d enaturation and a ggregation

The structural unfolding of proteins, commonly known as denaturation (Figure 3�3), is often done deliberately in the course of modifying proteins (Hardy et al�, 2008)�

In the initial steps of the denaturation mechanism, native proteins undergo intra- molecular transitions that differ from the native state only by minor conformational changes and then, protein unfolds, exposing hydrophobic amino acids buried in the native protein (Nishinari et al�, 2014)� These changes can be correlated with confor- mational changes found in protein secondary structures, which can be analyzed by self-deconvolution and curve fitting of Fourier transform infrared (FTIR) spectros- copy (Guerrero et al�, 2013; Ramos et al�, 2013)� Afterwards, through noncovalent interactions, such as ionic, van der Waals, and hydrophobic interactions, or covalent bonds, such as disulfide bonds, denatured proteins take part in irreversible intermo- lecular interactions, which result in the formation of aggregates (Pereira et al�, 2011)� The extent of these reactions can be controlled by several factors, such as pH, tem- perature, application of electric fields, or treatment time, and the behavior of proteins during those treatments is essential to control their characteristics and the desired properties for specific applications�

Proteins incorporate both acid and basic functional groups and thus, the net charge of the protein in solution depends on solution pH� At acid pHs, both carbox- ylate and amine functions are protonated, so the protein has a positive net charge� At basic pHs, amino groups exist as neutral bases and carboxyl groups as their con- jugate bases, so the protein has a negative net charge� At intermediate pHs, positively charged groups are balanced by negatively charged groups and the protein, on aver- age, has no net charge� This characteristic pH is called the isoelectric point (Chou and Morr, 1979)� At the isoelectric point, the proteins ability to interact with water is lower and thus, their solubility (Mojumdar et al�, 2011)� At extreme acidic and alka- line conditions, the strong repulsive forces or highly negative (pH > 12) or positive (pH < 1) charges are present along protein chains, preventing protein molecules from

Native protein Denaturated protein

associating and forming films (Flint and Johnson, 1981)� Mauri and Añón (2006) investigated changes in the solubility of soy protein films prepared at pH 2, 8, and 11 and observed that protein networks were maintained by the same type of interac- tions (disulfide bonds, hydrophobic interactions, and hydrogen bonds) at the different pH values analyzed, but films prepared at extreme pHs had a denser microstructure, indicating the formation of aggregates during film formation� Therefore, proteins are a kind of polyelectrolytes, and pH values influence their association and dissociation behavior in aqueous solution (Song et al�, 2011)�

In addition to pH, the functional properties of the films and coatings depend on other preparation conditions, such as temperature and time (Denavi et  al�, 2009; Pérez-Gago and Krochta, 2000)� Heating modifies the three-dimensional structure of proteins and the interactions in the native protein, exposing functional groups engaged in intramolecular bonding and thus, these groups become available for intermolecular interactions (Subirade et  al�, 1998; Wang and Damodaran, 1991)� Pérez-Gago et al� (1999) found that transparent films from native and heat-denatured whey proteins can be formed; however, some functional properties of films, such as solubility, are different due to the differences in intermolecular bonding upon dry- ing in both cases� Native whey proteins maintain their globular structure with most of the hydrophobic and sulfhydryl groups buried in the interior of the molecule and thus, cohesion relies mainly on hydrogen bonding, which leads to soluble films in water� In contrast, the intermolecular forces that promote cohesion in heat-denatured films involve hydrophobic bonds among the unfolded protein chains and covalent S–S bonding during drying, leading to insoluble films in water� Since some applica- tions of protein-based films may require water insolubility to enhance product integ- rity, whereas others may require water solubility before consumption of the product, the degree of protein denaturation and unfolding can be controlled by heating time and temperature, depending on the desirable film properties�

Although temperature is one of the key parameters when analyzing denaturation and aggregation processes and thus, a particular emphasis has been given to its effect, more recently the use of emergent processing technologies, such as elec- tric fields, has opened new perspectives for the development of innovative protein structures� By the use of electric fields, heating occurs by the transformation from electric to thermal energy, providing more uniform and rapid heating within the material in comparison with conventional technologies (Pereira et al�, 2010)� This approach avoids inducing an excessive denaturation of proteins� Xiang et al� (2011) analyzed the effect of pulsed electric fields (PEFs) on structural modification of WPI by using fluorescence spectroscopy� They found that PEF treatment increased the intrinsic tryptophan fluorescence of WPI, indicating changes in the polarity of tryptophan residues and resulting in a higher surface hydrophobicity� The effect of applying moderate electric fields (MEFs) has also been investigated on whey pro- teins� In comparison with PEF, MEF treatments use lower intensities� Pereira et al� (2011) found lower rates of WPI denaturation for MEF-treated samples compared with the samples treated by a conventional heating treatment� This behavior was kinetically supported by a lower rate constant (k) and denaturation reaction order (n)� Furthermore, the thermodynamic parameters obtained in the study indicated that the rate-determining stage was protein unfolding over aggregation, as reflected

by high activation energy (Ea) and enthalpy change (ΔH) together with a positive

entropy change (ΔS)� Although this treatment seems to be less aggressive for protein structure and shows a great potential for the manufacture of innovative protein- derived products with enhanced functional and technological properties (Rodrigues et al�, 2015), further research is still needed for a better understanding of the effect of electric fields on conformational changes in native proteins and interactions between denatured proteins�

