The Influence Of Covalent Cross-linking On Whey Protein Film Formation And Its Mode Of Action

With the rapid growth of the world population, the stress upon the environment thereof, and thegradual depletion of natural resources, the use of biodegradable and recyclable materials to replacenon-biodegradable petroleum-based plastics in food packaging has received considerable attention. As anexample, whey protein isolate （WPI） has been used to prepare edible films and coatings, and the novelpackaging materials appear to have great potential for food applications. To produce films with diverseproperties suitable for the specific food packaging needs, WPI is often subjected to physicochemicalmodifications. Several attempts have been made to modify proteins or promote protein-protein interactions,for example, cross-linking, thereby improving the mechanical properties of WPI films. A potential methodto produce cross-linked proteins potentially suitable for film formation is oxidation with reactive oxygenspecies, an approach commonly taken in the polymer industry where radical chain reactions lead to thepolymerization of ethylene, styrene, and other small molecules into plastic packaging sheets. Anotherpossible approach is to employ natural cross-linkers to induce WPI covalent cross-linking for filmsmodification.So far, little information related the effects of free radical reaction, malondialdehyde （MDA） andnatural cross-linkers on the film-forming properties of WPI. Therefore, the general objectives of thisresearch were:（1） to investigate the influence of hydroxyl radical （OH）-initiated oxidation, MDA andcross-linkers on the film-forming ability of WPI;（2） to explore the forces involved in the WPI filmformation under oxidative stress or cross-linkers; and （3） to establish the relationship betweenprotein-protein interaction or cross-linking and the mechanical and structural characteristics of WPI films.The ultimate goal of the dissertation research was to improve the theoretical understanding of protein filmformation by means of radical reactions and natural cross-linkers and the implications of the resultingedible films in food packaging.In experiment1, the Fenton oxidizing system was employed to evaluate the effect of hydroxyl radicalson the film-forming properties of whey proteins. Sequential heating （70-90°C） then oxidation （0.1mMFeCl3/mM ascorbate/0-20mM H₂O₂）（H�'O） or vice versa （O�'H） were conducted to oxidize/unfold WPIat pH6.8and8.0before casting to use OH cross-link protein and to simulate oxidative effect of WPIduring storage, respectively. The result of total sulfhydryls （SH）; disulfide bonds （SS）; and proteincarbonyls in samples and electrophoresis analysis indicated that oxidation promoted protein cross-linkingmainly though SS and partial non-reducing covalent bonds, and also lead to the loss of methionine andproline. The resulting films were characterized through mechanical, microstructural, protein electrophoretic,hydration, transparent, and color analyses. The obtained results showed that the1mM H₂O₂was a criticalconcentration to affect the above aforementioned functional properties of WPI films. Tensile strength （TS）,elongation at break （EAB） of films decreased for WPI oxidized by higher concentrations of H₂O₂（>1mM）.Meanwhile, protein leachability at pH3-7and protein solubility in water of films dramatically increased asH₂O₂increased. Electrophoresis identified the leached fractions from WPI films were protein polymers thatmainly maintained by SS. Moreover, the color and transparency of resulting WPI films also were slightlyaffected by oxidation. Higher heating temperature （e.g.:90°C） and pH can compensate the loss offunctional properties of films caused by oxidation. In conclusion, oxidation promotes cross-linking butimpairs film-forming properties of whey proteins.In experiment2, ployphenols were used because they are abundant in plants, which endowed with theproperties of anti-oxidation, low toxicity and reactive activity with proteins. The oxidized caffeic acid（OCA） at2%or4%concentrations, oxidized ferulic acids （OFA） and oxidized tannic acids （OTA） both at 2.5%and4%concentrations （based on protein content, w/w） were added to the preheated6%（w/v） wheyprotein solutions to make film-forming solutions and cast films. The physiochemical properties （SH, aminogroups, hydrophobic property, particle size, and ζ–potential） of cast solutions, degree of cross-linking,mechanical properties, thermal, optical （transmittance and transparency scanned at200-800nm）, watervapor permeability （WVP） hydration （swelling and leachability）, and digestible properties of WPI-basedfilms were systematically evaluated. The characteristics of cast solutions suggested that both of themreacted with SH in cysteine residue and amino groups at least, and consequently promoted proteincross-linking. However, the reactive activity of OCA or OTA was higher than that of OFA. OCA, especiallyfor OTA, leads to higher “hydrogen content” in WPI film matrix, whereas the roles of hydrogen bonds andhydrophobic property were decreased by OTA. The changes in force involved in film matrix could accountfor OCA or OTA-treated WPI films resulted in improving TS and stiffness whereas OFA-treated WPI filmsresulted improving EAB, and the former （OCA and OTA treatments） also highly improved thermalresistance and significantly decreased light transmittance at200-600nm and transparency （P <0.05）compared with of OFA, but the later treatment did not dramatically affect the transparency of WPI films.Both of OCA, OFA and OTA treatments slightly decreased water vapor permeability （WVP）.Microstructure characterized by atomic force microscopy （AFM） and scanning electron microscopy （SEM）unveiled OCA or OTA induced the porosity and enhanced roughness of WPI films which caused highprotein