radiation processing of food proteins e · food proteins exert various functional properties for...

Trends in Food Science & Technology 30 (2013) 105e120

Review

* Corresponding author.

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Radiation processing

of food proteins e

A review on the

recent developments

Yau-Hoong Kuana, Rajeev Bhata,
Ankit Patrasb andAlias A. Karima,*
aFood Biopolymer Research Group, Food Technology

Division, School of Industrial Technology, Universiti

Sains Malaysia, 11800 Minden, Penang, Malaysia

(Tel.: D60 4 653 2268; fax: D60 4 657 3678;

e-mail: [email protected])bDepartment of Food Science, University of Guelph,

Guelph, Ontario N1G 2W1, Canada

Food proteins exert various functional properties for various

food products on their textural, sensory and nutritional proper-

ties. However without modification, the applications of these

food proteins are limited. Food irradiation is a physical method

of food preservation to retain the freshness and quality of the

food products. To date, the application of irradiation is not

only limited to food preservation, but also widely used in other

applications suchas cross-linkingof synthetic polymers andbio-

polymers, the enhancement of health promoting phytochemi-

cals, etc. Application of radiation has been extended to the

modification of proteins. Depending on the adsorbed radiation

dose or radiation exposure time, various effects can be achieved

resulting in the polymerization or depolymerization of protein

molecules. This review paper summarizes the recent progress

made on the application and the effects of ionizing and non-

ionizing radiation on the conformational changes of food pro-

teins. These changes could subsequently exert improvement

on the functional properties of food proteins. The information

documented in this review will be beneficial for further com-

mercialization and exploration of this exciting novel technology

on a pilot scale in food industries world-wide.

IntroductionThere is an old Chinese proverb that says, “May you live ininteresting times.” This proverb is best characterized by theinteresting innovative technologies developed to ease hu-man life. The application of new innovations is being intro-duced and explored in the field of food preservationcorresponding to the increasing consumer demand forfood with high quality and longer shelf life. The food in-dustry is currently in need of these innovative processingtechnologies in order to meet consumers’ demand of fresherand safer ready-to-eat products. The interests and demandsin sourcing of novel techniques in food preservation are dueto the persistent high food losses from infestation, contam-ination and spoilage; mounting concerns over food-bornediseases and growing international trade in food productsthat must meet strict import standards on quality and quar-antine (Robertson et al., 2004). However, increase of re-stricted regulations and complete prohibition on the useof a number of chemical preservatives for microbial controlin food, safety and wholesomeness become the main con-straint (Havelaar et al., 2010). The use of chemical preser-vatives might prevent the microbial spoilage, but thewholesomeness is yet to be identified upon long term con-sumption. Therefore, major emphasis is placed on thesourcing of food preservation techniques that ensure equalquality and safety, which has undergone minimal process,high nutritional quality and natural with minimal or nochemical preservatives. Technologies that essentially relyon non-thermal physicochemical characteristics to foodpreservation without the collateral effects of heat treat-ments while optimizing biological and sensory propertiesare being intensely studied and tested (Farkas, 2006;Wan, Coventry, Swiergon, Sanguansri, & Versteeg, 2009).One such procedure is the irradiation of foods.

Food irradiation is the process of exposing food to ion-izing and non-ionizing radiation to destroy microorgan-isms, bacteria, viruses, or insects that might be present inthe food (Farkas, 1998; Farkas & Moh�acsi-Farkas, 2011;Lacroix & Outtara, 2000). Concurrently, the chemicalchanges induced by radiation are minimal (Farkas, 2006).Further applications include sprout inhibition, delay in rip-ening, increase in juice yield, and improvement of re-hydration. In addition, radiation processing has been shownto reduce anti-nutritional components along with improvingthe nutritional and functional properties, and overall qualityof plant produce (Bhat, Ameran, Karim, & Liong, 2011;

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http://dx.doi.org/10.1016/j.tifs.2012.12.002



106 Y.-H. Kuan et al. / Trends in Food Science & Technology 30 (2013) 105e120

Bhat & Sridhar, 2008; Bhat, Sridhar, & Yokotani, 2007).Treatment of foods and agricultural products with ionizingradiations (e.g., gamma rays, X-rays, and electron beams) isincreasingly being accepted in Europe as well as AsiaPacific region in order to meet sanitary and phyto-sanitary requirements in the international trade (Kume,Furuta, Todoriki, Uenoyama, & Kobayashi, 2009;Luckman, 2002). Therefore, many countries have recog-nized irradiation as a useful technology for the reductionof pathogens for public health significance as part of overallgood manufacturing practice (GMP) and hazard analysiscritical control points (HACCP) systems (Luckman,2002). In the USA, a mandatory was set to label irradiatedfood with “Treated with irradiation” or “Treated by irradi-ation” and is required to use the RADURA-logo at the pointof sale (Ehlermann, 2009). However, a few countries allowits optional use; particularly the European Union.

The ionizing irradiation sources which are permitted foruse in food processing are gamma rays produced from ra-dioisotopes cobalt-60 (1.17 and 1.33 MeV) and cesium-137 (0.662 MeV), machine-generated electron beams(maximum energy 4e10 MeV), and X-rays (maximum en-ergy 5 MeV) (Codex Alimentarius Commission, 2003).Meanwhile, the non-ionizing irradiation refers to the elec-tromagnetic radiation that does not carry enough energyto ionize atoms or molecules; including ultraviolet rays(UV-A, B, and C), visible light and infrared. The most re-cent available statistical data on the status of food irradia-tion in the world was reported in 2009 (Kume et al.,2009); based on a survey performed by the Japan AtomicEnergy Commission Cabinet Office in 2005. The surveywas carried out using published data, questionnaire survey,and direct visits. Countries involved were divided into 4 re-gions: (1) America, (2) EU, (3) Asia and Oceania, and (4)Africa and others; these countries include Germany, France,Belgium, the Netherlands, Croatia, USA, Canada, Mexico,South Africa, India, China, Malaysia, Vietnam, Thailandand Ukraine. They reported that the quantity of irradiatedfoods in the world in 2005 was 405,000 ton and comprisedof 186,000 ton (46%) for disinfection of spices and dry veg-etables, 82,000 ton (20%) for disinfestations of grains andfruits, 32,000 ton (8%) for disinfection of meat and fish,88,000 ton (22%) for sprout inhibition of garlic and potato,and 17,000 (4%) of other food items that included healthfoods, mushroom, honey, etc. The processing of thesefood items by ionizing radiation in specific direction hasbeen regulated by the European Union since 1999; andthe relevant documents and reports can be accessed online(EU: Food Irradiation e Community Legislation). Mean-while, the analytical methods to identify irradiated foodhave been reviewed by Dr. Henry Delinc�ee (Delinc�ee,2002). With these methods, it is expected that the consumerconfidence and acceptance on irradiated food would be en-hanced. On the other hand, the history and future of foodirradiation have just been reviewed by Farkas andMoh�acsi-Farkas (2011).

Apart from disinfestations, irradiation can cause subse-quent changes in food components; especially in carbohy-drates, proteins, lipids and vitamins. However, theprimary changes on the physicochemical properties, suchas the involvement of direct versus indirect effect, are notunderstood. Therefore, studying the radiation chemistry offood components in model systems would provide a rationalbasis for extrapolating the results obtained to the complexsituation existing in food (Kempner, 2001; de Pomeraiet al., 2003). The advances in the radiation chemistry en-tailed the uniformity of the reaction mechanisms and per-mitted reasonable predictions to be made on the changesthat are likely to occur in food or food products. In lipids,it has been found that a relatively high dose of irradiationwould give rise to a relatively milder decomposition com-pared to decomposition produced by normal cooking tem-perature (Nawar, 1983). Concurrently, the reaction onglobular and fibrous proteins was also found to differupon irradiation (Delinc�ee, 1983; Stewart, 2001). The prog-ress in the radiation chemistry of carbohydrates showedthat degradation often occurred. A review on the effectsof radiation processing on starch has been reported byBhat and Karim (2009a). However, less degradation wasfound to occur in food than that of model systems(Stewart, 2001). The effects of irradiation in protein wouldbe discussed in more detail in this paper.

