Understanding and predicting storage stability of UHT milk

UHT milk is gaining market share worldwide for being a healthy, safe and convenient food product with a long shelf life at room temperature. However, the UHT treatment (usually 140- 150 °C for 2-10 s) contributing to its long shelf life may induce some changes, which may make it unstable during storage. Considerable levels of sedimentation (up to 1 % dry weight basis) of proteinaceous material at the bottom of the storage container, or formation of a gel (age-gelation), or both, can mark the end of shelf life of UHT milk, and hence affect its market potential and incur financial losses to the UHT dairy processors. Irrespective of a mechanism, gelation or sedimentation appears to be governed and preceded by changes in the extent and nature of interactions among milk proteins leading to their aggregation. Though recent investigations have added significantly to the understanding of the different protein interactions, the exact mechanism involving changes in these molecular interactions leading to sedimentation and gelation, is not clear yet. Moreover, less is known about the role of proteinlipid interactions in the development of a storage instability. In addition, the need of a rapid technique which can detect the shelf life of UHT milk has been emphasised in recent years. This is mainly because applying full-length shelf life tests in a product such as UHT milk would be time- and resource-intensive. Thus, the overall objective of this project was to understand and rapidly predict the storage stability of UHT milk. Accelerated shelf life testing is one such rapid alternative applied to a variety of food products to save time. The industry has not been successful in applying this approach to ultrahigh temperature (UHT) milk because of the chemical and physical changes in the milk proteins that take place during its processing and storage. Thus, the first objective of the study was to investigate the feasibility of applying accelerated shelf life principles to UHT milk samples with different fat levels to elucidate changes in interactions of milk proteins at ambient temperature using electrophoretic analysis (Native- and Sodium dodecyl sulfatepolyacrylamide gel electrophoresis). Samples of UHT skim (SM) and whole milk (WM) were stored at 20, 30, 40, and 50 °C for 28 days. Irrespective of fat content, UHT treatment had a similar effect on the electrophoretic patterns of milk proteins. At the start of testing, proteins were bonded mainly through disulphide and noncovalent interactions. However, storage at and above 30 °C enhanced protein aggregation via covalent interactions. The extent of aggregation appeared to be influenced by fat content, implying aggregation via melted or oxidized fat, or both. Based on the reduction in loss in the absolute quantity of individual proteins, covalent crosslinking in WM was facilitated mainly by products of lipid oxidation. Whereas, Maillard and dehydroalanine products appeared to be the main contributors to protein changes in SM. Protein crosslinking appeared to follow a different pathway at higher temperatures (≥40 °C), making it difficult to extrapolate these changes to lower temperatures. The changes identified under the accelerated shelf life conditions using electrophoretic analysis assisted in evaluating the potential of using another rapid technique, Fourier transform infrared spectroscopy (FTIR) in detecting changes in structure and interactions of milk proteins (the second objective). The feasibility of using FTIR to detect changes in conformational rearrangements, protein-protein and protein-lipid interactions was studied with accelerated shelf life protocols. WM and SM were stored at 20, 30, 40 and 50 °C for 28 days. The changes in FTIR spectra were observed concomitant with increased sedimentation in SM (by 50 %) and WM (by 20 %) at higher temperatures (40 °C) after 14 days of the storage period. Milk samples stored at 40 and 50 °C showed marked changes in the bands corresponding to the conformations of milk lipids and formation of intermolecular β-sheets, indicating protein-lipid interactions and aggregation. Dried sediment contained fat confirming protein-lipid participation in the sedimentation. FTIR was also able to detect changes that led to increased sedimentation in SM at temperatures lower than 40 °C, but only after 28 days. However, to establish appropriateness of accelerated shelf life testing and FTIR as a tool for prediction of stability of UHT milk, the observed correlation between spectral changes and sediment formation at accelerated temperatures has to align with that at normal storage temperature (third objective of the study). SM and WM were stored at 20 °C for 9 months to investigate the feasibility of using FTIR to predict sedimentation in UHT milk. Identified spectral marker variables corresponding to changes in the structure and interactions of lipids, proteins and carbohydrates successfully predicted sedimentation in SM (R2 -0.92) and WM (R2 - 0.60). Low predictability in WM may be due to the influence of fat. These markers were similar to those observed during accelerated shelf life testing, hence implying that the accelerated shelf life testing could be used in UHT milk. Among several changes in milk protein interactions during its heating and storage, conformational changes specific to an interaction remain largely unknown. Hence, the fourth objective of the project evaluated the possibility of fingerprinting two selected changes, i.e., deamidation and dephosphorylation, using FTIR. Enzymatic deamidation and dephosphorylation were carried out prior to heat treatment. Principal component analysis revealed that the heat treatment induced different changes in the secondary structure of control, deamidated and dephosphorylated milk samples. In contrast to a significant (P<0.05) decrease in β-sheet (1624 cm-1) and a rise in β-turns (1674 cm-1) in heated control samples, both deamidation and dephosphorylation of SM before heat treatment created more ordered secondary structure (significant (P<0.05) increase in α-helix (1650-52 cm-1) and β-sheet at the expense of 310-helix (1661 cm-1), random (1645-46 cm-1) and β-turn (1674 cm-1). The only difference between heated deamidated and dephosphorylated samples was decrease in large loops (1656 cm-1) in the latter opposed to the increase in the former. The project therefore established that the accelerated shelf life testing in combination with FTIR spectroscopy has a potential as a rapid tool to forecast sedimentation and other instabilities in UHT milk, and hence the shelf life of UHT milk. Further, a complete understanding of the conformational changes affecting the storage stability of UHT milk could also be attained by studying the structural changes specific to different known interactions using FTIR spectroscopy