Heat treatment of milk signifies a certain degree of protein denaturation, which modifies the functional properties of dairy products. Traditional methods for detecting and quantifying the denaturation of whey proteins are slow, complex and require sample preparation and qualified staff. The world’s current trend is to develop rapid, real-time analytical methods that do not destroy the sample and can be applied on/in-line during processing.
The milk-protein fraction consists of a large variety of proteins. They are classified into two big groups: caseins and whey proteins. The group of proteins that precipitate at pH 4.6 are known as caseins, representing approximately 80% of milk proteins. Serum or whey proteins are proteins that do not precipitate and remain in the supernatant, representing about 20% of total protein 
. Four major proteins constitute whey proteins: β-lactoglobulin, α-lactalbumin, both being the most abundant 
, bovine serum albumin (BSA) and immunoglobulin.
In the dairy industry, raw milk goes through several processes, including standardization and heat treatment. Pasteurization and ultrahigh-temperature (UHT) sterilization, the most commonly used heat treatments, affect the quality, safety and shelf life of milk and dairy products, inducing physical and chemical changes in protein structure and functionality 
. Thermal processing above 65 °C produces the denaturation of whey proteins such as BSA, while the denaturation of β-lactoglobulin and α-lactalbumin occurs above 70–74 °C 
. When whey proteins are denatured, their hydrophobic groups are exposed, leading to interactions with themselves, forming aggregates or binding to the casein micelles through irreversible disulphide bonds (e.g., between β-lactoglobulin and κ-casein on the surface of the casein micelle). If the heating process is intensified, irreversible denaturation of α-lactalbumin occurs, enhancing the formation of a complex with denatured β-lactoglobulin 
. Consequently, heat denaturation of proteins induces changes in the functional properties of milk, which may be of significant impact on the dairy industry. In particular, changes in the structure of milk proteins have an significant effect on milk structure and stability 
, which might be undesirable or advisable depending on the dairy product being manufactured. For instance, extensive denaturation of whey proteins and subsequent interaction with caseins is pursued in several dairy products due to functional or yield advantages. This is the case on the intensive thermal stabilization of milk required to ensure good heat stability during sterilization of unsweetened condensed milk, as well as to withstand the evaporation processes in sweetened condensed milk. Another example is the intensive whey-protein denaturation prior to coagulation of Mató cheese, a traditional cheese from Catalunya (Spain), to obtain its typical moist and soft texture. Furthermore, the low pasteurization of milk increases the performance in cheese processing and reduces nitrogenous matters of whey, but high pasteurization and UHT milk impairs milk coagulation 
, yielding high moisture and soft gels that are undesirable for cheese manufacture, while intense denaturation of whey proteins is very desirable in yogurt manufacture, as it aids in improving its texture, preventing wheying-off 
Control and quantification of heat-induced protein denaturation are important in the dairy industry. There are different methods and techniques for determining proteins in milk and dairy products. The Kjeldahl, Dumas and Amido Black methods are three chemical procedures to quantify total protein content. The advantages of standard methods such as Kjeldahl or Dumas are that they are precise and have good reproducibility. However, when it comes to quantifying whey-protein content from milk, whey-protein precipitation becomes necessary since these methods quantify the total protein content from the sample. Consequently, this adds more time to the procedure and is not suitable for in-, on-, or at-line determination. The Kjeldahl method can be performed within 30 min to 2 h, while the modern Dumas instrument takes around 5 min 
. In order to analyze the individual proteins, several classical techniques are used at the laboratory scale: electrophoresis, chromatography and immunochemistry. These techniques need complex sample preparations before carrying out the analysis. Previously, it is necessary to fractionate the caseins and whey proteins by precipitation, and then these must be filtered to obtain a pure preparation of protein 
. Moreover, they also need rather expensive reagents and equipment, which means that they are slow protein-detection and quantification methods. Since classical techniques are time-consuming methods, new rapid technologies are being studied for in-, on-, or at-line determination and real-time process control in the dairy industry 
Thus, it is obvious that at least for small processing plants where more than just one dairy food is manufactured, e.g., cheese and yogurt, knowing if the protein is native or denatured at real time, rather than later in the lab after applying conventional off-line methodologies, will aim with industrial decision making during manufacturing. In other words, rapid methods to directly quantify whey-protein denaturation (i.e., without the need of sample manipulation), and with the potential for in-, on-, or at-line measurements, are compatible with real-time assessment of milk-protein functionality, allowing milk batches to be used for their most suitable purpose, increasing quality of the final product. It would also avoid the need to take samples from the processing line and submit then to either an internal or even external quality-control laboratory.
2. Rapid Methods for Determining the Thermal Denaturation of Whey Proteins in Milk
This entry is adapted from 10.3390/dairy3030036