copyright Springer Science+Business Media New York 2015.
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With permission of Springer.
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These studies show that there is no agreement on whether extrusion decreases or increases the
292
β-sheet content of zein since the findings show different results. However, this difference may be
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due to the different operating conditions of the extrusion processes. The β-sheet content might be
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increasing up to a temperature of 80⁰C as a result of decreasing α-helix content, which is the
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operating temperature in holt melt study30. Above that temperature, heat might be disrupting the
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bonds of both α-helix and β-sheet resulting in a loss of both structures as seen in the previous
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study29. Clearly, more studies should be conducted for a better understanding.
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2.9 Structural changes in zein during non-thermal applications
299
FTIR has also been employed to understand the effects of non-thermal processes on zein
300
structures. For instance, Pankaj et al. studied zein films after atmospheric cold plasma treatment31.
301
Atmospheric cold plasma treatment was supplied by applying the dielectric barrier discharge
302
(DBD) method on zein films in this study. The results of FTIR analysis at amide I band indicate
303
that atmospheric cold plasma treatment increased α-helix content which was observed by an
304
intensity increase at 1650 cm-1 (Figure 3). The examination of amide II band also supports this
305
result by showing a content shift from β-sheet to α-helix structures. These findings were also
306
consistent with wide angle X-ray Scattering Spectroscopy (WAXS) results which will be discussed
307
in a separate part of this review.
308 309
Figure 3. FTIR spectra of DBD plasma treated and control zein films. Treatments are shown as
310
voltage (kV)-time (min). Reprinted from ref 31. Copyright 2014 Wiley Periodicals, Inc., with
311
permission from John Wiley & Sons.
312
The effects of gamma irradiation and graft copolymerization on zein secondary structure was
313
analyzed by FTIR32. In this study, the blends of zein and polyvinyl alcohol (PVA) were treated 16
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with gamma irradiation and the chemical and physical changes were observed with FTIR. Also
315
the changes occurring during graft copolymerization with acrylic acid were analyzed. The
316
comparison between unirradiated zein and PVA and their blend showed that O-H stretching band
317
of the blend gave a lower intensity due to dehydration of PVA with the increasing content of zein.
318
Also the result indicated that gamma irradiation had a significant effect on both the crystallinity of
319
PVA and on the secondary structures of zein. Also the increasing effect of irradiation on both O-
320
H stretching and C-H stretching band intensities indicated a fragmentation of proteins which is
321
considered to have a positive effect on the distribution of PVA in the zein matrix. The successful
322
grafting of acrylic acid into zein/PVA blend was also confirmed with FTIR by the appearance of
323
new peaks due to acrylic acid interactions.
324
Studies covered in this section discuss the latest applications of FTIR spectroscopy and the
325
importance of FTIR in understanding the chemical changes occurring in zein under different
326
treatments. This coverage offers a comprehensive review and discussion of the behaviour of zein
327
during plasticization, crosslinking, dough formation, dissolvation or extrusion processes so that
328
engineering zein properties and functionalities would be easier to design improved products.
329 330
3. Raman studies of characterization of zein structures and its chemical interactions with other components
331
Raman spectroscopy is another commonly used spectroscopic technique for characterization of
332
organic materials. Similar to infrared spectroscopy, Raman spectroscopy is also used to observe
333
the vibrational, rotational and other types of motions occurring at the molecular level of samples
334
when the samples are excited by coherent laser light. In general, in the analyses of proteins, Raman
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is a very commonly used technique. However, there is only a limited number of studies that uses
336
Raman spectroscopy on zein and other zein based materials.
337
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3.1 Characterization of electrospun pristine zein fibers
338
Electrospun zein fibers have been analyzed by Raman spectroscopy. Selling et al. studied the
339
effects of different solvents on the structure of electrospun pristine zein fibers by using both FTIR
340
and Raman spectroscopy33. Raman spectra of zein fibers produced by glacial acetic acid solution
341
and 80% ethanol solution showed different peak formations suggesting that there are differences
342
in the environment of tryptophan and tyrosine amino acids. Also different solvent use was
343
considered to result in different secondary and tertiary structures in dissolved zein which also
344
changes the morphology of the fibers after drying. Plane polarized Raman results obtained from
345
parallel and perpendicular directions of the fibers did not show any differences indicating that the
346
environment around tryptophan and tyrosine are the same through the entire matrix. Raman results
347
are consistent with the birefringence results of fibers from acetic acid and ethanol solutions. Fibers
348
produced from acetic acid solutions had birefringence through the entire thickness while fibers
349
from ethanol solutions showed higher birefringence on the surface of the fibers. Faster drying on
350
the surface of the fibers with ethanol increased the birefringence of the outer layer of the fibers
351
impeding the solvent movement from inside out. This delay in drying allowed time for chains to
352
relax to a more un-oriented form causing significant structural differences between fibers produced
353
from acetic acid solutions.
354
3.2 Characterization of zein-chitosan films
355
The characterization of zein films are usually investigated by FTIR supported with additional
356
information from Raman spectroscopy. For instance, composite zein-chitosan edible films were 18
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357
characterized by using Raman spectroscopy34. Raman spectra of chitosan-zein films were
358
compared to those of pure chitosan and pure zein and no new peaks were observed in the spectra
359
of the blend. So, the interaction between chitosan and zein was explained through hydrogen
360
bonding between the amide groups of glutamine groups of zein and hydroxyl groups of chitosan.