In the present review, we have focused on the effects ofradiation on protein properties that are relevant for food andfood-based industries. The previous reviews were eitherpublished more than 30 years ago, not up to date or not spe-cific to food applications (Delinc�ee, 1983; Simic, 1978). Atpresent, there is no recent systematic review on the impactof radiation on food proteins has been reported. Researchpertaining to the functional/physical characterization offood proteins exposed to radiations is highlighted in thepresent review, to provide insight into the present scenario,as well as to provide information for future research appli-cations by employing radiation treatments. The review hasbeen constructed as follows: a brief description on the func-tional properties of food proteins (gelling, foaming andemulsifying properties), a brief description on the modifica-tion of food proteins, and a detailed discussion on the radi-ation chemistry of proteins. For each type of radiations onproteins, discussions on the effects of gamma, electronbeam and UV irradiation were reported considering all sig-nificant articles published in the field. Finally, a reflectionon the important aspects of the process, its limits and poten-tials, the lack of scientific information to fill up, and a direc-tion to orientate the research will be evidenced.

Functionality of proteinsProteins are the basic functional components of various

high protein processed food products and thus determinethe textural, sensory and nutritional properties. Functionalproperties of protein are those physicochemical propertiesof proteins which affect their behavior in food systems

107Y.-H. Kuan et al. / Trends in Food Science & Technology 30 (2013) 105e120

during preparation, processing, storage, and consumptionas well as the contribution to the quality and sensory attri-butes of food systems (Kinsella, 1976). Food proteins com-monly exist together with other food constituents andcontribute significantly in building or stabilizing gels,foams, doughs, emulsions and fibrous structures as wellas biodegradable films. The most important functionalproperties of proteins in food applications are (1) hydro-philic, i.e. protein solubility, swelling and water retentioncapacity, foaming properties, and gelling capacity; (2) hy-drophilicehydrophobic, i.e. emulsifying; and foaming;(3) hydrophobic, i.e. fat binding properties (Zayas, 1997).

Gel formationThe gelling capacity of food proteins is an important

functional attribute in food manufacturing. Proteins areimportant gelling ingredients in large number of food prod-ucts.Together with pectins, starches and gums form strong gels(Belitz, Grosch, & Schieberle, 2009). Gelation is the basicprocess using these food proteins to produce gels or gel-containing products which exhibit various vital rheologicalproperties, appearance and gel point (Zayas, 1997). A com-prehensive review on protein gel formation was performedby Ziegler and Foegeding (1990). The gelation process de-pends on the formation of a three-dimensional protein net-work as a result of proteineprotein and proteinesolvent(water) interactions (Hermansson, 1979). Protein gel for-mation is a result of intermolecular interactions resultingin the three-dimensional network of protein fibers whichdevelop high structural rigidity (Zayas, 1997). A gel matrixis a dispersion made up of at least two components in whichthe disperse phase in the dispersant forms a cohesive net-work. They are characterized by the lack of fluidity andelastic deformability (Belitz et al., 2009). Tanaka (1981)has defined a gel as a form of matter intermediate betweena solid and a liquid, consisting of “strands” or “chains”cross-linked to create a continuous network immersed ina liquid medium. Hence, gels are space-filling and three-dimensional structures. Generally, gels can be categorizedas polymeric networks and aggregated dispersions, al-though intermediate forms are found.

Polymeric or fine-stranded networks are well repre-sented by gels formed by gelatin, and polysaccharidessuch as agarose and carrageenan (Belitz et al., 2009). For-mation of a three-dimensional network takes place throughthe aggregation of unordered fibrous molecules via partlyordered structures, e.g., formation of double helices struc-ture (Belitz et al., 2009; Koning & Visser, 1992). Character-istic for this type of gels is the low polymer concentration(w1%) as well as transparency and fine texture (Tanaka,1981). The gel formation is initiated by setting a certainpH, adding certain ions, or heating/cooling. Since aggrega-tion takes place mostly via intermolecular hydrogen bonds,which break easily when heated, polymeric networks arethermo-reversible, as in the gels are formed when a solution

cools, and they melt again when reheated (Belitz et al.,2009). This type of gel with the small pores can effectivelybind more water and is less prone to syneresis compared tothe aggregated dispersions (Koning & Visser, 1992;Tanaka, 1981).

On the other hand, aggregated dispersions are the gelsformed by globular proteins after heating and denaturation(Belitz et al., 2009; Koning & Visser, 1992). The thermal un-folding of the protein leads to the release of amino acid sidechains which may enter into intermolecular interactions. Thesubsequent association occurs while small spherical aggre-gates form which combine into linear strands to form the gelnetwork. This type of gel formation requires comparativelyhigh protein concentrations (50e10%) due to the unorderedtype of aggregation (Belitz et al., 2009). Therefore, thesegels are opaque (Koning & Visser, 1992). However, the rateof aggregation should be smaller than that of unfolding ratein order to avoid the formation of coarse and less structuredgels (Belitz et al., 2009); especially in the area of the iso-electric point (Koning&Visser, 1992).A certain degree of un-folding is necessary to initiate aggregation, and this processdepends on the specific protein (Rector, Kella, & Kinsella,1989). Since partial denaturation releases primarily hydropho-bic groups, intermolecular hydrophobic bonds generally pre-dominate, resulting in thermoplastic (thermo-irreversible)gels (Belitz et al., 2009). In contrast, the thermo-reversiblegels are stabilized by hydrogen bonds (Clark, Saunderson, &Suggett, 1981). Thermoplastic gels do not liquefy during heat-ing, but they can soften or shrink (Belitz et al., 2009). Otherthan hydrophobic bonds, the disulfide bonds formed from re-leased thiol groups may contribute to cross-linking of gels, aswell as ionic bonds betweenproteinswith different iso-electricpoints in heterogeneous systems (e.g. egg white protein)(Belitz et al., 2009; Koning & Visser, 1992).

Foaming propertiesAerated food products are found in great varieties and have

been known for a long time elsewhere. They are highly appre-ciated by consumers because of their light structure and texture(Kammesheidt, 2003). In several foods, proteins function asfoam-forming and foam stabilizing components to providethe texture to many traditional (ice cream, whipped topping,breads, cakes, meringues, beers and champagne) and new(cheese, butter, spreads, confectionary, and sausages) aeratedfood products (Rodr�ıguez Patino, Carrera S�anchez, &Rodr�ıguez Ni~no, 2008). Moreover, gas bubbles can be usedto replace fat to make low caloric food products, which is animportant trend in making healthier food products (Campbell& Mougeot, 1999; Z�u~niga & Aguilera, 2008).

Foaming properties of proteins have been extensively re-viewed by Campbell and Mougeot (1999), Halling (1981)and Wilde (2000). These properties are manifested by thefoaming power and the foam stability. Foaming power is de-termined by measuring an increase in foam volume upon theintroduction of gas into the emulsifier solutions. Foams arethermodynamically unstable. Therefore, foam properties


vary with time as a result of shifts in the distribution of gasand liquid in the foam (Dickinson, 1992). Foam stability isdetermined by measuring the rate of liquid drainage fromfoam or the rate of decrease in foam volume with time. Theinstability of foams is attributed to three processes, whichare drainage, coalescence and disproportionation (Prins,1988). According to Bisperink, Ronteltap, and Prins(1992), drainage is the liquid flow from foam as a result ofgravity and capillary forces. Foams change from sphericalfoams to polyhedral foams as a consequence of drainage. Co-alescence is the merging of two bubbles resulting from therupture of the film between the foams. As a consequence,large bubbles appear in the foam and the number of bubblesdecreases. Lastly, disproportionation refers to the inter-bubble gas diffusion. Larger bubbles grow at the expense ofsmaller bubbles resulting from the gas diffusion. The smallerbubbles then shrink and may finally disappear.

The foaming properties of proteins depend on a variety offactors, such as protein concentration, pH, ionic strength,aqueous phase composition, temperature, and methods offoamproduction (Rodr�ıguez Patino et al., 2008).Modificationofproteins such as chemical andphysicalmodificationmay in-crease the foaming properties (Kuan,Bhat,&Karim, 2011). Inaddition, partial enzymatic hydrolysis produces smaller mole-cules of higher diffusion rate, better solubility, and higher sur-face hydrophobicity in whey proteins (Chen, 2002). Improvedfoaming ability and foaming stability were also observed onegg white proteins upon heat or high pressure treatment(Van der Plancken, Van Loey, & Hendrickx, 2007). They ex-plained that the heat-induced conformational changes by en-hancing the proteineprotein interactions at the interface,have given rise to the improved foaming properties.

