Atmospheric Pressure Cold Plasma and Ozone Technologies: Unconventional Treatments in the Dairy Field. Study of effects on milk proteins as model systems and relevant case studies

Among non-thermal technologies, atmospheric pressure cold plasma (ACP) and ozone treatments have gained enormous pace especially for their safety assurance and sustainability. Many studies and data regarding ACP inactivation of food-borne pathogens are already available in the literature. Most of them concern the decontamination by microorganisms in buffer or food matrices. However, ACP provides opportunities in several other applications including the modifications of food properties for improved functionality and healthy products. The interaction between ACP and whey protein isolate (WPI) and alkaline phosphatase (ALP) enzyme model solutions were investigated in two different experiments. The aims were to study the effects on structure and functionality of the whey protein solutions and structure and residual enzymatic activity of the enzyme solution. In both cases, the findings covered a gap in the dairy field where the general information and research were insufficient. In the first experiment, the results showed an increase in yellow colour and a minor reduction in the pH value in the whey protein solutions (2% w/v). These results were attributed to the reactions of reactive oxygen and nitrogen species of the plasma diffusing into the liquid. The carbonyl groups and the surface hydrophobicity increased, in addition to the reduction of free sulfhydryl (SH) groups, probably due to mild oxidation, which occurred in the proteins following the ACP treatments for 15 min. The protein structure modifications revealed a certain degree of unfolding, which improve foaming and emulsifying capacity. Upon extended treatment for 30 and 60 min, the changes were quite pronounced: the carbonyl and sulfhydryl groups reached equilibrium, the surface hydrophobicity remarkably increased and small aggregates were formed. Overall, the foaming and emulsifying capacity dramatically decreased against an increase of foam stability. In the second experiment, the ALP subjected to the ACP was inactivated in a few seconds and the Weibull model was found to best describe the observed variance in residual activity for all the voltages applied. The results from the circular dichroism spectroscopy revealed a predominance of the α-helix structure, with a tendency to decrease with increasing treatment time and voltage. The ACP treatment did not affect the temperature and pH of the enzyme solution. Since it is well known that protein functionality and protein structure are strictly connected, in the third experiment possible modifications in the secondary structure of WPI in powder treated with ACP at different voltages and times were studies, throughout the Fourier Transform-Infrared (FTIR) spectroscopy. The second derivate of the amide I region of the spectra is obtained and the results show that the β-structures are relatively more stable compared to the α-helix. Changes in the α-helical structure are evident, and significantly affected by treatment time, at the lower voltage applied than the higher, for which no definite trend is recorded. The study on model systems of whey proteins is also extended using solely the gaseous ozone at a high concentration in order to evaluate the changes in protein structure and their consequences on selected functional properties (fourth experiment). The results show a reduction of free SH groups and an increase in surface hydrophobicity, indicating a self-rearrangement in the protein structure following ozonation. Thus, ozonation allows for the creation of a more flexible structure without forming a strong network of disulphide bonds or aggregations (confirmed by the turbidity analysis and SDS-PAGE). Moreover, ozone processing induces modifications that improve foaming capacity and foam stability, however, a slight reduction in the solubility is encountered. It is a well-known fact that ozone has a strong oxidant potential and the main applicability is related to the decontamination of foods, so the fifth and sixth experiments were focused on relevant case studies in the dairy field. The fourth one aimed to evaluate ozone effectiveness in reducing viable spoilage bacteria load throughout the high moisture (HM) Mozzarella cheese-making process. Many approaches in different phases of processing were tested but the ozone (gaseous and also solubilised in water) was not effective in surface microbiological decontamination of cheeses. However, it can be successfully applied in Mozzarella cheese processing only to decontaminate water contaminated by potential spoilage bacteria before coming into contact with the product. Finally, the last experiment aimed to reduce the microbial load of cheese brines contaminated by the reuse. The different ozone concentrations tested obtained a sufficient Log reduction. Moreover, considering the intermediate concentration tested (0.40 ± 0.2 ppm), the treatment time was extended up to 240 min to obtain a 4-Log reduction of the total microbial count (TMC) and other microorganisms considered. The longer the treatment time, the better the sanitisation effect. When the cheese brine samples with different protein contents (from 0.20 to 1.30 g/100 g) were treated, different levels of microbial inactivation were obtained: the higher the protein content, the lower the microbial reduction.