361
Since the formation of these new hydrogen bonds did not create new functional groups, it only
362
increased the intensities of the peaks of C=O and N=H2 in the spectra of the blend. Also, the
363
characteristic peaks of cysteine and lysine in zein spectra disappeared in the chitosan-zein spectra
364
indicating that these amino acids are reacting with active groups of chitosan during blending.
365
Raman results of the blend films with different zein concentrations showed that increasing zein
366
concentration caused significant changes in the structure of the film as can be seen in Figure 4.
367
This structural change due to the increasing concentration of zein in the films decreased the
368
hydrophilicity of the films which also decreased the water vapor permeability of the films.
369
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Figure 4. Raman spectra of zein-chitosan edible films with changing zein concentration. Reprinted
371
from ref 34. Copyright (2013), with permission from Elsevier.
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372
Increasing concentration of zein in the films also decreased the elasticity of the films caused by
373
increasing discontinuity in the films34. This increased the chain mobility in the films resulting in
374
lower elastic modulus. On the other hand, it appears from the data that the zein-chitosan composite
375
becomes more extensible.
376 377 378
To our knowledge, there is no other study that investigated zein chitosan blend films with Raman spectroscopy. More studies will surely help to analyze zein-chitosan interaction. 3.3 Detecting the components of encapsulation systems
379
Raman spectroscopy also helps to analyze encapsulation processes. Citral that was encapsulated
380
within zein nanoparticles was detected with Raman spectroscopy35. To detect the existence of an
381
encapsulated core material in the nanoparticles, Raman spectra of the particles were compared to
382
those of pure citral and pure zein. The identification of citral was done based on the peaks
383
appearing at 1682 cm-1 and 1629 cm-1 and the identification of zein was interpreted from the peak
384
at 1656 cm-1. The results indicated that the broad peak appearing around 1645 cm-1 was forming
385
due to the presence of the peaks at 1682 cm-1 and 1629 cm-1 which validated the presence of citral
386
within zein matrix. In this paper Raman spectroscopy was strictly used to understand the existence
387
of a specific compound, citral. There were no structural analyses done on zein35.
388
Hu et al. also characterized zein nanoparticles formed by electrostatic deposition36. Pectin, a
389
hydrophilic polysaccharide was used as the shell material and the hydrophobic core material zein
390
was fortified with another hydrophobic bioactive molecule, curcumin. Raman was used to provide
391
more information about the molecular interactions of curcumin within the system in addition to 20
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FTIR. The Raman spectra of pure curcumin was compared to the spectra of both physical mixture
393
of curcumin-zein-pectin and nanoparticles formed with curcumin-zein-pectin. The results showed
394
that the intensity of the peak at 1600 cm-1 that is responsible for C=C stretching of aromatic rings
395
in pure curcumin spectra decreased during nanoparticulation. Also the peak at 1625 cm-1 which
396
shows C=C and C=O stretching of inter-ring chains changed its location to 1640 cm-1 after
397
encapsulation36. These results indicate that curcumin was interacting with zein through its aromatic
398
rings and inter-ring chains. Intensity changes at the peaks responsible for in-plane bending or
399
aromatic rings also supported the suggestion that curcumin was interacting with zein through
400
hydrophobic bonds. Raman results were consistent with FTIR results of the same samples. These
401
studies show that Raman is a reliable technique for the verification of encapsulation of materials.
402
3.4 Analyses of samples with Raman microscopy
403
Raman imaging technique is also used in the literature to analyze zein based products.
404
Electrospun zein fibers were investigated with Raman for the controlled release of the antibiotic
405
tetracycline37. The release of tetracycline from zein/Poly-ε-caprolactone fibers and the distribution
406
of the components within the fibers were analyzed by taking their images by Raman microscopy.
407
The images suggested a uniform distribution of all the components within the fiber matrix.
408
In another study, confocal Raman images of electrospun hordein/zein fibers fortified with
409
surface-modified cellulose nanowhiskers were analyzed in addition to their Raman spectra38. The
410
mechanical properties and stability of these fibers were related to the structural changes occurring
411
in films formed by either random alignment or uniaxial alignment. The results showed that while
412
randomly aligned fibers were having an almost uniform structure distribution, uniaxial aligned
413
fibers had a lower β-sheet content in some regions within the measured area, as measured using
414
FTIR. The distributions are shown in Figure 5. 21
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415 416
Figure 5. Confocal Raman images of randomly aligned hordein/zein (left) and uniaxially aligned
417
hordein/zein (right) fibers mapping the signal intensity at 1670 cm-1 (red: greater signal intensity
418
and β-sheet content, black: lower signal intensity and β-sheet content). Reprinted with permission
419
from ref 38. Copyright 2014 American Chemical Society.
420
Similarly α-helix content in uniaxially aligned fibers was lower than randomly oriented fibers
421
due to partially restricted rearrangement of extended hordein molecules which also showed
422
consistency with the FTIR results. The Raman spectra taken from the highest (*) and lowest (**)
423
signal intensity points of the Raman images from randomly aligned and uniaxially aligned fibers
424
respectively are shown in Figure 6.
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425 426
Figure 6. Raman spectra of * and ** defined in Figure 5. Reprinted with permission from ref 38.
427
Copyright 2014 American Chemical Society.
