Alan L. Kelly

Activities of plasmin, cathepsin D, cathepsin B, and tripeptide aminopeptidase were measured in milk from 41 individual cows, with raw milk and corresponding pasteurized aliquots incubated at 37°C for 0, 1, and 7 d. Associations between individual protease activities and SCC, DIM, lactation number (LN), and protein content were also examined. In fresh raw milk, SCC was positively correlated with tripeptide aminopeptidase and cathepsin B activities but not with plasmin or cathepsin D activities. Plasmin activity, in contrast, was positively correlated with LN, whereas no clear association was observed with DIM. Protein content was positively associated with tripeptide aminopeptidase activity. During incubation of raw milk at 37°C, all 4 proteases exhibited a significant decrease in activity over the 7-d period. High-temperature short-time pasteurization (72°C for 15 s) significantly reduced the activity of all 4 proteases at d 0, although the extent of the reduction differed substantially between enzymes. Tripeptide aminopeptidase and cathepsin B were highly heat-sensitive, with activities reduced by 97.9% and 94.0%, respectively. Cathepsin D displayed intermediate heat resistance, with a reduction of 77.0%, whereas plasmin showed only moderate heat sensitivity, with activity decreasing by 45.9% following pasteurization. The behavior of the enzymes during subsequent incubation of pasteurized milk also differed. Tripeptide aminopeptidase activity remained negligible throughout the entire incubation period, indicating almost complete inactivation by heat treatment, and activities of cathepsins B and D continued to decline further during incubation. In contrast, plasmin activity increased significantly during incubation of pasteurized milk, rising by ∼131% by d 7. Overall, these findings highlight the complexity of proteolytic systems in bovine milk and demonstrate that both cow-associated factors and thermal processing influence individual proteases in markedly different ways, with shifts in proteolytic balance having potential consequences for dairy product quality and shelf life.

This study elucidated the role of specific milk proteases in generating distinct proteolytic patterns and peptide profiles in bovine milk. Specifically, the activities of plasmin and the lysosomal proteases cathepsin D and cathepsin B were analyzed in both raw and pasteurized bulk bovine milk. Samples were incubated at their enzymes' optimal pH levels (6.7 for plasmin; 5.0 for lysosomal proteases) at 37°C. Protease activities, together with casein breakdown monitored by urea-PAGE and UPLC, were followed over 3 d, with urea-PAGE used to assess breakdown of intact caseins and larger proteolytic fragments, and UPLC to detect the accumulation of soluble low-molecular-weight peptides. Peptide profiles were further characterized by LC-MS after 7 d to allow greater peptide accumulation and improved resolution of protease-specific hydrolysis patterns. Overall, pH had a dramatic effect on proteolysis; both UPLC and LC-MS analyses revealed a marked reduction in total peptide abundance under acidic conditions compared with neutral pH. However, the impact of pH was not limited to a simple decrease in proteolytic extent. Rather, acidification induced a shift in the balance among active proteases, which translated directly into changes in substrate preference and casein breakdown patterns. In raw milk at neutral pH, plasmin activity declined gradually over time, whereas lysosomal protease activity decreased sharply after 24 h at 37°C. Under these conditions, α_s1- and β-caseins were the primary substrates. At neutral pH, lasmin predominantly hydrolyzed β-casein to generate γ-caseins, while cathepsin D was likely associated with the formation of α_s1-I-casein. At acidic pH, despite lower overall peptide yields, the proteolytic profile shifted toward preferential breakdown of α_s2- and κ-caseins, driven mainly by cathepsin D and potentially by AprX, a bacterial metalloprotease. In contrast, cathepsin B exhibited minimal involvement in intact casein hydrolysis across both pH conditions, suggesting a more specialized functional role. Heat treatment further modulated protease dynamics. Plasmin activity was not significantly reduced following high-temperature short-time pasteurization (72°C for 15 s) and increased during incubation, whereas lysosomal protease activities were markedly reduced by pasteurization and further declined throughout the incubation period. Collectively, these findings demonstrate that both pH and heat treatment regulate milk proteolysis not only by affecting total enzymatic activity but also by altering protease balance, thereby redefining casein substrate utilization and peptide generation patterns.

The consumption of cheese is rapidly increasing, driven by its diverse nutritional and functional benefits. Consumers are shifting toward functional, minimally processed cheese products, enjoyed both on their own and as ingredients in various foods. As cheese undergoes heating processes like grilling and baking, its functionality (e.g., melt, flow, and stretch) become crucial factors in evaluation of quality and desirability with respect to cooking performance and sensorial attributes, such as mouthfeel, visual appeal, and texture. Heat-induced structural changes, such as disruption of the casein network and milk fat globule membrane (MFGM), and fat redistribution, can accelerate digestion, modify nutrient and bioactive release, and influence lipid absorption. This review examines how different heating methods impact the physicochemical, functional, and health properties of cheese, particularly in relation to the cheese matrix effect. It further explores how temperature-induced changes influence the digestion, absorption, and bioavailability of nutrients and bioactive compounds, offering valuable insights into how these alterations affect the overall health benefits of cheese.

