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peer-reviewed Microfiltration (MF) of skim milk, when combined with diafiltration (DF), facilitates the manufacture of liquid micellar casein concentrate (MCC), which can be spray-dried into high-protein (≥80% protein, dry-basis) powders. MCC powders rehydrate slowly, which is typically considered a defect by end-users. This study compared the impact of cold (

The aim of this study was to determine the effects of calcium chelating agents on the dissolution and functionality of 10% (w/w) milk protein concentrate (MPC) powder. MPC powder dissolution rate and solubility significantly (P > 0.05) increased with addition of sodium phosphate, trisodium citrate (TSC) and sodium hexametaphosphate (SHMP), compared to MPC dispersions alone. Trisodium citrate and SHMP addition increased viscosity as a result of micelle swelling. However, dispersions containing SHMP showed a decrease in viscosity after prolonged time due to micelle dissociation. Overall, MPC powder dissolution was aided by the addition of calcium chelating agents.

Whey proteins are widely used in the food industry due to their excellent nutritional and functional qualities. Nevertheless, numerous applications of whey proteins in foods are affected by heat treatments that are commonly used in food processing to ensure microbiological safety. Commercial thermal processing conditions often result in denaturation and aggregation of whey proteins. This chapter describes the fundamental theory of whey protein denaturation and aggregation during thermal processing, with emphasis on β-lactoglobulin, the major protein in whey. It also examines how thermal processing impacts the complexity of whey protein interactions, whey protein size and structure, as well as whey protein functional properties. Finally, novel approaches/methods to prevent denaturation and aggregation of whey protein are summarized in this chapter.

β-Lactoglobulin (β-Lg) is relatively unstable during thermal processes used in the dairy industry. One of many ways to improve the heat stability of β-Lg is by chemical modification by protein–reagent binding. Potential reagents, such as 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) and N-ethylmaleimide (NEM), bind to the exposed free sulfhydryl (–SH) group of denatured β-Lg and prevent it from being involved in protein aggregation reactions. However, no study has shown the specific binding of these reagents to β-Lg. Furthermore, the question of how many molecules of these reagents bind to β-Lg remains unanswered. In this research, the reactions of DTNB or NEM with the reactive monomers of β-Lg were identified by the presence of bands for 5-thio-2-nitrobenzoyl (TNB)- or NEM-modified monomers on Native-PAGE and Non-Reducing (NR) sodium deodecyl sulphate (SDS)-PAGE gels, and by an additional mass of β-Lg monomers corresponding to a molecule of TNB or NEM in mass spectra.

The effects of sulphydryl (-SH) reagents on protein aggregation reactions in heated whey protein isolate (WPI) and pure α-lactalbumin (α-La) were investigated. In contrast to its previously reported effect with pure β-Lg, dihydrolipoic acid (DHLA) markedly reduced the heat stability of WPI, especially the α-La component, which aggregated much more readily in the presence of DHLA than in WPI alone. Whilst pure α-La is quite stable to heat, it is much less stable in the presence of DHLA. An effect similar to DHLA was observed with reduced glutathione (GSH). N-ethylmaleimide (NEM), and to a lesser extent, dithio(bis)-p-nitrobenzoate (DTNB), improved the heat stability of WPI; these reagents had little effect on α-La.

Whey proteins have many benefits due to their high nutritional value and their various applications in food products. A drawback of whey proteins is their instability to thermal processing, which leads to their denaturation, aggregation, and, under some conditions, gelation. As thermal processing is a major treatment in the processing of milk and milk products, its influence on whey proteins has been extensively studied. Understanding the mechanisms involved during each stage of denaturation and aggregation of whey proteins is critical to devising ways of improving their stability. These aspects are reviewed in this paper. Also covered are approaches to preventing or reducing heat-induced aggregation of whey proteins. Inhibition of aggregate formation has considerable potential for alleviating the problems that arise from the instability of whey proteins.