428
The intensity differences between the peaks that are responsible for tyrosine (850 cm-1 and 827
429
cm-1) revealed that in randomly aligned fibers tyrosine residues were mainly exposed on the
430
surface of the fibers allowing it to interact through weak or moderate hydrogen bonds while in
431
uniaxially aligned fibers they were mainly buried within the matrix leading to a stronger hydrogen
432
bonding38. The reason of this structural change is considered as the extra stretching force in
433
uniaxial alignment. The mechanical property results showed that, the structural changes occurring
434
during the uniaxial alignment of the fibers also increased the tensile strength of the fibers
435
significantly. This increase in tensile strength also increased the stability of uniaxially aligned
436
fibers when compared to randomly aligned fibers. Thermogravimetric analyses results showed that
437
increased phase separation of prolamins due to uniaxial alignment of the fibers decreased the size
438
of the protein aggregates, resulting in nano-sized hydrophobic behavior of these aggregates which
439
increased the stability of the fibers. These results show consistency with both Raman and FTIR
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results such that the mainly buried tyrosine residues were increasing the hydrogen bonding strength
441
which is also expected to increase the stability of the fibers.
Page 24 of 74
442
Raman imaging of electrospun zein fibers has also been studied by Fernandez and co-workers39.
443
The encapsulation efficiency of β-carotene in electrospun zein fibers and its oxidation stability was
444
studied. Even though Raman spectra of non-encapsulated and encapsulated β-carotene did not
445
show any differences, obvious differences were observed between the spectra of oxidized and
446
encapsulated β-carotene spectra indicating a successful encapsulation process. Confocal Raman
447
images (Figure 7) of zein/ β-carotene fibers at the characteristic wavenumber of β-carotene (1149
448
cm-1) were compared with the optical image of the fibers to investigate the distribution of β-
449
carotene. The results showed that β-carotene was well entrapped and was uniformly distributed
450
throughout the fibers. These results were also confirmed by Raman spectra taken from points A
451
and B, which are outside of the fiber limits, and point C, which is within the fiber limits in the
452
Raman images (Figure 7). The characteristic β-carotene peaks were better observed in point C
453
indicating a good dispersion inside the fibers. The presence of peaks in the spectra from point B
454
and the absence of them in the spectra from point A showed that there is an inhomogeneous
455
distribution of β-carotene outside of the fibers. The oxidative stability results also proved that
456
encapsulation of β-carotene in the zein matrix was achieved successfully.
24
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457 458
Figure 7. Confocal Raman image (top) of zein/β-carotene fiber mat mapping the intensity area of
459
the β-carotene band at 1149 cm-1 encapsulated in zein ultrafine fibers and the corresponding optical
460
microscope image (bottom). Reprinted from ref 39, Copyright (2009), with permission from
461
Elsevier.
462
The encapsulation of β-carotene in plasticized zein films was also studied by the same group in
463
comparison with other biodegradable films such as starch, soy protein and whey protein
464
concentrate40. Confocal Raman images taken at 1149 cm-1 showed that β-carotene was
465
agglomerating more in the zein films in comparison to the other films (Figure 8). This outcome,
466
with the assumption of β-carotene accumulation in the glycerol phase regions, indicated a phase
467
separation of glycerol within the zein matrix due to poor interactions between glycerol and zein. 25
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However, the assumption is contradictory since β-carotene should not be interacting with glycerol
469
due to its high hydrophobicity.
Page 26 of 74
470 471
Figure 8. Confocal Raman image of zein film with β-carotene and corresponding Raman spectra
472
of defined points. Reprinted from ref 40, Copyright (2011), with permission from Elsevier.
473
The studies show that Raman spectroscopy is an effective technique especially when it is
474
coupled with Raman microscopy. The information obtained from this technique helps getting
475
spectra from specific points of the samples which enables a thorough examination of the samples.
476
3.5 Analyzing zein with Fourier Transform Raman Spectroscopy
477
Zein films have also been characterized using Fourier Transform Raman (FT-Raman)
478
Spectroscopy due to its ability to reduce fluorescence of zein films. This allowed better reading of
479
spectra in some studies. For instance, FT-Raman was used to study the differences in the structure
480
of zein films when benzoic acid was incorporated into the films41. The results showed that the 26
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addition of benzoic acid did not cause any significant changes in the positions of the bands that
482
are responsible for the zein structure, indicating that secondary structure of zein remained stable
483
in the presence of benzoic acid. The analysis of the tyrosine peaks also showed that benzoic acid
484
is physically entrapped within the zein matrix through the process and not interacting with zein
485
through chemical bonding. This study also suggested that FT-Raman can be used to measure the
486
thickness of zein films. As can be seen in the Figure 9, even though the intensity of the peak at
487
1003 cm-1 was increasing with increasing thickness of the films, the peak at 84 cm-1 was found to
488
be inert to thickness changes. The correlation of these intensities to film thicknesses were also
489
validated with micrometer measurements.
490 491
Figure 9. FT-Raman spectra normalized at the band of 84 cm-1 for different concentration of zein
492
films: (a) 8%, (b) 10%, (c) 12%, (d) 14%, (e) 16%, and (f) 18%. Spectral intensity between 900-
493
1100 cm-1 was enlarged at the same proportion to observe easily the change of Raman intensity
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494
upon different zein thicknesses. Reprinted with permission from ref 41. Copyright 2005 American
495
Chemical Society.