Emulsifying propertiesProteins are able to stabilize emulsions on account of

their amphiphilic nature. These proteins molecules concen-trate at the oil and water interface; with lipophilic portion inthe non-polar phase (oil) and the hydrophobic portion in thepolar (water) phase (Wilde, 2000). The stabilization of pro-tein is effective when the proteins form a solid visco-elasticlayer. The proteins adsorb, partially unfold and form stronginteractions, which in turn result in a visco-elastic adsorbedlayer that has been well correlated with emulsion stability(Halling, 1981; Mitchell, 1986). The unfolding of proteinsat interfaces is influenced by the structure in solution, suchthat flexible proteins will unfold quickly and rapidly lowerthe interfacial tension (Kinsella & Whitehead, 1989;Mitchell, 1986), whereas globular proteins unfold moreslowly as they have more intramolecular bonds stabilizingtheir structure (Wilde, 2000). The unfolded proteins tendto form stronger intermolecular interactions and stabilizeagainst coalescence very effectively (Mitchell, 1986).Therefore, changing the structure of proteins by variousmeans has been used as a tool for improving protein func-tionality, probably by inducing a change in adsorbedconformation.

Protein film propertiesBiodegradable films (i.e., films made with proteins, poly-

saccharides and lipids) are gainingmarket interest momentumin many countries as biodegradable materials. Among thesebio resources, proteins have long been used for the applicationof protein film (e.g., traditional lipoprotein skin termed tou-fu-pi in China) (Krochta, Baldwin, & Nisperos-Carriedo, 1994).In nutritional aspect, films made from protein can supplementthe nutritional value of the food (Gennadios &Weller, 1990).Moreover, films made from proteins are substantially betterthan that with filmsmade from polysaccharides and lipids, be-cause protein have a unique structure (based on 20 differentmonomers) which confers a wider range of functional proper-ties, especially a high intermolecular binding potential (Cuq,Aymard, Cuq, & Guilbert, 1995). Great varieties of proteinsfrom animal (e.g., milk proteins, collagen, gelatin, keratin,and myofibrillar protein) and plant (e.g., corn zein, wheat glu-ten, and soy protein) resources have received attention for theproduction of biodegradable films (Gennadios, 2002). Reviewon the protein films pertaining their formation and propertiescould be obtained from the articles published by Zhang andMittal (2010) and Bourtoom (2009).

Generally, there are two process pathways used for buildingup protein films, i.e., the dry and the wet process (Zhang &Mittal, 2010). The dry process (e.g., thermoplastic extrusion)is based on thermoplastic properties of biopolymerswhenplas-ticized and heated above their glass transition temperature un-der low water content. The wet process (e.g., solvent process)or casting is based on the drying of a film-forming solutionor dispersion. Thewet process is generally used for performedfilms and coatings (Khwaldia, Perez, Banon, Desobry, &Hardy, 2004). The film-forming properties of protein varyamong different protein types and sources, as different proteinstypes impart different physical and chemical characteristics(Zhang &Mittal, 2010). Moreover, the variables (i.e., temper-ature, solvent, plasticizers, and incorporation of additives) ap-plied in the protein film-forming process are greatly affectedthe protein film-forming ability. Protein film-forming abilityhas been reported along with some functional properties,such as barrier properties (i.e., water vapor permeability), me-chanical properties (i.e., tensile strength, elongation, deform-ability, and elastic modulus) as well as microstructuralproperties (i.e., dough andfiber formation and texturizingcapa-bility) (Wihodo &Moraru, 2012). These functional propertiesare crucial on improving the quality of food products, espe-cially extending the shelf life of processed fruits and vegetablescoated with the films. In addition, the incorporation of antimi-crobial agents into the film could further extend the shelf lifeand improved the safety of package foods (Cha & Chinnan,2004). Other than this, the addition of additives such as antiox-idants, anti-browning agents, nutraceuticals, texture enhancers,flavor, and color ingredients could further enhance the organo-leptic properties of the films (Wihodo & Moraru, 2012).

Various physical and chemical methods used to enhancethe structure and mechanical properties of protein filmshave been reported (Wihodo & Moraru, 2012). These


methods include the incorporation of plasticizers, pH alter-ation, and lipid addition as well as cross-linking of proteinfilms (i.e., chemically or physically cross-linking, enzy-matic cross-linking and the application of radiation). Themodification of the protein films significantly altered thefilm functional properties. Discussion on the effects of radi-ation on protein films will be detailed in the following text.

Modification of food proteinsModification of proteins is still far from common in food

processing, but is increasingly being recognized as essential(Belitz et al., 2009).Modification of protein is essential to al-ter the microstructure and physical performances of the bio-polymers used for food, medical, and industrial applications.In the food industry, proteins are often modified in order toobtain the desired functional traits at industrial levels. Theseinclude the improvement on texture, flavor, color, solubility,foam stability, whippability and digestibility (Ball, 1987;Hoogenkamp, 2001). Food proteins are usually modifiedchemically, enzymatically, physically, or at combination ofthese methods. Typically, the modification of food proteinsis either performed by cross-linking or by hydrolysis; de-pending on the application. Proteins cross-linking involvesthe formation of covalent bonds between polypeptide chainswithin a protein (intramolecular cross-linking) or betweenproteins (intermolecular cross-linking) (Feeney &Whitaker, 1988). The major pathways of protein cross-linking involving amino acid residues during food processingare reported by Gerrard (2002). The most common types ofcross-linking found in food processing are disulfide bonds,cross-linking derived from dehydroprotein, from tyrosine,from the Maillard reaction and by enzymatic reaction. Onthe other hand, the hydrolyzed protein is obtained usuallyby prolonged boiling in a strong acid or strong base or usingenzymes such as the pancreatic protease enzyme to simulatethe naturally-occurring hydrolytic process (Wanasundara,Amarowicz, Pegg, & Shand, 2002).

Basically, the methods of protein modification can becategorized as, (1) physical methods, (2) chemical methods,and (3) enzymatic methods. However, there is a concern re-garding the safety of chemically modified food proteins;and also the time-consuming treatments by using enzymaticmethods. Currently, there is a mounting interest in the ap-plication of physical methods for protein modification,which is considered safer than chemical modification.Some of these physical methods include radiation treat-ments, pulsed electric field (Fernandez-Diaz, Barsotti,Dumay, & Cheftel, 2000), heat treatment (Keerati-u-rai &Corredig, 2009), ultrasonic treatment (Tang, Wang, Yang,& Li, 2009) as well as elevated pressure treatment(Torrezan, Tham, Bell, Frazier, & Christianini, 2007). Radi-ation processing has been highlighted to provide a low-costand environmental-friendly alternative to alter the physical,chemical, and/or biological characteristic of a food product;other than being applied in food preservation. Moreover, ir-radiation treatments do not induce a significant increase in

temperature, require minimal sample preparation, are fast,and have no dependence on any type of catalysts (Diehl,2002; Farkas, 1998).

Radiation chemistry of proteinsFood components are usually composed of elements

such as carbon, hydrogen, oxygen, and nitrogen. These el-ements consist of characteristic atoms containing a rela-tively small nucleus and a number of electrons. Whenionizing radiation passes through matter such as food, itloses energy. At the same time, the energy is absorbedand consequently leads to the ionization or excitation ofthe atoms and molecules of the matter (Stewart, 2001).Therefore, this results in chemical changes when food is ir-radiated. Although ultraviolet radiation is a non-ionizingradiation, higher frequency of ultraviolet radiation cancause ionization and break chemical bonds. The chemistryof food irradiation has been extensively studied and re-ported in detail by various researchers (Delinc�ee, 1983;Hou�ee-Levin & Sicard-Roselli, 2001; Stewart, 2001).