3.3.2 PlastiCizing

Protein-based films and coatings without any additive have a brittle behavior, which makes processing difficult, but plasticization is often used for the modification of proteins to improve their processability and/or other properties demanded by food packaging materials (Aguirre et al�, 2013; Lian et al�, 1999)� Mechanical properties are extremely important, since protein-based materials must show adequate resis- tance and deformability to maintain their integrity during processing, handling, transport, and storage (Paes et  al�, 2010; Vieira et  al�, 2011)� For example, casein films produced without plasticizers are so brittle that it is not possible to determine tensile properties (Ghosh et  al�, 2009)� However, the addition of 10wt% glycerol imparts flexibility due to the fact that unordered random coil structures, predomi- nant in casein proteins, are transformed into helical structures, resulting in a more open molecular network (Siew et al�, 1999)�

Three theories have been proposed to explain the mechanism of the plasticizer effect (Sears and Darby, 1982)� According to the lubricity theory, a plasticizer is con- sidered as a lubricant to facilitate the movements of the macromolecules over each other; related to the gel theory, a plasticizer disrupts the polymer–polymer interac- tions, including hydrogen bonds, van der Waals, and ionic forces; and regarding the free volume theory, a plasticizer may depress the glass transition temperature by increasing polymer-free volume� The fundamental concept underlying these theories is that a plasticizer can interpose itself between the polymer chains and decrease the forces holding the chains together� Plasticizers exchange the intermolecular bonds among protein chains to bonds between the protein and the plasticizer, promoting conformational changes and resulting in higher deformability (Imre and Pukánszky, 2013)�

Although water is a very efficient plasticizer, compounds with a higher boiling point are preferred because they lead to more stable properties (Shi and Dumont, 2014)� The most commonly studied plasticizers are polyols (Bergo and Sobral, 2007; Gontard et al�, 1993; McHugh and Krochta, 1994; Park and Chinnan, 1995), such as glycerol, sorbitol, ethylene glycol, propylene glycol, polyethylene glycols, and polypropylene glycol� The differences in composition, size, structure, and shape of plasticizers directly influence their ability to plasticize proteins (Orliac et al�, 2003)� At the same plasticizer concentration, Jongjareonrak et  al� (2006) found that fish gelatin films plasticized with ethylene glycol showed the highest tensile strength, whereas glycerol-plasticized films showed the greatest elongation at break� Many studies have reported that plasticizing effect depends on the length of the plasticizer chain� Irissin-Mangata et al� (2001) observed that wheat gluten films plasticized with

high-molecular weight (Mw = 1500 and 3400 Da) polyethylene glycols were opaque, indicating that these plasticizers were not miscible with protein and resulting in brittle films; however, low-molecular weight (Mw = 200 and 400 Da) polyethylene glycols resulted in continuous and homogeneous films, although not flexible enough�

Other hydrophilic compounds, such as diethanolamine or triethanolamine, which contain amino functional groups and not only hydroxyl groups, have also been tested to plasticize protein-based materials (Rahman and Brazel, 2004)� However, glyc- erol is currently regarded as one of the most efficient plasticizers for proteins (Cao et al�, 2009)� Generally, smaller molecules are more easily incorporated into the pro- tein matrix and exhibit a more efficient plasticizing effect (Sothornvit and Krochta, 2001)� Plasticizers with different chemical structures produce different effects on film properties� The effect of different types and contents of plasticizers on mechani- cal properties of some vegetal and animal protein-based films are shown in Table 3�1� As can be seen, the combination of different plasticizers can cause a further improvement of mechanical properties� To explain the synergistic effect of oleic acid and glycerol on zein films at a molecular level, Xu et al� (2012) examined the sec- ondary structure of zein proteins by FTIR� Since the band corresponding to amide

TABLE 3.1

Mechanical Properties (Tensile Strength, TS, and Elongation at Break, EB) of Plasticized Protein-Based Films

Protein + Plasticizer TS (MPa) EB (%) References

Casein + 30wt% glycerol 6�5 ± 1�0 58�1 ± 9�0 Cho et al� (2014)