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496
FT-Raman was also used to investigate the effects of the production of oral controlled-release
497
tablets42. The tablets, which contain calcium hydrogen orthophosphate, polyvinyl pyrrolidone,
498
theophylline and magnesium stearate, were prepared with both unground and ground zein to see
499
the effects of grinding on tablet preparation. Also the effects of compression during tablet
500
production were tested using FT-Raman. The results showed that in the characteristic peaks of
501
zein (Figure 10), no significant changes occurred. Also, both grinding and compression did not
502
alter the secondary structure of the proteins as indicated by the amide I and amide III bands. Lastly,
503
lack of change in tyrosine and phenylalanine peaks validated the structural stability of the tablet
504
during processing.
505 506
Figure 10. FT-Raman spectra of unground zein, compressed unground zein, ground zein and
507
compression ground zein. Reprinted from ref 42, Copyright (2008), with permission from Elsevier. 28
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508
FT-Raman was used to observe the differences in zein structure occurring during nutraceutical
509
product development43. FT-Raman experiments were conducted on different forms of tetracycline-
510
zein mixtures to understand the structural changes occurring in spray dried polylactidecoglycolic-
511
zein microparticles loaded with tetracycline. The spectra were compared to those of pure zein and
512
pure tetracycline separately. The comparison of spectra between zein films dispersed in water and
513
tetracycline loaded zein films dispersed in dichloromethane showed that the characteristic peaks
514
of zein side chains, such as tyrosine and phenylalanine, appear in all. The presence of the peak at
515
520 cm-1, which shows the existence of S-S bonds and the absence of a peak at 2500-2700 cm-1,
516
which shows the absence of S-H bonds suggested that the thiol groups in zein were in oxidation
517
form. This indicates that the disulfide bonds that gives stability to the structure of zein were not
518
disrupted and both spray drying and drug insertion do not influence the structure of the protein and
519
do not cause protein denaturation. Also the intensity ratios of peaks at 850 cm-1 and 830 cm-1
520
indicated that tetracycline is interacting with the tyrosine groups of zein through hydrogen
521
bonding. This bond formation was also validated by observing the spectra of the physical mixture
522
of tetracycline and zein.
523
Even though there is only a limited number of Raman spectroscopy studies on zein based
524
biopolymers, it is an effective method for analyzing the presence of specific bonds between
525
materials and validation of encapsulation success by detecting the encapsulated bioactive.
526
Secondary structure analysis can also be done using Raman spectroscopy, however, it is not as
527
prevalent as other methods like FTIR or CD, since the information obtained from specific peaks
528
in Raman are sometimes partially lost due to high fluorescence of proteins and the necessary
529
baseline correction steps.
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4. Circular dichroism studies that investigate the secondary structure of zein in different temperature, pH and solvent conditions
532
In circular dichroism (CD) spectroscopy, both left- and right- circularly polarized light is used
533
as the light source to induce absorption by the molecules studied. Depending on the chirality of
534
the tested material, some of the polarized light gets absorbed by the sample and some of it does
535
not. Therefore, it is important to have a sample with chirality to be able to get information about
536
the conformation. Also the solvent choice must be done carefully, such that the solvent does not
537
absorb the circularly polarized light.
538
Page 30 of 74
4.1 Understanding the structural organization of pristine zein
539
CD spectroscopy is usually used in parallel with FTIR and/or Raman spectroscopies to study
540
secondary structures. The classical study of Argos et al. is one of the earliest studies of zein
541
secondary structure with CD9. Far UV light was used as the light source and 70% methanol was
542
used as the solvent. Argos et al. used the results of Greenfield and Fasman with eight commonly
543
known proteins, whose secondary structures were well studied with X-ray crystallography, as
544
reference to identify the secondary structures of zein9,44. The results showed that zein has a high
545
α-helix content around 59% with 41% β-turn structures and 0% β-sheet structures. Their findings
546
of high helical content were validated with many other studies later on, but their claim of almost
547
zero β-sheet structure does not agree with later studies. In the model they proposed, zein is
548
composed of rod-like structures formed by α-helices which are connected by glutamine turns.
549
Probably the reason why the model includes only rod-like structures of zein is their zero β-sheet
550
content assumptions9. This is a major limitation of this model and while it is being continuously
551
referred to in the literature clearly it is not correct when the totality of the data is considered. 30
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552
Tatham et al. proposed an alternative structural model for zein, based on their CD data45. In their
553
study, they also used far UV range as the wavelength range and 70% methanol as the solvent for
554
zein. The results with 50% α-helix content were similar to those of Argos et al.’s9. However, the
555
remaining 50% of the structure was not discussed in that study probably because it would have
556
contradicted Argos et al.’s study9. The model these authors proposed has different dimensional
557
ratios (9.9 nm x 0.35 nm prolate ellipsoid) than that proposed by Argos et al., even though the
558
findings of CD experiments gave similar percentages of helical content of zein9. Later Cabra et al.
559
investigated the purity of α-zein obtained by ethanol extraction from corn flour by comparing it to
560
pure Z19 (19 kDa fraction of zein) protein46. Their CD data (Figure 11) gave a high α-helix content
561
in both pure Z19 zein and the α-zein solution in 70% aqueous methanol. The low β-sheet content
562
of α-zein Cabra et al. found is consistent with the study of Tatham et al. but not consistent with the
563
findings of Argos et al.9,45. Also, Cabra et al. did not assign a percentage for β-turn structures but
564
rather identified them as “undetermined” structures46. Also this study quantified the random coil
565
structures and found that while α-zein solution had around 8%, pure Z19 had 15.4% random coils
566
(Figure 11).