Radiation chemistry of proteins has been studied formore than 30 years (Hou�ee-Levin & Sicard-Roselli,2001). The understanding on the changes occurring inamino acids and peptide upon irradiation is crucial whenstudying the effects of irradiation on the most complex pro-teins or protein-based foods. Under irradiation, proteins areaffected by direct and indirect effects of ionizing radiation.When these macromolecules are in liquid solution, directeffects can be neglected and the indirect effects are pre-dominant. However, in solid state, proteins are ionizedmainly by direct interaction (Kempner, 2001). Upon irradi-ation, the generation of primary water free radicals (hy-drated electron, hydrogen atom, and hydroxyl radical)reacts very efficiently with proteins (Fig. 1). Hence, variousforms of modifications are expected, e.g., polymerization(dimerization) and fragmentation. These reactions havebeen studied extensively; especially those of OH free radi-cals because of their importance in biological oxidative dis-orders (Audette-Stuart, Hou�ee-Levin, & Potier, 2005).Also, these reactions have been suggested to be the mainmechanism underlying physicochemical changes in proteinfoods, like the increase and reduction of viscosity and watersolubility of protein pastes (Ishizaki, Hamada, Iso, &Taguchi, 1993a; Ishizaki, Hamada, Tanaka, & Taguchi,1993b; Al-Assaf, Phillips, Williams, & du Plessis, 2007;Bhat & Karim, 2009b), the emulsifying and foaming prop-erties of protein emulsion (Kuan et al., 2011; Song et al.,2009), and the tensile strength as well as the water vaporpermeability of protein films (Micard, Belamri, Morel, &Guilbert, 2000; Cie�sla, Salmieri, & Lacroix, 2006a, 2006b).

The chemical reactions that occur during irradiation of pro-teins are exerted byvarious factors suchas the structure and thestate of protein (e.g., fibrous or globular, native or denatured,the composition, presence of foreign substances, wet, dry, insolution or whether in liquid state or frozen state) and the con-ditions of irradiation (e.g., dose, dose rate, temperature,

Fig. 1. Effect of irradiation on protein showing possible depolymerization (fragmentation) and polymerization (cross-linking) of protein chains.


presence of oxygen) (Audette-Stuart et al., 2005; Delinc�ee,1983; Hou�ee-Levin & Sicard-Roselli, 2001). Irradiationcauses the folding of the peptide chains, intramolecular disul-fide chains, and secondary binding forces such as hydrogenbonds, hydrophobic bonds, ionic bonds, or bonds that holdseveral subunits together as a functional protein (Stewart,2001). The irradiationmechanism entails a series of reactions,whichwill substantially lead to the formation of ionic and freeradical intermediates; and ultimately into the stable products(Delinc�ee, 1983). The schematic representation in Fig. 1shows the generation of water free radicals upon radiation ex-posure. Reduction and/or oxidative reactions take place de-pending on the various factors mentioned above. Therefore,it is in general interest to have more research carried out onthe elucidation of the chemical reactions; especially in the un-derstanding of long range intra- and intermolecular electronand radicals transfer mechanisms.

Irradiation of proteins can cause certain permanent changessuch as deamination, decarboxylation, reduction of disulphidelinkages, oxidationof sulfhydryl groups,modificationofaminoacid moieties, valance change of coordinated metal ions,peptide-chain cleavage, and aggregation. The deamination

occurs upon irradiation via the scission chain reaction(Rustgi & Riesz, 1978), especially by the reduction of the car-bonyl groups of peptide bonds via the attack of hydrated elec-trons (Diehl, 1995; Hou�ee-Levin & Sicard-Roselli, 2001).However, not all irradiated proteins undergo degradation.Cross-linking induced by the unfolding and aggregation via in-termolecular bonds would have occurred with globular pro-teins, whereas with fibrous proteins, degradation mainlyoccurs (Delinc�ee, 2002; Stewart, 2001). The splitting and ag-gregation of proteins that occur with irradiation are related tothe disturbances of the secondary and tertiary protein structuresthat expose reactive groups to the action of free radicals (hy-drated electron, hydrogen atom, and hydroxyl radical) as a re-sult from water radiolysis. The cross-linking or aggregation ofirradiatedproteinshas beenverifiedby themeanof gel filtrationsuch as SDS-PAGE assay, where, irradiated proteins were sep-arated on the basis of differences inmolecular size (Kuan et al.,2011; Stewart, 2001).

Effects of gamma irradiation on protein functionalitiesGamma radiation is obtained through the use of radio-

isotopes, generally cobalt-60 or cesium-137; which emits


high-energy gamma ray or photons capable of intruding in-depth into the target product. Radiation treatment has beenshown to either cross-link (polymerization) or degrade (de-polymerization) food proteins, which is dependent on thedose delivered, exposure time, exposure conditions andthe type of food proteins used; as discussed in the previoussection. In terms of exposure to the irradiation doses, thisdepends on the amount of the dose applied (usually lowand medium doses have a non-significant effect on the pro-teins), and the sensitivity of the proteins toward irradiation(types of amino acids). Table 1 summarizes the effects ofgamma irradiation and the dose delivered on various typesof food proteins. In the following text, some of the studiespertaining to food proteins and the effect on their functionalproperties are detailed.

The application of gamma irradiation on the cross-linking of proteins was mainly on the production of proteinfilms or biodegradable films. In a research article reportedby Brault, D’Aprano, and Lacroix (1997), gamma irradia-tion (doses of 4, 8, 12, 15, and 20 kGy at a mean doserate of 2.18 kGy/h) was applied to produce free-standingsterilized edible films based on milk protein, i.e., sodiumcaseinate and calcium caseinate. Based on their work, irra-diation of a solution based on calcium caseinate has foundto exhibit more cross-links than sodium caseinate and con-tributed to better mechanical strength in films made withcalcium caseinate. They envisaged that upon exposure togamma irradiation, calcium caseinate would generatemore bityrosine than sodium caseinate, i.e., an increase ofcross-linking on calcium caseinate was observed. In theirresearch study, they also found out that the effect of irradi-ation on the mechanical properties was strongly dependent

Table 1. (a). Some reports on the effects of g-irradiation on plantproteins. (b). Effect of g-irradiation on animal proteins.

Protein source Application Reference

(a)Caseinate Protein films Ressouany et al. (1998),

Vachon et al. (2000),Lacroix et al. (2002)

Caseinateewheyprotein isolate

Protein films Cie�sla et al. (2006a),Cie�sla, Salmieri, andLacroix (2006b),Sabato et al. (2001)

Corn protein(zein)

Protein films Soliman et al. (2009)

Soy protein Protein films Lacroix et al. (2002),Lee et al. (2005a)

Wheat gluten Protein films Micard et al. (2000)Food system/bakery products

Koksel et al. (1998)

(b)Beef myoglobin Food system Clarke and Richards (1971)Collagen Food system Al-Assaf et al. (2007)Egg white Food system/

bakery productsJosimovi�c et al. (1996),Song et al. (2009)

Gelatin Protein films Jo et al. (2005)Meat Food preservation Losty, Roth, and Shults (1973)

on the glycerol/protein ratio during the film formation. Theaddition of glycerol has significantly increased the forma-tion of cross-links within protein chains. This effect is ex-plained by the preferential binding concept, whereby theproduction of bityrosine that attributes to the proteincross-linking would be enhanced in the presence of glyc-erol. Similar findings were reported on the addition of pro-pylene glycol and triethylene glycol (Lacroix, Jobin,Mezgheni, Srour, & Boileau, 1998).

A study by Ressouany, Vachon, and Lacroix (1998)showed that the addition of CaCl2 to the basic formulationof calcium caseinate film-forming solution prior to gammairradiation (doses of 0, 8, 16, 32, 64, 96, and 128 kGy) hadincreased the fluorescence signal of bityrosine at all doses.They suggested that the enhancement in protein cross-linking was attributed by the indirect effects of calciumon the formation of bityrosine, shortening the moleculardistances between polypeptides, and consequently contrib-uted to the feasibility of bityrosine formation. In their study,gels were formed in solutions containing CaCl2 at irradia-tion doses � 16 kGy. Moreover, maximum gel strengthwas obtained at an irradiation dose of 64 kGy for all formu-lations, suggesting a maximum cross-linking density.