During microfiltration (MF) of skim milk, diafiltration (DF) is often performed to facilitate the additional removal of target constituents. Water is widely used as a DF media in industry; however, ultrafiltration (UF) permeate also provides an ideal medium as it can minimise changes in mineral equilibria. Therefore, in this study, the effect of different DF media, namely water and simulated milk ultrafiltrate resembling the mineral profile of UF permeates obtained at 10 and 50 °C (i.e., SMUF 10 and 50, respectively), on processing performance, protein partitioning, and heat stability of the resulting retentates was investigated following the MF and DF of skim milk at 8 °C. The use of water resulted in significantly lower (P < 0.05) conductivity and higher pH of retentates compared to use of SMUF 10 or 50. The different DF media had no significant impact on the transmission of β-lactoglobulin, α-lactalbumin or β-casein throughout filtration. However, significantly longer (P < 0.05) heat coagulation times were measured for retentates generated using SMUF 10 and 50 as the DF medium which corresponded to significantly lower (P < 0.05) concentrations of ionic calcium, compared to when water was used. The results of this study indicate that the use of model lactose-free UF permeate (i.e., SMUF) provides similar protein partitioning characteristics to when water is used; however, a final micellar casein retentate with superior thermal stability can be produced.

Understanding salt diffusion is essential for optimizing salt uptake during cheese production. Although multiple interacting factors complicate this process, the utilization of a model cheese system offers a controlled platform to evaluate their relative influence. This study developed a model using renneted gels prepared from micellar casein concentrate and investigated the use of salty whey, a secondary byproduct of Cheddar production, as an alternative salting medium. We assessed the effects of varying the pH, fat, and calcium contents of the model cheese and salting temperature on salt migration. Salt diffusivity was modeled using Fick's second law. Increasing calcium content during formulation of the model system (0%-1% wt/wt, weight basis) significantly increased salt penetration. Similarly, salting at 30°C significantly enhanced salt diffusion compared with salting at 20°C or 40°C. Diffusion coefficients of salt in the model cheese systems ranged from 3.1 × 10^-9 to 8.4 × 10^-9 m²/s. Increasing calcium level promoted a more continuous compact protein matrix, which in turn increased salt diffusion; in contrast, fat globules hindered salt movement within the matrix. Analysis of Fourier transform infrared spectra indicated that higher levels of salt migration corresponded to more ordered protein secondary structures with stronger hydrogen bonding. The diffusivity of salty whey, however, was less than that of brine, likely due to the higher osmotic pressure and viscosity of the former.

The rennet and acid coagulation properties of reconstituted micellar casein concentrate prepared using cold or warm microfiltration (MF), at similar casein contents, were investigated, with low-heat skim milk powder (LHSMP) as a control. The MF retentates had higher casein content (as % of total protein) compared with LHSMP, and heat-induced whey protein-casein aggregates were only present in LHSMP. All MF retentates showed shorter rennet coagulation times and higher gel strengths than LHSMP, which may be linked to lower levels of whey protein (either native or denatured). At similar casein contents, longer rennet coagulation times were evident for cold MF retentates compared with warm MF retentates, as the ratio of κ-CN as a function of total increased with the depletion of β-CN. In terms of acid-induced coagulation, all MF retentates coagulated at a pH >5, higher than the gelation pH of LHSMP (4.7-4.9), which was confirmed by microscopic and textural analysis. An inflection point (increase, followed by a decrease) in the storage modulus value was seen during the acidification of warm MF retentates, but not cold MF retentates; this may be related to structural rearrangements of the gel initiated by release of colloidal calcium phosphate and compacting of the structure of warm MF retentate gels as pH decreased. Both warm and cold MF retentates exhibited shorter rennet coagulation times, stronger rennet-induced gels and higher acid-induced gelation pH compared with LHSMP, which might influence their use for the manufacture of cheese or yogurt with tailored functionalities.

The heat‐stable metalloprotease AprX, secreted by psychrotrophic Pseudomonas spp., is a major cause of quality deterioration in dairy products, particularly ultrahigh temperature (UHT) milk. This review synthesizes the evolution and current state of detection and quantification techniques for AprX in bovine milk, covering traditional immunological assays, enzymatic activity measurements, and zymography, alongside modern molecular approaches such as PCR‐based methods, mass spectrometry, and advanced biosensors. Highly sensitive systems, including indirect ELISA (LOD 21.0 ng/mL), multiplex qPCR, and biosensor platforms, have enhanced the ability to detect AprX activity at low concentrations. Comparative analysis evaluates these methods in terms of sensitivity, specificity, turnaround time, cost, ease of use, and industrial applicability. Emerging directions such as multiomics integration, biosensor miniaturization, and artificial intelligence‐driven data interpretation are also discussed. By critically assessing available and emerging tools, this review supports dairy scientists and industry stakeholders in selecting optimal strategies to detect, monitor, and mitigate AprX‐associated spoilage in milk and dairy products.