FOOD PROTEINS/ PRE-PROBIOTICS INGE GAZI, THOM HUPPERTZ* *Corresponding author NIZO food research, PO Box 20, 6710BA, Ede, The Netherlands Inge Gazi Thom Huppertz Casein-whey protein interactions for optimizing milk protein functionality KEYWORDS: Whey protein denaturation, distribution of proteins, rennet coagulation, acid gelation, heat stability. Heat treatment of milk typically results in denaturation of whey proteins and their association with caseins. Abstract The functionality of the proteins can be affected by the presence of these aggregates and the distribution of the aggregates between the casein micelles and the serum phase. Casein-whey protein interactions are detrimental in (rennet-)cheese making, whereas the presence of aggregates in the serum phase during yoghurt making improves gelation properties and strengthens gel texture; micelle-bound aggregates increase the heat stability of milk, particularly in concentrated systems. The current knowledge and a strict control of the processing conditions create functionality of the dairy proteins that covers a very broad range of applicability. INTRODUCTION Caseins and whey proteins co-exist in fresh milk, but do not interact. Both have specific nutritional and functional Milk proteins have always played a crucial role in the properties. The whey proteins are a readily digestible source human diet. Mother’s milk provides essential proteins to the of branched-chain amino acids, whereas the caseins are neonate, and at later stages in life, milk proteins are found slowly digested and are essential in the transport of calcium in a vast range of popular and widely-consumed dairy and phosphate. Likewise, the caseins are crucial in cheese non-dairy food products. In addition to a crucial nutritional making, whereas the whey proteins possess unique heat role, milk proteins also have an important role from a gelation properties. Casein-whey protein interactions can physicochemical perspective. They provide the texture of occur as a result of heat treatment. These interactions can cheese and yoghurt, and also stabilize air bubbles in ice provide highly desired functionality of milk proteins, e.g., for cream and whipped cream. Two different classes of milk the structure and stability of yoghurt, heat stability or proteins are distinguished, which show vastly different foaming. Whey protein denaturation and casein-whey behaviour. Caseins represent the major class of proteins protein interactions can also be induced through non- (~80% of total protein in cows’ milk). These phosphoproteins thermal technologies, such as high pressure treatment. In are organized in milk in spherical colloidal structures this article, we focus on the heat-induced casein-whey (diameter ~200 nm) called casein micelles, consisting of protein interactions, considering the factors influencing the thousands of casein molecules linked together, amongst interactions and their effect on the functional properties of others, through calcium phosphate nanoclusters. The milk proteins. casein micelles are rather heat-stable, but coagulate at pH <5 or after addition of rennet. Casein micelles are crucial in converting milk into e.g., cheese or yoghurt. In contrast, the HEAT-INDUCED WHEY PROTEIN DENATURATION AND CASEIN- whey proteins (~20% of total protein in cows’ milk) occur WHEY PROTEIN INTERACTIONS mostly as individual proteins. These whey proteins, of which α-lactalbumin (α-La) and β-lactoglobulin (β-Lg) are the Caseins have relatively little secondary and tertiary main representatives are heat-labile and can start structure. As a result, caseins have what is sometimes denaturing and aggregating at temperatures >65°C. referred to as a natively-unfolded structure and specific However, native whey proteins are stable on acidification. amino acids are rather easily accessible for interactions (1). Whey proteins are widely applied for their nutritional and On the other hand, the high degree of secondary and technological functionality, e.g., in beverages, infant tertiary structure of whey proteins results in a tightly-folded formula, etc. and poorly-accessible structure at ambient temperature (2). Monographic special issue: Food Proteins / Pre-Probiotics - Agro FOOD Industry Hi Tech - vol 26(2) - March/April 2015 11 However, increasing temperature results in the denaturation of whey proteins, which involves the following steps (3): Figure 2. Denaturation of - Dissociation of the β-Lg dimers into monomers; α-La in skim milk - Unfolding of the globular protein structure and exposure as a function of the reactive free thiol group of β-Lg and the 2 and 4 of heating time and temperature disulphide bridges in β-Lg and α-La, respectively; (70°C; 75°C; - Aggregation via thiol-thiol oxidation and thiol-disulphide 80°C▲; 85°C; 90°C; 95°C). interchange reactions. From NIZO In this last step, casein-whey protein interactions occur. β-Lg