Another study by the same research group reported thatthe production of sterile free-standing biodegradable ca-seinate films by employing gamma irradiation (doses of 4and 64 kGy) (Mezgheni, Vachon, & Lacroix, 2000). The ef-fect of irradiation doses (degree of cross-links) on the bio-degradability of these films was investigated using a strainof Pseudomonas aeruginosa. They concluded that thecross-linked caseinate films had improved resistance to mi-crobiological degradation and were less soluble in water.This outcome is interesting for the application on foodpackaging since the shelf life of these biomaterials couldbe tailor made by adjusting the radiation doses (degree ofcross-linking).

The gamma irradiation at a dose of 32 kGy has been re-ported to induce cross-linking on film made with calciumcaseinate; based on the results obtained from increased mo-lecular weight by employing size-exclusion chromatogra-phy, and increased protein insolubility via gravitationalmethod (Vachon et al., 2000). However, they reportedthat only very little molecular weight changes were ob-served on the film made with whey protein. They explainedthat the globular whey proteins are more prone to intramo-lecular cross-linking, leading to little change in its molecu-lar weight. In term of the mechanical properties of the films(calcium caseinate and whey protein), the puncture strengthwas significantly improved (P < 0.05), as a result of thecross-linking of protein chains. When the microstructureof these films was viewed under the transmission electronicmicroscopy (TEM), the pore size of the irradiated films wasfound to be denser than the un-irradiated films.

This Canadian research group further explored the ef-fects of gamma irradiation (dose of 32 kGy at a meandose rate of 31.24 kGy/h) on films made with soy protein


isolate, and a 1:1 mixture of SPI and whey protein isolate(Sabato et al., 2001). The effects of the incorporation ofcarboxymethylcellulose (CMC) and poly(vinyl alcohol)were also examined. Based on the data obtained fromsize-exclusion chromatography, an increase peak was foundon the irradiated samples, indicating the occurrence of pro-tein cross-linking (i.e., formation of bityrosine). In terms ofthe mechanical properties of all types of films, they re-ported that gamma irradiation had significantly improvedthe puncture strength and puncture deformation. The re-searchers pointed out that the irradiated formulations con-taining CMC, for both irradiated SPI films and un-irradiated SPIewhey protein isolate films, had improvedwater vapor permeability.

Lee, Lee, and Song (2005a) studied the effects ofgamma irradiation (doses of 0, 4, 16, 32, and 50 kGy) onthe film-forming solution of soy protein isolate. Based ontheir results on SDS-PAGE, it was found that at doserange � 16 kGy, cross-linking of protein molecules oc-curred. However, a significant decrease in the viscosity ofthe gamma-irradiated SPI solution was observed. They ex-plained that this reduction of viscosity was attributed to theconformational change of protein molecules by oxygen rad-icals generated by the water radiolysis. They also reportedthat gamma irradiation had substantially improved the wa-ter vapor permeability of the soy protein film produced; i.e.,at 50 kGy, the water vapor permeability decreased by 13%.Moreover, an increase Hunter b color value upon increasingof irradiation dose was observed, indicating the occurrenceof browning/darkening reaction.

The effects of gamma irradiation (doses of 0, 4, 16, 32, and50 kGy) on the physicochemical properties of gluten filmswere also studied (Lee, Lee, & Song, 2005b). Based on theSDS-PAGE results, they reported that gamma irradiation ongluten solutions caused disruption on the ordered structureof the gluten molecules, as well as degradation and aggrega-tion of the polypeptide chain. Gamma irradiation below16 kGy decreased the viscosity of film-forming solution dueto the cleavage of the polypeptide chains and increased againabove 32 kGy due to protein aggregation. At 50 kGy, tensilestrength was found to have increased 1.5 fold and water vaporpermeability reduced by 29%. They concluded that gamma ir-radiation may be a useful tool as a cross-linking agent to im-prove the functional properties of gluten films.

The effects of gamma irradiation (doses of 10, 20, and40 kGy) on wheat gluten films were also studied byMicard et al. (2000). They reported that gamma irradiationincreased the tensile strength and decreased the elongationat break; i.e., a 10 kGy dose resulted in a 32% decrease inelongation. They explained that this effect was attributed toa decrease in insoluble glutenin; i.e., depolymerization and/or breakdown of covalent linkages. However, the improve-ment on the mechanical properties was attributed to the for-mation of bityrosine upon irradiation. In terms of filmproperties, an increase in the water vapor permeability by19% was observed on gluten film treated at 10 and 20 kGy.

Soliman, Mohy Eldin, and Futura (2009) evaluated theimpact of gamma irradiation (doses of 0, 10, 20, 30, and40 kGy at rate of 10.5 kGy/h) on the film properties ofcorn zein. Results from far-UV CD and FTIR showedthat an increase in irradiation dosage had contributed tothe decrease in helical content and an increase in b-sheetcontent. The conformation changes were more pronouncedat 10 and 30 kGy. In terms of viscosity measurement, theyreported that the viscosity of the zein film-forming solutiondecreased significantly by the action of gamma irradiationat 10 kGy, increased at 20 kGy, and subsequently decreasedagain with a further increase for radiation dosage up to40 kGy. They explained that these results were attributedto the impact of gamma irradiation, which caused changesin aggregate formation and unfolding, scission, or cross-linking of the polypeptide chains of zein. In their study,they also observed improvements in color, surface density,mechanical properties and water vapor permeability of thefilms produced.

Mixture of proteins was also attempted to produce bio-films with the aid of gamma irradiation. Lacroix et al.(2002) studied the application of gamma irradiation (dosesof 4e128 kGy) to produce sterilized cross-linked films withcalcium caseinate and whey proteins [whey protein isolate(WPI) and commercial whey protein concentrate (WPC)]or mixture of soya protein isolate (SPI) with WPI. Resultsshowed that the irradiation-induced cross-linking treatmentsignificantly improved the mechanical properties (i.e.,puncture strength) for all types of films. The X-ray diffrac-tion analysis showed that gamma irradiation modified theconformation of protein to a certain extent, where the pro-tein molecules will adopt a more ordered and more stablestructure upon irradiation. Apart from this, the microstruc-ture and water vapor permeability of the films were consid-erably improved. Biodegradation study also showed that theirradiated cross-linked film slowed the biodegradation ofthe material used.

In a study reported by Cie�sla, Salmieri, Lacroix, and LeTien (2004), Brookfield viscometry, FTIR, and measure-ments of mechanical as well as barrier properties of filmwere applied on the films prepared by using calcium casein-ate (CC)ewhey protein isolate (WPI)eglycerol (1:1:1) af-ter irradiated with gamma rays (dose of 32 kGy at a doserate of 7 Gy/s). Based on their study, increases in the b-sheet and b-strand contents were observed, resulting fromthe protein cross-linking and the modification of proteinconformations upon irradiation. These changes subse-quently contributed to the improvement in the film proper-ties of the CCeWPI films. Furthermore, in another studywith the application of the same film making materialsand the same irradiation condition; the addition with cal-cium salt contributed to the formation of more “particulate”irradiated gels compared to the control which was more“fine-stranded” (Cie�sla et al., 2006a). This effect was attrib-uted to the higher amount of the regular b-structure in thegels obtained after irradiation, that are more capable of


joining more calcium ions and are more affected than thoseobtained from control solutions.

They also studied the effects of gamma irradiation (doseof 32 kGy) followed by the addition of polysaccharides(i.e., sodium alginate and soluble potato starch) on thesame CCeWPI mixture of films (Cie�sla et al., 2006b).They reported that the radiation induced an improvementon the mechanical and barrier properties of all films. Theaddition of polysaccharides was reported to show enhance-ment on the gel and film properties upon irradiation. Theauthors explained that the addition of polysaccharidesslightly altered the protein structure in terms of the compat-ibility between the protein and polysaccharide gel net-works. The presence of alginate stabilizes the structure inthe irradiated CCeWPI because of the entrapment ofcross-linked proteins in the stiff alginate gel network.Upon observation under TEM, a strongly bonded chain ma-terial was observed, indicating an improvement in the filmproperties. They concluded that the irradiation ofCCeWPIesodium alginate composition has led to the for-mation of the cross-linked proteinepolysaccharidenetwork.

