Development and characterization of bioactive, edible whey protein films and coatings to improve quality and safety of food products

In the last decade major efforts have been made by packaging and food industries to reduce the amount of residual waste associated with food consumption, in particular the development of edible packaging materials. Viable edible films and coatings have already proven to be successful when manufactured from whey proteins. Their ability to serve other functions, e.g. vehicles of antimicrobials, antioxidants or other nutraceuticals, without significantly compromising their desirable primary barrier and mechanical properties as packaging films, will add value to eventual commercial applications. The study reported in this thesis consisted of the development and characterization of bioactive edible films and coatings using whey proteins at 10 %(w/w) as feedstock, in order to improve the quality and safety of selected food products, using cheese as a case study. Hence, various whey protein products available commercially, including one whey protein isolate (WPI) and three whey protein concentrates (two forms of WPC 80, and WPC 50), were characterized in terms of their chemical and protein composition. Microdifferential scanning calorimetry (μDSC) and rheometry were used, as the main analytical techniques, to ascertain how composition of such products affects the thermal and gelation properties as a function of pH. Acidic conditions increased the temperature of the dominant endothermic transition associated to β-lactoglobulin (β-Lg), but decreased the temperature attributed to α-lactalbumin (α-La) transition thus increasing the thermal stability of WPI, WPC 80A and WPC 80B. WPI, WPC 80A and WPC 80B exhibited the highest gel strength at pH 5. In addition, WPI (followed by WPC 80A) led to the stronger gels, irrespective of pH values, which may be attributed to the higher purity, the lower content of insoluble protein and aggregates and the higher β- Lg/α-La and Ca/Na ratios. In the case of WPC 50, gelation did not occur at all. Consequently, WPI and WPC 80A products, that exhibited the greatest gel properties, were used at 10 %(w/w) to produce edible films. Afterwards three levels of glycerol were added as plasticizer, on a protein basis, and such films were extensively characterized. The molecular structure, barrier, tensile, thermal, surface hydrophobicity and optical properties of the films obtained, were determined in attempts to provide a better understanding of the effects of the proteinaceous feedstock and the glycerol content. WPI films exhibited statistically lower (p < 0.05) moisture content (MC), film solubility (S), water activity (aw), water vapor permeability (WVP), oxygen and carbon dioxide permeabilities (O2P and CO2P, respectively) and color change values, as well as statistically higher (p < 0.05) density (ρs), surface hydrophobicity, mechanical resistance, stiffness, extensibility and transparency values than WPC 80A films, for the same content of glycerol. The above results were in turn consistent with thermal and Fourier transform infrared spectroscopic (FTIR) analyses, which indicated that the WPI films were stronger and more stable, presumably because of higher thermal properties, along with a higher degree of cross-linking via non- covalent and covalent bonds (associated with a higher content of β-sheet structures). On the other hand, a significant increase (p < 0.05) was observed in MC, S, WVP, O2P, CO2P, weight loss and extensibility of both protein films when glycerol content was increased; whereas a significant decrease (p < 0.05) was obtained in thermal properties, as well as in mechanical resistance and stiffness, thus leading to weaker films. From the previous tests, 10 %(w/w) WPI added with 50 %(w/w) glycerol, was chosen as the base formulation for development of antimicrobial edible films and coatings, since it proved the best compromise of technological features (particularly in terms of film extensibility). Several antimicrobial compounds, including citric and lactic acids, nisin, chitooligosaccharides (COS) with a MW <3 kDa and sodium benzoate, were tested for their minimum inhibitory and lethal concentrations (MICs and MLCs, respectively) against a number of relevant food-borne microorganisms frequently found in cheese: Gram-negative bacteria (e.g. Escherichia coli, Salmonella spp. and Pseudomonas fluorescens); Gram-positive bacteria (e.g. Listeria innocua and Staphylococcus aureus); and a yeast (e.g. Yarrowia lipolytica). Afterwards, the antimicrobial agents were incorporated in formulated coatings at their MLCs. Finally, the formulation with the highest activity against all microorganisms was assessed for its efficacy for coating cow’s cheeses, and compared with commercially available coatings. All agents proved efficient in terms of antimicrobial activity; lactic acid and COS, when combined, yielded the strongest effects against all microorganisms. COS showed the highest