Casein + 40wt% glycerol 1�6 ± 0�2 101�0 ± 13�7

Bovine gelatin + 30wt% glycerol 17�0 ± 1�8 30�8 ± 4�1 Guerrero et al� (2014)

Bovine gelatin + 40wt% glycerol 7�8 ± 0�7 47�2 ± 5�1

Soy protein + 30wt% glycerol

Soy protein + 40wt% glycerol

Soy protein + 50wt% glycerol

4�1 ± 0�4 1�6 ± 0�3 1�5 ± 0�2 105�4 ± 13�3 145�5 ± 22�6 170�2 ± 18�5 Guerrero et al� (2010)

Soy protein + 30wt% sorbitol

Soy protein + 40wt% sorbitol

Soy protein + 50wt% sorbitol

7�5 ± 1�0 5�3 ± 0�6 2�9 ± 0�3 29�7 ± 9�1 105�6 ± 5�2 117�8 ± 22�9 Garrido et al� (2014)

Wheat gluten + 25wt% glycerol 3�1 ± 0�1 346�0 ± 10�7 Rafieian et al� (2014)

Whey protein + 25wt% glycerol 3�1 ± 1�0 19�2 ± 2�8 Wagh et al� (2014)

Whey protein + 50wt% glycerol 1�6 ± 1�0 61�7 ± 1�6

Whey protein + 30wt% sorbitol 12�1 ± 1�3 4�4 ± 0�5 Osés et al� (2009)

Zein

Zein + 10wt% tributyl citrate

Zein + 20wt% tributyl citrate

Zein + 30wt% tributyl citrate

Zein + 40wt% tributyl citrate

6�7 ± 0�4 17�8 ± 4�3 5�5 ± 0�6 5�4 ± 1�3 4�3 ± 0�2 2�0 ± 0�2 4�5 ± 0�5 3�0 ± 0�2 3�3 ± 1�0 2�4 ± 0�1 Shi et al� (2012) Zein Zein + 10wt% glycerol

Zein + 10wt% oleic acid

Zein + 10wt% glycerol + 10wt% oleic acid

5�7 ± 0�3 11�2 ± 0�9 7�6 ± 0�2 14�4 ± 0�9 1�4 ± 0�0 1�7 ± 0�0 2�1 ± 0�0 2�4 ± 0�0 Xu et al� (2012)

I depends on the secondary structure of the protein backbone, it can be used for the analysis of different secondary structures; specifically, the bands at 1620 and 1680 cm−1 _{are assigned to β-sheets (Guerrero et al�, 2014)� Xu, Chai, and Zhang also}

observed a decrease in the intensity of those two bands with the incorporation of a glycerol and oleic acid blend, indicating the change of the conformation of the pro- tein toward helical forms at the expense of β-sheets, as has also been shown in other works (Gillgren et al�, 2009)�

Wan et al� (2005) used scanning electron microscopy (SEM) to evaluate the cross sections of soy protein films plasticized with different combinations of plasticizers� They showed that the presence of large pores within the films resulted in elevated water vapor permeability (WVP)� Therefore, plasticizers must be added at a certain amount to obtain films with improved flexibility without significant decrease in bar- rier properties (Sothornvit and Krochta, 2001)� Recently, Shi et al� (2012) plasticized zein films with tributyl citrate to improve not only mechanical properties but also water resistance� They observed microstructural changes by atomic force micros- copy (AFM), which indicated a significant decrease in surface roughness with the addition of tributyl citrate up to 20wt%; however, phase separation started at higher contents of plasticizer� Commonly, fatty acids are also incorporated into the formu- lations with the aim to improve not only mechanical properties but also WVP� In those cases, caution must be taken when measuring WVP values, since the measure- ments are dependent on temperature� For example, samples formulated with oleic acid show lower WVP at low temperatures due to the crystallization of the fatty acid (Shi and Dumont, 2014)�

Regarding other barrier properties such as resistance against ultraviolet (UV) light, it is worth noting that films derived from proteins, such as casein, whey pro- teins, albumin, wheat gluten, and soy proteins, can resist UV light due to the pres- ence of disulfide bonds within the network and the presence of amino acids such as tyrosine, tryptophan, and phenylalanine, well-known sensitive chromophores (Li et al�, 2004)� Guerrero et al� (2011) observed that soy protein films show better bar- rier properties to UV light than synthetic films such as low-density polyethylene (LDPE) or orientated polypropylene (OPP; Figure 3�4)� Therefore, protein-based films could effectively prevent UV light transmission and product oxidation induced by UV light�

In addition to mechanical and barrier properties, the incorporation of phenolic acids as plasticizers can provide films with antioxidant and/or antimicrobial prop- erties� Arcan and Yemenicioglu (2011) incorporated gallic acid into zein films and observed that this phenolic acid could increase the film flexibility, maintain film integrity following hydration, and show antimicrobial activity