The effect of shredding and freezing on Mozzarella cheese microstructure and functionality

© 2018 Dr. Anita Penelope PaxMozzarella cheese is the third most produced cheese in Australia. A significant proportion of Mozzarella cheese contributes to the 34% of dairy products in Australia that are destined for export, making the extension of shelf life important. Processes that occur after cheese is manufactured (post manufacture) are of great importance to product quality and diversifying product range as well as the optimised storage and handling that will enable the supply of distant export markets. In this thesis the effect of storage format was first investigated, including storage as a block or shredded Mozzarella cheese. The aim was to determine whether the functionality and microstructure were altered between formats or as a function of storage time. Shredding is also a common process prior to the use of Mozzarella as an ingredient, such as on pizza, however the effects of shredding on Mozzarella cheese structure and function are not fully understood. Cheese was shredded at day fifteen or week eight and stored alongside block cheese for a total storage time of eight weeks, based on manufacturer guidance on shredding times. Shredding resulted in alterations to the microstructure examined after eight weeks; the alignment of protein was reduced and the fat globule surface area increased. Interestingly, after eight weeks, shredded samples also had increased proteolysis and a higher level of two bacterial proteases. Between block and both shredded treatments the profile of volatile compounds was slightly altered. The microbial viability was, however, unaffected and the functionality of the cheese was preserved. This work showed that shredding at either time (i.e., two or eight weeks) causes only slight changes in product properties, producing a product with similar microstructure and functionality. This study may provide dairy manufacturers with the flexibility to optimise shredding processes whilst potentially reducing storage and product handling costs. The effect of extended frozen storage was considered next. Temperatures below freezing may be used to extend the shelf life of cheese; however, such storage is known to affect the functionality of Mozzarella cheese. Studies to date have had limited applications as they did not use commercially relevant samples or cooling conditions, as well as the majority of studies testing functionality not assessing biochemical changes in parallel. The effect of frozen storage (-18 °C) as well as tempering at 4 °C for one and three weeks after frozen storage was compared to storage at 4 °C over a six month period using commercially relevant conditions and samples. Frozen storage prevented proteolytic breakdown, however, tempering for longer allowed some proteolysis to occur. In contrast to prior studies, structural damage due to the industrial freezing conditions used was not detected by confocal microscopy. The majority of key measures, including microstructure, proteolytic parameters, meltability and stretchability were altered as a function of time at 4 °C, with tempering leading to a positive improvement in properties. Interestingly, two measures of shreddability were also altered as a function of frozen storage time, suggesting structural or rheological changes with extended frozen storage. These results provide a greater understanding on the link between freezing and tempering and functional properties not previously measured, as well as contributing to the understanding of refrigeration and freezing time on key parameters. This study may provide dairy manufacturers with the guidelines to optimise Mozzarella cheese functionality after frozen storage as well as frozen conditions that make reaching distant export markets possible. The suitability of synchrotron FTIR (S-FTIR) microspectroscopy as a label-free technique to examine cheese microstructure was also assessed in this thesis. S-FTIR microspectroscopy has not been widely applied to dairy products despite the additional molecular information this technique can provide. S-FTIR microspectroscopy in conjunction with multivariate data analysis was successfully able to spatially resolve the characteristic pasta filata Mozzarella cheese microstructure with areas of both high lipid and high protein identified in both transmission and attenuated total reflectance (ATR) modes. The heterogeneity typical of pasta filata Mozzarella cheese was captured with S-FTIR microspectroscopy, highlighting the advantage of the technique over laboratory based spectroscopy for encompassing sample variation. Similar information was obtained with both sampling modes and sample preparations, allowing technique selection to be based on available sample preparation methods, equipment configurations and the time available for experiments. Despite the limited availability of synchrotron based FTIR microspectroscopy, this technique provides a complementary tool that may be used by dairy manufacturers to differentiate cheese with diverse ages and thermal histories. Finally, the S-FTIR microspectroscopy technique in transmission mode was applied to Mozzarella cheese stored for six months at 4 °C and -18 °C, as well as cheese tempered for three weeks after frozen storage and cheese aged at 4 °C for four weeks prior to frozen storage in order to assess the effect of time and treatment on protein secondary structure. This technique has not been applied spatially resolve the secondary structures of cheese samples with commercially storage conditions. When Mozzarella cheese was aged at 4 °C there was a significant increase in α-helical conformation in the first month. A reduction in sample variation, i.e., an increase in sample homogeneity was observed after six months storage, consistent with the greater extent of proteolysis at this time. Freezing initially induced random coil formation; however, frozen storage halted structural and biochemical changes such as proteolysis when assessed over a six month storage period. Ageing prior to frozen storage and tempering after frozen storage caused different effects on secondary structure conformation, confirming that extensive structural changes occur in Mozzarella cheese in the first month post manufacture. Analysis of secondary structures using PCA provided significant information on the effect of frozen storage, tempering and ageing that may be used to improve our understanding of storage and handling conditions. Further study of protein secondary structure will assist the development of guidelines to optimise cheese ageing and freezing based on expected structural conformations. The results presented in this thesis provide the Australian dairy industry and dairy researchers with greater knowledge on the effect of shredding and freezing processes on Mozzarella cheese. They increase our understanding of the effect of these processes on the microstructure, biochemical processes including proteolysis and lipolysis and functionality of Mozzarella cheese. A new S-FTIR microspectroscopy method has been developed for application to dairy products and the ability of this technique to detect structural changes in the protein network as a function of ageing and freezing demonstrated. This technique will be broadly applicable to develop new knowledge on the structure of proteins and lipids in cheese