leachability （especially at pH6and7） and swelling rate, however of OFA was contrary. SDS-PAGEanalyzed the leached fractions at pH6.0were protein polymers that mainly maintained by SS. Lastly, OTA,digestibility of WPI films can be inhibited when incorporated with OCA or OTA at higher concentrationswhereas no remarked influence on it to OFA. Generally, these effects can be attributed to the polymerizednetwork due to partial cross-linking and physical influence induced by them. Therefore, at least,3types ofthese oxidized polyphenols can be applied in food to replace toxic protein cross-linkers.In experiment3, with regard to the availabilities of SH/SS exchange reaction and transglutaminase（TG） cross-linking protein, cysteine （Cys） and dithiothreitol （DTT） concentrations both at1%or4%（basedon protein content, w/w） were used alone or combined with4U TG treatments to investigate their effectson the functional properties of WPI films. A cast film was made by the solvent cast way. TheCys/DTT-treated cast solutions samples were performed by SDS-PAGE, and the result showed WPI wasinhibited to cross-linking due to loss of SH�'SS reaction caused by N-ethylmaleimide （NEM）, wasincreasing cross-linked by Cys with its adding amount, and was initially increasing cross-linked by DTT tomaximum degree at its1%concentration then was turned down till to4%. When TG was integrated to Cysor DTT, protein cross-linking was further cross-linked; however no impact of concentration of Cys or DTTon cross-linking when TG applied can be observed. Solubility in various solvent （S1-S5） indicated Cyspromoted the role of SS in WPI film matrix whereas DTT enhanced the role hydrophobic force. When TGwas combined to Cys or DTT, the effect of such combination increased the role of SS in Cys+TGcross-linked WPI film matrix but reduced hydrophobic force in DTT+TG cross-linked WPI films matrix.The changes of these forces involved in WPI film matrix correspond to mechanical properties of resultedWPI films. The Cys or DTT treatments improved TS and EAB and Cys+TG or DTT+TG treatmentsdecreased TS （compared to Cys or DTT use alone）, however Cys or DTT combined TG treatment enhancedEAB regardless of their concentrations （except1%Cys）. Moreover, Cys or DTT treatments improvedthermal properties and decreased protein leachability of WPI films as improved or decreased by theirconcentrations increased and TG added. In other words, duplicate effect of Cys or DTT and TGco-improved thermal stability and decreased protein leachability of resulting films. Similarly, the behaviorwas of thermal stability. Cys or DTT use alone significantly decreased swelling rate of resulting films（except1%Cys）, however when TG was combined to Cys or DTT, the swelling rate remarkably increased（compared to Cys or DTT use alone）. The microstuctural analyses suggested the more rough structure of resulting films could account for increasing swelling rate.In experiment4, cinnamaldehyde （CA）, which is a natural product derived from cinnamon andendowed with the antimicrobial/antifungal properties, was incorporated into whey proteins to make films.Another two types of aldehyde （formaldehyde, FA; malondialdehyde, MDA） was also used in this sectionto serve as a reference （FA） and to simulate the effect of product of lipids oxidation （MDA） on the proteinfilm-forming properties, respectively. CA and FA concentrations both at1-4%（based on protein content,w/w） were added to90°C preheated WPI reacting with protein for1h, whereas1-4%MDA （based onprotein content, w/w） was added to native WPI solution for reacting1h and was then heated at90°C. Theprepared solutions were to measure contents of SH and free amines and to make cast films. The changes inchemical content were reflected to Cys and NH2involved in aldehyde-protein reaction and consequentlylead to WPI cross-linking through non-reducing covalent bonds. The sequence of reactive activities ofabove3types of aldehyde, which based on the amount of non-reducing covalent bonds formed from thereaction, was MDA> FA> CA. Solubility in various solvent （S1-S5） indicated CA enhanced the role of SSin the resulting films, whereas FA and MDA treatments improved the roles of newly non-reducing covalentbonds. Both of them strengthened mechanical properties of resulting films exclude4%MDA decreasingEAB. CA treatment gave birth to slight greenness but trace yellowness in the film, decreased lighttransmittance at200-400nm, MDA impairs sensory properties of WPI films whereas FA treatment did notaffect color, light transmittance and transparency. Thermal stability of resulting films characterized bydifferential scanning calorimetor （DSC） was not affected by the treatments of CA, FA, or MDA both at2%concentrations, however, WVP and protein leachability were greatly decreased by them which orders wereFA> CA> MDA and MDA> FA> CA, respectively. AFM and SEM concealed the microstructuralcharacteristics showed that CA or MDA-treated WPI films were porous but rough, whereas FA-treated WPIfilms were refined; however, these changes seemly increased swelling rate of resulting films less than bynon-reducing covalent bonds.Overall, the results from this research indicated that protein covalent cross-linking plays a critical rolein WPI-based films, and the mechanism in different cross-linkers to WPI is diverse. Therefore, the proteinsubjected to cross-linking could form a film endowed with diverse functions. Generally, cross-linking canpromote WPI forming a improved functional film. The understanding on protein cross-linking obtainedfrom this research provides fundamental theory and guides to develop or explore new protein cross-linkersfor foods. Moreover, the developed protein covalent cross-linking theory guides protein applied in otherfood systems （e.g.: gels or emulsions） for pursuing improved functional properties.