Effects of gamma irradiation (doses of 0, 10, 20, and30 kGy; temperature, 14 � 1 �C at a dose rate of10 kGy/h) on the mixture of pectinegelatin (1:1) basedfilm were studied by several researchers (Jo, Kang, Lee,Kwon, & Byun, 2005). They found out that the tensilestrength of the 10 kGy-irradiated film was the highestamong the treatments but the elongation at break, water va-por permeability, and swelling ratio were the lowest. TheHunter b color value was found to increase with an increas-ing dosage of irradiation. The results of SEM indicated thatthe irradiation of the pectinegelatin film at 10 kGy qualita-tively decreased the interlayer space of the film. In term ofbiodegradability, it was found that film casted on irradiationdose of 10 kGy was the lowest among all. They concludedthat the irradiation of the film casting solution at 10 kGy isthe optimum condition to give the increased mechanicalproperties of the films.

The application of gamma irradiation was also extendedto bakery products and food systems. Koksel, Sapirstein,Celik, and Bushuk (1998) employed gamma irradiation(doses of 2.5, 5.0, 10.0, and 20.0 kGy) on gluten proteinsof two bread wheats and one durum wheat cultivar. Basedon the results obtained from the mixograph, a weakeningeffect and the deterioration of rheological properties onthe dough mixing properties were observed at irradiationdosage of �10 kGy. They pointed out that irradiationcaused a significant deteriorating effect on the gluten pro-teins. Moreover, SDS-PAGE profiles showed a noticeablereduction in band intensities of both high (HMW) andlow molecular weight (LMW) glutenin subunits (GS)with an increasing irradiation dosage greater than 5 kGy.The effect was then further quantified with reverse phaseHPLC and the results shown a progressive decrease in thequantity of subunits with increasing irradiation dose level.

The total insoluble glutenin was found to have reducedby 34e49% at 20 kGy, depending on the cultivar. The in-creasing irradiation dosage had also progressively reducedthe ratio of HMW:LMW-GS by up to 13e15% at20 kGy. They concluded that the increasing levels ofgamma irradiation are consistent with the degradation ofglutenin to a lower average molecular size by depolymer-ization and/or disaggregation.

Gamma irradiation (doses of 0, 1, 2, and 5 kGy; temper-ature, 10 � 0.5 �C at a dose rate of 20 kGy/h) was reportedto improve the foaming properties of egg white [liquid eggwhite (LEW) and egg white powder (EWP)] which led toan increased volume in angel cake produced (Song et al.,2009). A significant decrease in viscosity was observedon LEW upon irradiation, whereas that of EWP was not af-fected. They explained that the reduction of LEW was dueto the breakdown of protein, and the resistance of change inEWP was due to the low water content that protects the pro-tein from breakdown by radiolytic products of water. Theangel cakes produced with irradiated egg white samples ex-hibited increased volume than that of control samples. Theyexplained that this effect was contributed by the increasedfoaming ability of gamma-irradiated LEW and EWP asa dose-dependent manner (P < 0.05). Gamma irradiationwas also found to reduce the hardness, chewiness, and gum-miness and increase the Hunter L (lightness) value in theangel cakes. The results obtained were in good agreementwith the findings reported by Josimovi�c, Radoj�ci�c, andMilosavljevi�c (1996), where gamma irradiation (doses of10, 30, 60 kGy at a dose rate of 51.5 Gy/min) induced frag-mentation of protein molecules which led to the reductionof viscosity of fresh egg white.

Apart from being applied on films and bakery products,Al-Assaf et al. (2007) demonstrated the use of gamma irra-diation (doses of 8 and 16 kGy) on the controlled modifica-tion of collagen with the presence of a mediating alkynegas. In their study, the viscosity increased with the radiationdose. The high molecular weight materials produced after8 kGy were completely soluble with viscosity 100 timesgreater than that of control samples, and increase furtherup to 1000-fold was observed after 16 kGy. They concludedthat the molecular weight as well as the rheological param-eters of the collagen can be controlled with the aid ofgamma irradiation. Hence, the process allowed the produc-tion of tailor made and reproducible materials with en-hanced functionality.

As a concluding remark on the effects of gamma irradi-ation on food proteins, it is noteworthy that most food pro-teins undergo irradiation-induced cross-linking andsubsequent improvement on the film properties. Thesefood proteins include soy protein isolate, corn zein, wheatgluten, caseinates, whey protein, egg white, collagen, andgelatin (Table 1). However, for wheat gluten sample, depo-lymerization was found on the glutenin with the reductionin average molecular size (Koksel et al., 1998). Weakeningeffect and deterioration of rheological properties were then


observed on the dough mixing properties. It has been pre-viously stated that cross-linking usually occurs in globularprotein while degradation usually occurs in fibrous protein(Stewart, 2001). Since glutenin is a typical fibrous protein,therefore depolymerization occurred.

Films treated with gamma irradiation showed substantialimprovement on the film properties especially water vaporpermeability (Lee et al., 2005a; Soliman et al., 2009;Vachon et al., 2000). The microstructure of the gamma-irradiatedfilmswas found tohave denser and smoother surfacethan the control film viewed under scanning electron micro-scope. Gamma irradiation caused the disruption of the orderedstructure of the protein molecules, as well as degradation,cross-linking, and aggregation of the polypeptide chains, re-sulting in the improvement of the film properties.

Effects of electron beam irradiation on proteinsfunctionalities

Electron-beam radiation is a form of ionizing energythat is generated by the acceleration and conversion of elec-tricity. Electron beam is similar to gamma processing inthat, upon contact with the exposed product, electrons altervarious chemical and molecular bonds. However, the studyof the application of electron-beam irradiation on food pro-teins is rather scarce.

Huang, Herald, and Mueller (1997) studied the effect ofpost-electron beam irradiation (doses of 2.3e3.0 kGy withbeam power of 8.1 kW and energy level of 10 MeV) onphysical, physicochemical, and functional properties of liq-uid egg yolk stored at �15 �C. Analyses were undertakenat 0, 1, 7, 15, 30, and 60 days of storage. They reportedthat the development of storage modulus (G0) and proteinsolubility of irradiated samples showed a delayed reductionwithin the first 7 days of storage compared to control sam-ple, indicating less destruction was developed upon irradi-ation. On color measurement, the saturation index ofirradiated samples was lower than that of control samples.They explained that this radiation-induced discolorationwas attributed by the destruction of carotenoid in eggyolk. However, no significant results were observed onthe SDS-PAGE analysis. They concluded that electronbeam could serve as a preservation method for liquid eggyolk since no significant physical, chemical or functionalchanges were found.

Vieira and Del Mastro (2002) compared the effects ofgamma and electron-beam irradiation (doses of 0, 5, 10,20, and 50 kGy at dose rate of 7 kGy/h and 11 kGy/h forgamma and electron-beam irradiation, respectively) onthe viscosity of gelatin produced from powdered bovinegelatin. In their study, the viscosity of gelation solutions re-duced as a consequence of irradiation in both cases. The re-lationship between the decrease in viscosity of gelatinsolutions and radiation presented close comparable valuesfor both irradiation processes, indicating no difference onchemical reactions conducting to physical effects betweenboth types of irradiation.

The effects of electron-beam irradiation on instrumentalcolor, texture, microbial inactivation, and proteineproteininteractions on surimi seafood were investigated byJaczynski and Park (2004). Whitening (increased b value)was observed, indicating the bleaching of yellow hue byozone that generated during electron-beam treatment. Elec-tron-beam irradiation up to 6e8 kGy also increased thestrength of surimi gels. The D10 value for Staphylococcusaureus was 0.34 kGy. Modeling of microbial inactivationdemonstrated that two-sided electron beam may control S.aureus if the surimi seafood package is thinner than82 mm. Results from SDS-PAGE showed gradual degrada-tion of myosin heavy chain upon increasing dosage of radi-ation. They also reported that the integrity of actin wasslightly affected by electron-beam irradiation.