bactericidal effect against the Gram-negative bacteria, whereas lactic acid exhibited the greatest activity against the Gram-positive bacteria; the yeast was, in turn, strongly inhibited by sodium benzoate and COS. When applied on the surface of cow’s cheese, edible coating containing a combination of antimicrobial agents, lactic acid and COS, was more powerful towards bacteria but less effective against yeasts and molds than their commercial counterpart. In addition, several antimicrobial compounds, including lactic and propionic acids, COS with a MW <3 kDa and natamycin, were assessed for their MICs and MLCs against a model Gram-negative bacterium (e.g. E. coli), a model Gram-positive bacterium (e.g. S. aureus), and a model yeast (e.g. Y. lipolytica). Subsequently, the compounds were incorporated in edible films at their MLCs. The resulting antimicrobial features were evaluated using two complementary assays: one more qualitative and another more quantitative in nature, i.e. agar diffusion and viable cell counting, respectively. The effect of incorporating the compounds on the thickness, MC, S, ρs, aw, WVP, tensile and optical properties of those films was also evaluated. Films containing lactic and propionic acids or COS exhibited antimicrobial activity against all microorganisms tested. Moreover, the viable cell count assay was more sensitive and reproducible than the agar diffusion one. COS was the most active agent against Gram-negative bacteria, whereas lactic acid was the most active against Gram-positive ones. Natamycin did not exhibit activity against bacteria, but displayed the strongest effect against yeast. Incorporation of the antimicrobial compounds did not significantly increase (p > 0.05) film thickness; however, it significantly (p < 0.05) reduced mechanical resistance. Incorporation of lactic acid and natamycin did not significantly (p > 0.05) affect MC, S, ρs, WVP, extensibility and stiffness values. Nevertheless, a statistically significant increase (p < 0.05) of MC, S and WVP, together with a statistically significant decrease (p < 0.05) of ρs were attained upon incorporation of propionic acid or COS. Moreover, propionic acid produced the highest variation (p < 0.05) in mechanical resistance, stiffness and extensibility, whereas COS produced the highest change (p < 0.05) in optical properties. Finally, in order to improve the coating adherence to the surface of cheese and reduce the drying time of the edible coating base matrix (i.e. 10 %,w/w WPI with 50 %,w/w glycerol, on a protein basis) to wrap cheese, specific food-grade compounds at pre-selected concentrations (i.e. guar gum (0.7 %,w/w), sunflower oil (10 %,w/w) and Tween 20 (0.2 %,w/w)) were added to promote an increase of viscosity, hydrophobicity and stability, respectively. Following the results obtained with the antimicrobial edible coatings and films, combinations of antimicrobial compounds (i.e. natamycin and lactic acid, natamycin and COS, and natamycin, lactic acid and COS), were incorporated in the edible coating developed and the resulting solutions were applied on the surface of commercial Saloio cheese. The effectiveness of the antimicrobial coating solutions was evaluated by measuring the physicochemical, microbiological and sensory properties of coated cheese over a period of 60 days storage. Those properties were latter compared with cheese coated with commercial coatings and uncoated cheese. The physicochemical and microbiological analyses showed that application of the coating decreased water loss (ca. 10 %,w/w), hardness and color changes, as well as microbial development on cheese throughout storage. Statistically significant (p > 0.05) differences were not obtained in terms of weight loss, moisture, fat and salt contents, as well as of aw, pH and hardness between cheese samples bearing edible or commercial coatings, which demonstrate that the antimicrobial edible coatings developed, could be used as a suitable alternative to their commercial counterpart(s) regarding these properties. On the other hand, color analysis showed that cheese samples with any of the antimicrobial edible coatings exhibited a lower color change than cheese coated with commercial coating or without coating at all. In terms of microbiological properties, the antimicrobial edible coatings did not display growth (<100 CFU g-1) of Staphylococcus spp., Pseudomonas spp., Enterobacteriaceae, yeasts and molds which demonstrates their ability to assure the safety of cheese for, at least, 60 days storage. In addition, they did not inhibit lactic acid bacteria growth and these bacteria were present at high constant levels throughout the storage period. Commercial coatings inhibited growth of only yeasts and molds. With regard to sensory analysis, the antimicrobial edible coating containing natamycin and lactic acid was the most accepted by the panellists throughout the storage period, whereas the remaining edible and commercial coatings showed a similar (p > 0.05) acceptability.