567
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568
Figure 11. CD spectra of pure Z19 (solid line) and for α-zein mixture in 70% (v/v) aqueous
569
methanol (dashed line) at 25⁰C and their secondary structure percentages. Reprinted with
570
permission from ref 46. Copyright 2005 American Chemical Society.
571
The purity of zein was also studied by using CD28. To understand the level of purity, zein was
572
analyzed before and after column separation. CD experiments were conducted with zein dissolved
573
in 90% ethanol solution. The authors suggested that the purification step increased the helical
574
content of zein in 90% ethanol. It must be noted that 90% ethanol might have also changed the
575
configurational distribution of zein. Their CD results were in agreement with their FTIR results.
576
Studies summarized in this section serve as the basis for explaining the structural organization.
577
Most of these early studies based their conclusions only on CD results, so there are inconsistencies
578
with subsequent studies which use multiple spectroscopic techniques. However, the contribution
579
of these early studies to the literature cannot be denied.
580
4.2 Analyzing the effects of solvent on zein conformation
581
In addition to FTIR studies, CD was also used to investigate the behavior of zein in different
582
solvents. The effects of water/ethanol concentration on the secondary structure of zein were
583
investigated by Bugs et al.47. The results showed that when the water content was increased from
584
30% to 72%, α-helix content slightly increased from 82% to 96% in one zein cultivar (CO3HS)
585
while another cultivar (BR451) did not show any differences in secondary structures (89%).
586
Overall, only minor changes were observed in the helical content of zein with increasing water
587
content of the solution. The stability in the secondary structure content of zein was explained with
588
the main-chain hydrogen bonds being protected from the solution and therefore remaining
589
unaltered. 32
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590
The effects of solution pH on the oligomerization of Z19 protein were also investigated with
591
CD48. When pH was increased from 3 to 6 in 70% ethanol solution, α-helix content decreased
592
while β-turn and random coil content increased. However, above pH 6 the opposite trend was seen
593
where the α-helix content of Z19 increased significantly while β-turn and random coil contents
594
decreased. At higher pH values than 6 the helical content significantly dominated the secondary
595
structures. This behavior was associated with the isoelectric point of the Z19 prolamine, which is
596
6.8. It was concluded that α-helix structure is favored while proteins have a less positive charge
597
and the reverse is true when there is more positive charge. The effects of temperature on
598
oligomerization of Z19 was also investigated with and without the effects of pH change48. The
599
sharp reduction in the ellipticity of α-helix band when the zein solutions were heated from 25°C
600
to 90°C indicated a complete loss of secondary structure through thermal folding at 90°C. Also,
601
when the solution was cooled back down to 25°C, the original CD spectrum could no longer be
602
obtained indicating an irreversible secondary structure loss. When the temperature was increased
603
by increments of 10°C each time, however, secondary structures were not completely lost but α-
604
helix content decreased while β-sheet, β-turn and random coil contents increased. The overall
605
conclusion of the authors was that heat induced the formation of disulfide bonds between the
606
polymers and therefore caused secondary structure changes during this irreversible
607
polymerization.
608
Selling et al. studied the effects of temperature, solvent and pH on secondary structure of zein
609
and showed that increase in temperature decreased the α-helix content in the secondary structure
610
which are located at 208 nm and 222 nm on the CD spectra49. Interestingly, after heat treatment
611
up to 70°C, the secondary structures were recovered when the protein was brought back to 25 °C.
612
These results are not consistent with the findings of Cabra et al., who found an irreversible 33
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613
secondary structural change after heat treatment to 90°C48. These results indicate that the
614
temperature where zein starts an irreversible denaturation begins between 70-90°C. The results
615
also showed that heat treatment caused some changes in the tertiary structures of zein proteins,
616
evidenced from the changes occurring at 268 nm on CD spectra49. Similarly, the changes occurring
617
in the tertiary structures were reversible.
618
The effect of different solvents on the secondary structures of zein were also examined 49. The
619
results showed that while the concentration of ethanol did not cause any differences in the
620
secondary structures, the type of solvent did because of changing polarity. Changing the solvent
621
did not have any significant changes in the tertiary structure of zein.
622
The effect of high pH on the secondary and tertiary structures of zein was also analyzed and the
623
results showed that high pH (12.7) caused a 60-90% reduction in helical content of α-zein as well
624
as a large (>90%) and non-denaturative reduction in the magnitude of CD ellipticity at 268 nm,
625
which are indications of secondary and tertiary structural changes, respectively49.
626
Acetic acid is not a commonly used solvent for far UV range CD experiments49. However, acetic
627
acid can be used in tertiary structure analysis of zein since the wavenumber range is above 250
628
nm. Li et al. showed that acetic acid can be used as a solvent for zein in CD measurements as long
629
as the wavelength is kept above 210 nm3. The peak responsible for α-helix structure usually
630
appears at 209 and 222 nm in CD spectra. However, with acetic acid α-helix peak appeared
631
between 227 and 237 nm3. With increasing zein concentration in the solution, the peak shifted to
632
237 nm and decreased in height which indicates that the α-helix content of zein decreased (Figure
633
12). Small Angle X-ray Scattering (SAXS) experiments that they conducted were in correlation
634
with these findings, suggesting that extra aggregation of zein with increasing concentration causes
635
unfolding of zein resulting in a loss of secondary structures. 34
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636 637
Figure 12. CD spectra of zein/acetic acid solutions at various zein concentrations. Reprinted with
638
permission from ref 3. Copyright 2011 American Chemical Society.