A study was conducted to compare the effects of gammaand electron-beam irradiation (doses of 0, 3, 5, 7, and10 kGy for both types of irradiation) for the inhibitionand reduction allergenicity of hen’s egg albumin (Lee,Seo, Kim, Lee, & Byun, 2007). SDS-PAGE patternsshowed that the intact egg albumin band disappeared andthat it was dependent upon irradiation doses regardless ofthe radiation types. Binding abilities of the irradiated eggalbumin against the monoclonal IgG and the egg allergicpatients’ IgE decreased due to a conformational changeof the epitope, but differences from using the two differentradiation types were not observed. They concluded bothtypes of irradiation had similar characteristic based on theresults obtained from SDS-PAGE, immunoblotting andELISA, hence could be used for an inhibition and a reduc-tion of a food allergy.

Herrero, Carmona, Ord�o~nez, de la Hoz, and Cambero(2009) evaluated the electron-beam irradiated (doses of0, 1, 2, 3, 4, and 8 kGy at energy level of 10 MeV)cold-smoked salmon with Raman spectroscopy. These re-searchers reported that the irradiation at 8 kGy originatedmodifications on the protein secondary structures witha decrease (P < 0.05) in a-helix protein backbone ar-rangement and the concomitant increase (P < 0.05) inb-sheet content, turns and unordered structure. They alsoemphasized that the irradiation dose of �1 kGy provokeda significantly decrease in 1518 cm�1 band intensity,which can be attributed to a decrease in carotenoid contentin cold-smoked salmon.

The application of electron beam was also extended tothe production of protein-based biodegradable films.Sabato, Nakamurakare, and Sobral (2007) evaluated themechanical and thermal properties of films made with Tila-pia proteins by applying electron-beam radiations (doses of25e200 kGy with beam current of 2.01 mA and accelera-tion energy of 0.550 MeV). They reported that a slight im-provement on tensile strength was observed at radiationdose of 100 kGy, most probably attributed to the radia-tion-induced cross-linking of protein molecules. However,thermal analysis by DSC showed no visible effect of theradiation doses on proteins.

Table 2. (a). Some reports on the effects of UV irradiation on plantproteins. (b). Effect of UV irradiation on animal proteins.

Protein source Application Reference

(a)Soy protein Protein films Gennadios et al., 1998Corn zein Protein films Rhim et al., 1999Wheat gluten Protein films Micard et al., 2000;

Rhim et al., 1999

(b)Collagen Food system/

pharmaceuticalCurran et al., 1984;Fujimori, 1965;Kano et al., 1987;Uchida et al., 1990

Sardine paste Food system Ishizaki et al., 1993a,1993b

Pork meat paste Food system Ishizaki et al., 1993a,1993b

Walleye Pollackpaste

Food system Ishizaki et al., 1993b

Flying fish paste Food system Ishizaki et al., 1993b,1994

Egg albumin Protein films Rhim et al., 1999Food system Kuan et al., 2011

Sodium caseinate Protein films Rhim et al., 1999Food system Kuan et al., 2011

Fish gelatin Food/pharmaceutical

Bhat & Karim, 2009b


Recently, a study was undertaken to produce caseinatebased biodegradable films with improved water resistanceby applying electron-beam irradiation (doses of 20, 30,40, 50, and 60 kGy) (Audic & Chaufer, 2010). They alsocompared the effectiveness of electron-beam irradiationover formaldehyde treatments on the films production. Re-duction on protein solubility was observed for both treat-ments indicating the occurrence of protein cross-linking.However, the protein cross-linking with formaldehydewas significantly more efficient than with electron beam.Therefore, they only employed formaldehyde treatmentson their continuous study. They studied the effect of surfacemodification on plasticizer exudation with triethanolamine(TEA) by employing pulse voltammetry. Surface modifyingadditives (SMA) based on sodium caseinate and organo-silicones were used to modify films surface properties.They found out that the SMAwere less efficient in control-ling the plasticizer exudation rates but could significantlyreduce surface energy to 42 mJ m2.

Effects of ultraviolet irradiation on proteinsfunctionalities

The use of UV radiation to induce cross-linking has beenapplied in food proteins to cause cross-linking in the DNAof microorganisms, leading to the lethal breakdown of mi-croorganism. It was observed that the UV irradiation couldenhance the physicochemical and functional properties offood proteins. It has been reported that under certain favor-able conditions, proteins could be cross-linked (Urbain,1977). These conditions include the use of physical, chem-ical and enzymatic treatments as previously discussed.However, the toxicity of chemical to induce cross-linkingremains unknown, and the use of enzyme is time consum-ing and costly. Therefore, of late, UV irradiation appears tobe the most convenient method to alter the properties offood proteins. Table 2 summarizes the effects of UV radia-tion and the doses delivered on various types of food pro-teins, as well as their activity. In the following text, somerecent reports on the application of UV radiation in variousfood proteins are detailed, including the plant and animalproteins.

Early studies of UV irradiation have been shown to mod-ify proteins in general (McLaren & Shugar, 1964) and col-lagen in particular (Cooper & Davidson, 1966). Theapplication of UV irradiation to modify and improve onthe skin has been interpreted as causing “aging” in collagenthrough cross-linking within the collagen fibrils (Shuster &Bottoms, 1963). Fujimori (1965) disclosed the applicationof UV irradiation to cross-link the collagen. Nakatsuka,Suzuki, Tanimoto, and Funatsu (1978) then patented theuse of UV light to produce a cross-linked edible film. Inthe year 1996, Kelman and Devore (1996) disclosed biolog-ical compatible reaction products from collagen which arepolymerized by exposure to UV radiation. The material ismolded to form useful medical implant articles. The pro-cess for strengthening the collagen casings for the

application in food industry using UV irradiation hasbeen patented (Miller & Marder, 1998). Resulted effectson these UV-irradiated casings improved the uniformityin tensile values, mechanical properties and thermal resis-tance for the application in encased sausages.

However, there are reports on the oxidative degradationon collagen upon UV irradiation (Curran, Ammoruso,Goldstein, & Berg, 1984; Kano, Sakano, & Fujimoto,1987; Uchida, Kato, & Kawasaki, 1990). This shows thatdegradation/fragmentation occurs simultaneously withcross-linking in protein. Although many studies on the frag-mentation of collagen with UV irradiation have been per-formed, however there are very less studies published onthe mechanism of its oxidative fragmentation of collagen.Kato, Uchida, and Kawakishi (1992) studied the effectsof UV irradiation on collagen and its model peptides.They found out that degradation and fragmentation of col-lagen were predominant in the system by employing gel fil-tration chromatography. They also found out that thefragmentation was presumably due to oxidation of proline,since collagen is a proline-rich protein and proline residueson collagen dictated marked decrease with irradiation treat-ment. They clarified this mechanism by using poly(L-pro-line) and (Pro-Pro-Gly)10 as models of a collagenmolecule. Glutamic acid, g-aminobutyric acid (GABA),and ammonia from the hydrolyzates of the irradiated propylpeptides were identified by amino acid analysis. It was pre-sumed that GABA was generated from a 2-pyrrolidonestructure by acid hydrolysis. To confirm this prediction,they exposed N-tert-butoxycarbonyl (Boc)-L-proline and


N-tert-Boc-L-prolylglycine were exposed to UV light, andthe irradiated products were isolated and characterized.Then, N-tert-Boc-2-pyrrolidone was identified from bothUV-irradiated N-tert-Boc-L-proline and N-tert-Boc-L-pro-lylglycine. They proposed that the formation of the 2-pyrrolidone compound contributed to the fragmentation ofprolyl peptide on the bases of its structural property.

A study was undertaken to investigate the possible im-pact of UV irradiation (exposure times of 30 and 60 minat wavelength 253.7 nm) on the gel strength, viscosity,and thermal properties of a commercially procured fish gel-atin samples (Bhat & Karim, 2009b). These authors re-ported that the irradiated samples exhibited significantimprovement in the gel strength, marked reduction in vis-cosity, and significant changes in the melting enthalpy byDSC study. They explained that the improvement in thegel strength was attributed to the occurrence of UV-induced cross-linking and the reduction in viscosity was at-tributed to the UV-induced fragmentation. Their resultsdemonstrated the prospects of employing UV radiation asan alternative method over conventional means of improv-ing some of the quality attributes of fish gelatin.