food research is the main source of free thiol groups in milk and drives – unpublished data. these interactions. The free thiol group of β-Lg can interact with disulphide bonds of other b-Lg molecules or other whey proteins, e.g., α-La, but also with αs2-casein (αs2-CN) and κ -casein (κ-CN), which can thus participate in thiol-disulphide Figure 3. Denaturation of interchange reactions (3). The location of κ-CN on the β-Lg in skim milk casein micelle surface makes it readily accessible for as a function interactions with denatured whey proteins. Although α-La is of heating time and temperature not believed to directly interact with κ-CN, it is also found in (70°C; 75°C; the whey protein-κ-CN aggregates, along with β-Lg. 80°C▲; 85°C; κ-CN-whey protein complexes are found in heated milk on 90°C ; 95°C ). From NIZO the surface of the casein micelles, but also in the serum food research phase of the milk (Figure 1). The distribution of these – unpublished data. complexes between the casein micelles and the serum depends on many factors, including heating time and temperature, milk pH and the salt composition. Functional The fraction of denatured whey proteins associated with the properties of milk, such as gelation, heat stability and casein micelles strongly depends on several factors, e.g., foaming can be tailored by controlling the casein-whey pH (Figure 1) and the salt composition of the milk serum. protein interactions. When milk is heated at its natural pH, i.e., 6.6-6.7, part of the denatured whey proteins is associated with the casein micelles and part of the denatured whey proteins is found in the milk serum (9,10). When the pH of the milk is lowered prior to heating, e.g., to 6.3-6.5, the level of denatured whey protein associated with the casein micelles increases. Conversely, at higher pH, the level of denatured whey protein associated with the casein micelles decreases. The κ-CN distribution follows similar trends (9,10). After heat treatment of milk at low pH, virtually all κ-CN is associated with the casein micelles, whereas after heat treatment at high pH, a considerable proportion of κ-CN is found in the milk serum, mostly associated with denatured whey proteins (11). The influence Figure 1. Schematic representation of the effect of skim milk pH of the mineral (A, D – pH 6.5; B, E – pH 6.8; C, F – pH 7.1) on the distribution of whey protein (WP) aggregates between the serum phase and the composition on casein (CN) micelles in skim milk heated for 30 min at 90°C (A, B, C – the distribution unheated skim milk; D, E, F – heated skim milk), based on data from of denatured Crowley et al. (4). whey proteins and κ-CN Heating time and temperature applied are the primary after heating determinants of the extent of whey protein denaturation was illustrated (Figures 2 and 3). The intensity of heat treatment of milk in a recent varies widely, from e.g., 15 sec at 65°C for thermization, study on the 14 sec at 72°C for a typical high temperature-short heat stability time (HTST) pasteurization, to e.g., 5 min at 95°C for of milk protein Figure 4. Effect of solution pH on the distribution preheating of yoghurt milk, 5 sec at 140°C for ultra-high concentrates of κ-CN and β-Lg in between the serum phase () and casein micelles () in skim milk powder temperature (UHT) treatment and 10 min at 121°C for (MPCs) (Figure (SMP) and milk protein concentrates (MPC80 in-container sterilization of milk (5). The extent of whey 4). These MPCs and MPC90) reconstituted at 3.5%, m/m, protein denaturation increases with increasing time and are made protein content and heated for 30 min at 90°C. Data from Crowley et al. (4). temperature. Thermization and HTST pasteurization result by removing in little or no denaturation of whey proteins (6,7), but the lactose and preheating of yoghurt milk and in-container sterilization soluble minerals and concentrating the proteins by ultrafiltration. denature virtually all whey proteins (6,7). UHT treatment of After heat treatment, the degree of whey protein denaturation milk denatures approximately 50% of the whey proteins in was comparable in skim milk and MPC suspensions, but the milk (7,8). distribution of denatured whey protein and κ-CN was strongly 12 MonographicMonographic specialspecial issue:issue: FoodFood ProteinsProteins // Pre-ProbioticsPre-Probiotcs - Agro FOOD Industry Hi Tech - vol 26(2) - March/April 2015 affected. In MPC suspensions, heat-induced dissociation of κ-CN in the serum or was found to be far lower than in milk and virtually all denatured their absence whey protein was found to be associated with the casein from the micelles (4). These studies illustrate that ingredient selection is a micellar surface key factor in determining casein-whey protein interactions. aid the acid coagulation process of IMPACT OF CASEIN-WHEY PROTEIN INTERACTIONS ON MILK milk, leading PROTEIN FUNCTIONALITY to a firmer gel.