639
Briefly, this part summarized the studies which investigated the effects of solvent. The results
640
of these studies show that CD results support the findings of FTIR, such that the concentration of
641
zein and the concentration of the solvents, as well as the pH of the solvents affect the secondary
642
structure content of zein.
643
4.3 Characterization of zein fibers
644
Crosslinking of zein has also been investigated by CD. The interaction between zein and the
645
crosslinking agent, glyoxal, was studied with CD to analyze secondary structures in electrospun
646
zein fibers50. Both α-helix and β-sheet structures decreased in content when glyoxal was used as a
647
crosslinking agent, resulting in a higher unordered and non-specific structure concentration. They
648
suggested that the addition of glyoxal disrupted the hydrogen bonds which hold the secondary
649
structures stable, resulting in a decrease in secondary structure content. This finding is consistent
650
with the findings of FTIR studies of crosslinked zein, indicating that crosslinking has a reducing
651
effect on ordered secondary structures. 35
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Page 36 of 74
4.4 Understanding the effects of extrusion on secondary structure of zein
653
The extent of secondary structure change in zein as a result of extrusion was also investigated51.
654
The first pass from the single-screw extruder caused a slight reduction in both α-helix and β-sheet
655
content of zein, and the more obvious changes started to be seen after the forth pass from the
656
extruder (Figure 13). The first and the second passes affected the structure of zein the most.
657
Secondary and tertiary structures changed because of the formation of new disulfide bonds during
658
extrusion.
659 660
Figure 13. CD spectra of as-is zein with 10% triethylene glycol after passing indicated number of
661
passes through an extruder (A) and the ellipticity at 208 nm. Reprinted from ref 51, Copyright
662
(2013), with permission from Elsevier.
663
Zheng et al. also examined the effects of extrusion on zein structures by using CD52. Zein was
664
extracted from corn gluten meals and was treated with either extrusion only or extrusion in addition
665
to removal of starch. Extrusion was shown to have a decreasing effect on the α-helix content while
666
starch removal did not have any significant effect on it. This reduction in α-helix structure content 36
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was considered to be due to the disruption of the disulfide bonds during extrusion. However, this
668
idea contradicts the suggestion of Selling and Utt who stated extrusion results in formation of new
669
disulfide bonds51. α-helix content increased when extrusion was followed by removal of starch and
670
extrusion had a decreasing effect on both β-sheet and β-turn structure contents.
671 672 673
These studies show that, there is no absolute conclusion of extrusion effects on secondary structures of zein, based not only on FTIR results but also on CD results. 4.5 Characterization of zein particles
674
Zein nanoparticles have been growing interest lately, especially after the discovery of zein
675
utilization in pharmaceutical field. Therefore, it is of high importance to investigate the structure
676
of zein during particle formation. The self-assembly mechanism of zein particles, produced by
677
evaporation-induced self-assembly (EISA) technique was investigated by Wang and Padua with
678
CD53. Sonicated zein solutions in ethanol-water mixture were analyzed over time to observe the
679
secondary structural changes occurring during self-assembly. The self-assembly driving
680
mechanism of zein particles appears to be due to their amphiphilic characteristics. This self-
681
assembly is also induced by the changing polarity of the solution as evaporation occurs. Their CD
682
measurements of the evaporating solution which were taken at different time intervals showed that
683
α-helix content decreased while β-sheet and random coil structure contents increased (Figure 14).
684
It was concluded that evaporation of the ethanol from the solution induced the transformation of
685
α-helix structures into β-sheet structures in zein.
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686 687
Figure 14. CD spectra of zein during EISA and corresponding secondary structures. Reprinted
688
with permission from ref 53. Copyright 2012 American Chemical Society.
689
CD was also used to investigate the formation of a food grade colloidal particle complex from
690
zein and tannic acid54. The mechanism of the formation of this complex was analyzed to further
691
understand the structural changes occurring during the hydrogen bonding between zein and tannic
692
acid. The zein-tannic acid solutions were poured into Millipore water and then the solvents were
693
evaporated by stirring or rotary evaporator. The results showed that α-helix structure content
694
depended on pH levels. Especially at pH values 3 and 7 as opposed to pH value of 5, α-helix
695
content was higher (Figure 15). Therefore it was suggested that at pH values of 3 and 7, ionization
696
of hydroxyl groups of tannic acid caused the formation of electrostatic interactions with unstable
697
colloidal dispersions. However, at pH 5 tannic acid was in protonated form which led to stronger
698
hydrogen bonding between tannic acid and zein and less α-helix structure. The results obtained
699
from the formation of zein/tannic acid complex at different concentrations of tannic acid also
700
showed that, the concentration of tannic acid in the solution had a significant effect of the
701
secondary structure formation of zein (Figure 15).
38
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702 703
Figure 15. CD spectra of zein in 70% ethanol solution with different pH values (A) and with
704
different tannic acid (TA) concentrations (B). Reprinted with permission from ref 54. Copyright
705
2015 American Chemical Society.
706
The findings of these studies revealed that the evaporation of solvents during the particle
707
formation as well as the pH of the solvents have great impact on the secondary structure content
708
of zein particles.