Other than being applied in cross-linking of collagenand gelatin, UV irradiation has been extended to the mod-ification of meat pastes from fishes and poultries. Ishizakiet al. (1993a) studied the effect of UV irradiation (at wave-length of 250e400 nm, and intensity up to 8000 mW/cm2)on the rheological properties of thermal gels from sardineand pork meat paste. Based on their study, an increase ingel strength was observed on the thermal gels preparedfrom sardine and pork meat upon increasing UV intensity.They explained that the improvement on gel strength wasdue to the increases in both elasticity and viscosity. SEMstudy showed that the microstructure of these UV-irradiated gels has a denser structure which is less porousthan the control. They suggested that these changes wereattributed to the denaturation of meat proteins caused byUV irradiation.

To further elucidate the conformational changes in thesemeat proteins, the authors later evaluated the hydrophobicinteraction and disulfide bond in the UV denaturation of ac-tomyosins in the samples (Ishizaki et al., 1993b). A DSCstudy was also carried out. Results showed that UV irradi-ation significantly increased the surface hydrophobicity andreduced the total SH content of all the samples. Data fromDSC evidenced that a conformational change or partial un-folding of actomyosins occurred during UV irradiation.Hence these changes were closely related to the enhancingeffect on the thermal gel formation on the paper reportedpreviously. However, they concluded that the relation be-tween denaturation states and increasing gel strength isnot fully understood yet.

They further extended their study to evaluate the UV-induced denaturation of meat proteins by using flying fishmyosin as a sample (Ishizaki, Ogasawara, Tanaka, &Taguchi, 1994). The UV denaturation of flying fish myosin

was examined by means of solubility test, circular dichro-ism (CD) measurement, 8-anilino-1-napthalene sulfonate(ANS) intensity, and tryptophan fluorescence intensitymeasurements. They reported that the solubilities of UV-irradiated myosin decreased with increasing irradiationtime and UV intensity. CD-curves illustrated that the a-he-lical content of myosin decreased rapidly with increase ofUV intensity and UV irradiation time. The ANS and trypto-phan fluorescence intensities of UV-irradiated myosin alsodecreased upon UV irradiation. The SDS-PAGE patternsshowed increased myosin polymers with increasing irradi-ation time, indicating the occurrence of UV-induced poly-merization and coagulation involving hydrophobicinteractions between the myosin molecules.

Jiang, Leu, and Tsai (1998) evaluated the gel strength ofmackerel surimi actomyosin brought by combined treat-ments of UV irradiation (exposure times of 0, 5, 10, 15,20, 25, 30 and 35 min at wavelength of 365 nm, and inten-sity of 850 mW/cm2) and microbial transglutaminase(MTGase) (0e0.52 unit g of meat). They reported thatthe gel strength of minced mackerel with MTGase aloneat a concentration of 0.47 unit/g reached 1789 g cm, whichwas 3 times greater than that of control. When MTGase-supplemented minced mackerel was exposed to UV lightfor the optimal irradiation time of 20 min, the gel strengthwas further increased by 25%. Results obtained from SDS-PAGE analysis suggested that UV irradiation acceleratedthe MTGase to catalyze the cross-linking of myosin heavychains in mackerel actomyosin.

The application of UV irradiation technique was also ex-tended on the production of protein films. Gennadios,Rhim, Handa, Weller, and Hanna (1998) have reportedthe production of soy protein film by exposure to UV irra-diation (doses of 0, 13.0, 25.9, 38.9, 51.8, 77.8, and 103.7 J/m2 or exposure times of 0, 6, 12, 18, 24, 36 and 48 h). Ac-cording to them, the efficiency of UV radiation depends onthe protein source and in particular on amino acid compo-sitions and molecular structures. In their study, films pro-duced were evaluated for tensile strength, elongation atbreak, water vapor permeability, and Hunter L, a, andb color values. They reported that the tensile strength sig-nificantly (P < 0.05) increased linearly while elongationat break decreased linearly with the increasing of irradia-tion exposure time. Yellowish coloration (increasing Hunterb values) was observed upon increasing UV exposure time.SDS-PAGE patterns revealed bands of aggregates, and thiseffect increased with increasing UV exposure time. Theyexplained that these changes were caused by UV-inducedcross-linking in films. However, the water vapor permeabil-ity was not affected by UV irradiation. They proposed thatthe reduction on water vapor permeability on soy proteinfilm may be reduced by applying greater UV dosages or ex-tended exposure time.

They further explored the possible changes of UV irradi-ation (dose of 51.8 J/m2 over 24 h of exposure with a wave-length 253.7 nm) on films made with wheat gluten, corn


zein, egg albumin and sodium caseinate (Rhim, Gennadios,Fu, Weller, & Hanna, 1999). They reported that UV treat-ment increased tensile strength of gluten, zein, and albuminfilms suggesting the occurrence of UV radiation-inducedcross-linking within film structures. For caseinates films,UV treatment did not affect tensile strength but substan-tially reduced total soluble matter. Small but significant de-creases in total soluble matter also were noticed for UV-treated zein and albumin films. UV irradiation reduced wa-ter vapor permeability of albumin films but did not affectwater permeability of other types of films. Gluten, albumin,and caseinate films had increased yellowness as a result ofUV treatment. In contrast, UV treatment decreased the yel-lowness of zein films, possibly due to the destruction ofzein pigments by UV radiation. They concluded that UV ir-radiation showed potential for modifying properties of pro-tein films.

Recently, UV irradiation has been shown to improve theemulsifying and foaming properties of egg white proteinand sodium caseinate (Kuan et al., 2011). UV irradiationof the proteins was carried out for 30, 60, 90, and120 min at wavelength of 253.7 nm. However, the sodiumcaseinate were subjected to extended UV irradiation for 4and 6 h as no difference was found on the initial UV expo-sure time. Results obtained from formol titration, SDS-PAGE, and FTIR analyses indicated that UV irradiationcould induce cross-linking on proteins and led to improvedemulsifying and foaming properties. They concluded thatthe UV-irradiated proteins could be used as novel emulsi-fiers and foaming agents in broad food systems.

Conclusions and future trendsIt is evident from the available literature that radiation

technology is a promising technique that could be em-ployed for modification of food protein, especially on theprotein films and gelation properties. Majority of the re-search works reported in this review are mostly on the ef-fects of irradiation on protein films properties but not onimproving protein functionality. Therefore, there is lackof information on the effects of irradiation on other proteinfunctionalities aside from being applied on the productionof film. Generally, irradiation is known as the non-thermal and cost effective technique for protein films mod-ification. Future studies might be expanded on the otherfood systems such as spreads, bakeries, emulsions, aeratedproducts, pharmaceuticals, etc. by applying this radiation-induced protein modification within the permitted doses.These studies could be designed to ascertain the effectsof irradiation on the gelling, foaming and emulsifying prop-erties for different irradiation time or doses within the per-mitted doses. Furthermore, finding the best conditions,doses, and combination treatments for protein irradiationare crucial in order to achieve the desired functionalproperties.

Reports available on the use of electron beams, X-raysand microwave irradiation on protein modification are

rather scarce and need to be investigated. Further studieson the effects of electron beams, X-rays and microwave ir-radiation could be designed to elucidate the chemicalchanges on proteins. Studies considering which type of pro-tein, or amino acid that is responsible for the irradiation-induced chemical and compositional changes on the mole-cule, as well as the relationship of irradiated proteins withother food components in a food system would be worth-while attempts. Information on the actual biochemicalmechanism causing the polymerization or depolymeriza-tion in the proteins is still obscure, which could be exploredin detail.

Furthermore, biodegradable polymeric materials havegained strong interest on the encapsulation of antioxidants,vitamins, minerals, fatty acids, probiotics, food additives aswell as the controlled-release of drugs and bioactive com-ponents to the targeted site. These materials can be derivedfrom polysaccharides and proteins. It has been reported thatthe application of these materials caused undesirable earlyrelease of drugs or core materials, as attributed to the po-rous structure. Irradiations have been shown to reduce thepore size on the bio-films by various researchers in this re-view. Therefore, it would be a worthwhile attempt to em-ploy the irradiation treatments on the improvement ofencapsulation efficiency.

AcknowledgmentsYau-Hoong Kuan gratefully acknowledges a postgradu-

ate fellowship and a postgraduate research grant (1001/PTEKIND/832048) from Universiti Sains Malaysia.

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