709
4.6 Understanding the effects of ultrasonication on zein structure
710
The effects of two different ultrasonication treatments on the conformation of zein was studied
711
using CD55. Specifically, the degree of hydrolysis occurring in zein was tested by using sweeping
712
frequency ultrasound treatment and compared to fixed frequency ultrasound treatment. The peaks
713
appearing at 195 nm, 208 nm and 222 nm were all attributed to α-helix structure. The overall
714
analysis showed that while the fixed frequency treatment caused only a slight increase in α-helix
715
content and a slight decrease in β-sheet, β-turn and random coil contents, sweeping ultrasound
716
treatment caused an increase in all of the secondary structures. These results proved that the
717
molecular weight changes occurring during ultrasound treatment can be explained by the
718
secondary structural changes occurring within zein molecules. In a following study, the same 39
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719
group also investigated the effects of dual-frequency frequency-sweeping ultrasound treatment
720
effects on zein structure56. Unlike their previous study, they assigned the peak at 208 nm to α-helix
721
structure while the peak at 222 nm was assigned to β-sheet structure. Similarly, in the overall
722
analysis of secondary structures, it was seen that dual-frequency frequency-sweep ultrasound
723
treatment had an increasing effect on α-helix and decreasing effect on β-sheet content.
Page 40 of 74
724
CD is mainly used to analyze the secondary structure content of zein-based materials in the
725
literature and the information obtained from it is considered to be more valuable than secondary
726
structure findings of FTIR. The first models proposed for zein’s organizational structure were
727
based on CD results, as explained above. Effects of many different applications like
728
ultrasonication, dissolvation, extrusion and electrospinning on zein have been analyzed by CD,
729
however, there are variations in the interpretation of the data and there is no clear agreement yet.
730
It is clear that there is a need for more and in-depth studies of CD on zein which should also be
731
conducted along with other techniques for comparison and validation.
732
5. 3D organization investigation of zein with X-Ray Diffraction
733
XRD gives information about the structure of particles at the atomic level due to the small
734
wavelength of X-rays, which is about 1 x 10-10 meters57. Therefore in understanding the distances
735
between the zein molecules and angle of the bonds, XRD is a highly preferred method.
736
5.1 Studying the molecular structure of pristine zein
737
One of the earliest characterizations of zein with Small Angle X-ray Scattering (SAXS) was
738
done by Tatham et al.45. They analyzed α-zein proteins in 70% methanol with both SAXS and CD
739
spectroscopy. Their Guinier plot calculations showed that the radius of gyration of zein was 4.41
740
nm which is higher than that of other globular proteins (~1.5nm). This study also showed that zein 40
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has an elongated asymmetric structure in the form of a coil-like structure. Based on the results of
742
SAXS and CD experiments, they proposed a 3D model which shows some differences compared
743
to the previous model proposed by Argos et al.9,45. In their model Argos et al. suggested a length
744
to width ratio of 2:1 for a zein molecule which is not consistent with Tatham et al.’s model who
745
found 9.9 nm x 0.35 nm prolate ellipsoid structure45. Argos et al. proposed a secondary structure
746
which is exclusively α-helix with no β-sheet secondary structure9. Consequently the turn-helix-
747
turn repeat model Argos et al. proposed is considerably different than Tatham et al.’s model which
748
shows some β-sheet structures at the N- and C-terminal regions of zein molecules and β-turn
749
structures only around the C-terminal regions in addition to α-helix structures9,45. The reason for
750
these differences appear to be Argos et al.’s assumption of a globular structure for zein. In their
751
study Argos et al. calculated the secondary structures of zein, based on other globular protein
752
secondary structures and did not show any β-sheet content in zein9. Even though Tatham et al.’s
753
model is a more recent model with more reliable techniques, Argos et al.’s model is interestingly
754
more widely referred to because of its simplicity.
755
Matsushima et al.’s results also indicate that zein molecules have asymmetric structures with a
756
length of 13 nm in 70% ethanol solutions which is consistent with SAXS results of Tatham et
757
al.10,45. Based on radius of gyration calculations and measured dimensions, Matsushima et al. also
758
proposed a model for the organization of zein molecules (Figure 16)10. In the model, a zero β-sheet
759
content was assumed, consistent with Argos et al.’s model, and repeat units were assumed to
760
consist of 20 residues. The tandem repeat units were connected to each other through glutamine
761
turns similar with Argos et al. and Garratt et al., who proposed a wheel structure for zein with a
762
more compact form of helix structures9,58. Also a linear stacking of repeat units were proposed in
763
Tatham et al.’s study, similar to Argos et al. but different than Garratt et al.’s study. Unlike 41
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764
Argos’s, Matsushima’s model does not have a globular structure and the length to width ratio is
765
6:1 with dimensions a=13 nm, b=1.2 nm, c=3 nm. The calculated radius of gyration values are
766
different than previously proposed models presumably due to using 70% ethanol instead of 70%
767
methanol like Argos et al. and Tatham et al. did and due to different experimental parameters9,45.
768
The model proposed by Matsushima et al. is one of the latest attempts to understand the tertiary
769
structure of zein and is also widely accepted even though it has considerable differences with the
770
models proposed previously10.
771 772
Figure 16. Zein structural model proposed by Matsushima et al. Reprinted from ref 10, Copyright
773
(1997), with permission from Elsevier.
774
SAXS technique has also been used to analyze the conformation of the α-zein prolamin fraction,
775
Z1959. The results showed that Z19 has a smaller radius of gyration (3.8 nm) compared to the
776
estimates of Tatham et al. and Matsushima et al.10,45. Based on the SAXS results, another model
777
for zein was proposed (Figure 17)59. According to this model, Z19 forms itself in a hairpin
778
structure, in which the α-helix structures are connected by loops and/or β-sheets. This model is
779
very different from the models of Argos et al., Garratt et al. or Matsushima et al., who all proposed
780
glutamine turns as the linkage between helical structures9,10,58. The reason for this difference is the 42
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781
high β-sheet content that was obtained from FTIR measurements. The length of the molecule in
782
this model is approximately the same as the dimensions of Matsushima et al.. The calculated
783
dimensions of the hairpin model is 12-13 nm in length and 2-4 nm in the transverse direction. This
784
model also provide hints on the fiber forming abilities of α-zeins.
785 786
Figure 17. Zein structural model proposed by Forato et al., (a) top view, (b) side view, (c) same
787
as in (b) rotated by 90⁰. Reprinted with permission from ref 59 Copyright 2004 American
788
Chemical Society.
789
Another structural model for zein was proposed based on SAXS results47. In this model, a
790
distribution of amino acids along the helical surfaces which are packed in an antiparallel structure
791
was proposed (Figure 18)47. Their FTIR and CD results indicated zero β-sheet content and
792
therefore in their model an α-helix packing in prolate ellipsoid conformation with dimensions
793
a=13.8 nm and b=3.4 nm was assumed. The molecular dimensions are similar with the results of 43
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794
Tatham et al. and Matsushima et al. but the model they proposed is very different from all the
795
models proposed previously10,45.
Page 44 of 74
796 797
Figure 18. Zein structural model proposed by Bugs et al. Reprinted from ref 47, Copyright EBSA
798
2003. With permission of Springer.
799
By using the findings of previously discussed papers, Momany et al. proposed yet another model
800
for zein60. They used computational methods to simulate a 3D structure of the zein molecules. The
801
complete structure of Z19 was proposed to be consisting of nine α-helix structures in addition to
802
an N terminal segment (Figure 19). The helices in this model are in coiled-coil heptad structure.
803
The carotenoid lutein is bound to the core of the superhelix and therefore gives the characteristic
804
yellow color of zein which is strongly bound and really hard to remove. Axial ratio was assumed
805
to be 6:1 or 7:1 which is different than the models of Argos et al.’s and Garratt et al.’s9,58. Also the
806
coiled-coil heptad model offers a contrast to the linear organization of helices which are linked by
807
glutamine-rich turns. This model includes a more detailed organization of amino acids compared
808
to previous models.
44
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809 810
Figure 19. Zein structural model proposed by Momany et al. Reprinted with permission from ref
811
60. http://pubs.acs.org/doi/abs/10.1021/jf058135h
812
In this part, the most commonly acknowledged models for zein structure have been summarized.
813
It is clear that, despite the similar techniques used in these studies, the results show discrepancies.
814
Even between those, in which similar dimensions were found, the alignment of the molecules
815
differ from one model to another. Despite these opposing models, however, the generally accepted
816
model is linear stacking of rod-shaped helical repeat units. This model also makes it easier to
817
understand the interactions of zein with plasticizers as well as its behavior on surfaces with
818
different hydrophilicities.
819
5.2 Studying the solution behavior of zein
820
To understand the effect of zein concentration on the secondary structure conformations and the
821
rheological behavior of the solutions, SAXS and CD spectroscopies were used in acetic acid
822
solution3. Based on the previous models of zein, another model for zein, in which the whole amino
823
acid sequence of zein and the secondary structure analyses from the previous studies were taken
824
into consideration, was proposed3. In the model, Z19 component of α-zein was assumed to have
825
an oblate ellipsoid shape when it is folded with dimensions a=5.9 nm b=4.5 nm and c=2.3 nm
826
(Figure 20). The dimensions of Z19 in extended form were also given which are consistent with 45
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827
the results of Momany et al.60. Z22 was reported to have a globular shape with dimensions a=5.8
828
nm, b=5.0 nm and c=3.4 nm (Figure 20).
829 830
Figure 20. Zein structural model proposed by Li et al. Reprinted with permission from ref 3.
831
Copyright 2011 American Chemical Society.
832
SAXS results indicated that the scaling behavior of zein in acetic acid solution is similar to that
833
of a rod-like molecule3. Zein in acetic acid solution was found to have two different scaling regions
834
for both particle size and viscosity both having a threshold at 43 mg/ml where zein is in the dilute
835
solution region. Both the conformation of zein and the viscosity of the solution was not affected
836
by concentration change in this range. In concentrations higher than 43 mg/ml, the increasing
837
concentration was found to cause unfolding and overlapping of zein molecules which also affected
838
the particle size of the molecules and the rheological behavior of the solution. The same group also
839
studied the behavior of zein in ethanol solution in comparison with its behavior in acetic acid
840
solution using SAXS61. The results confirmed that like in acetic acid solutions, zein has an 46
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elongated conformation in ethanol solutions as well. However, it was seen that in the dilute
842
solution region (