Prevention of the heat-induced aggregation of β-lactoglobulin (β-Lg) would improve the heat stability of whey proteins. The effects of lipoic acid (LA, or thioctic acid), in both its oxidised and reduced form (dihydrolipoic acid, DHLA), on heat-induced unfolding and aggregation of β-Lg were investigated. LA/DHLA was added to native β-Lg and the mixture was heated at 70, 75, 80 or 85 °C for up to 30 min at pH 6·8. The samples were analysed by Polyacrylamide Gel Electrophoresis (PAGE) and Size-exclusion HPLC (SE-HPLC). LA was not as effective as DHLA in reducing the formation of aggregates of heated β-Lg. Heating β-Lg with DHLA resulted in formation of more β-Lg monomers (due to dissociation of native dimers) and significantly less β-Lg aggregates, compared with heating β-Lg alone. The aggregates formed in the presence of DHLA were both covalently linked, via disulphide bonds, and non-covalently (hydrophobically) linked, but the amount of covalently linked aggregates was much less than when β-Lg was heated alone. The results suggest that DHLA was able to partially trap the reactive β-Lg monomer containing a free sulphydryl (−SH) group, by forming a ‘modified monomer’, and to prevent some sulphydryl−sulphydryl and sulphydryl−disulphide interactions that lead to the formation of covalently linked protein aggregates. The effects of DHLA were similar to those of N-ethylmaleimide (NEM) and dithio(bis)-p-nitrobenzoate (DTNB). However, the advantage of using DHLA over NEM and DTNB to lessen aggregation of β-Lg is that it is a food-grade compound which occurs naturally in milk.

Maillard reaction is a nonenzymatic browning reaction that involves the reaction of carbonyl groups, primarily reducing sugars with free amino groups which cause the changes of chemical and physiological properties of proteins (Labuza and Saltmarch 1981). It results in the development of complex mixtures of colored and colorless reaction products which range from flavor volatiles (low molecular weight compounds) to melanoidins, a series of brown pigments with high molecular weights (Carabasa-Giribet and Ibarz-Ribas 2000; Martins and Van-Boekel 2005); these effects could be either desirable or undesirable. Browning and the formation of aroma are desired in baking, roasting, or frying, while it is undesirable in the foods which have a typical weak or other color of their own such as browning in the products of condensed milk, white dried soups, tomato soup, etc. and generation of off-flavors in food during storage. Besides, Maillard reaction can also have negative effects on nutritional values such as the losses of essential amino acids, as well as the formation of mutagenic compounds (Belitz et al. 2004).

The instability of whey protein during heat treatment has always been a major problem limiting its use in food. On heating they denature and form aggregates, and can form gels at high concentration. The present study aimed to investigate ways of reducing formation of aggregates from heat-denatured whey proteins. Inhibition of aggregate formation has considerable potential for alleviating the problems that arise from the instability of whey proteins. The approach taken in this study to inhibition was intercepting the reactive sulfhydryl (-SH) group from the major whey protein, b-lactoglobulin (b-Lg), with -SH specific reagents and hence reducing its participation in the formation of aggregated protein complexes. The study of the modification of major whey proteins (b-Lg, a-lactalbumin (a-La) and bovine serum albumin (BSA)) was carried out on both single b-Lg and mixed whey protein systems at near neutral pH (pH 6.8) and low concentration of protein (1%, w/w). The heating conditions were set at low-moderate temperatures (55-85oC) for 10-30 min. Such conditions were chosen to allow the heat-induced changes of whey protein to be readily followed; higher temperature, different pH and higher concentration of protein would make this more difficult or impossible. The results of the modification were analysed using a combination of size exclusion-high performance liquid chromatography (SE-HPLC), reversed phase HPLC (RP-HPLC) and polyacrylamide electrophoresis (PAGE) techniques. To confirm the presence of interactions between b-Lg and -SH reagents, electrospray ionization/time of flight-mass spectrometry (ESI/TOF-MS) analysis was conducted. The results from the single b-Lg system showed that blocking the free -SH group with dithio(bis)-p-nitrobenzoate (DTNB), N-ethylmaleimide (NEM), dihydrolipoic acid (DHLA) or reduced glutathione (GSH) resulted in formation of a new type of monomer (beside the dissociated reactive monomers), referred as the modified monomers in this thesis, and significantly less of the larger b-Lg aggregates compared with heated b-Lg with no reagents. The modified monomers are monomers with reagents attached to them and were found to form via covalent interactions. DTNB, NEM, DHLA or GSH were able to trap the monomer containing a free -SH group, to varying degrees, and prevent -SH/disulfide (S-S) exchange phenomena that lead to formation of S-S-linked protein aggregates. Furthermore, as the presence of these reagents was able to reduce formation of covalently linked aggregates, mostly non-covalent aggregates were left in the system. This fact denotes the importance of non-covalent interactions during aggregation reactions and led to an investigation using biotin and lecithin that are able to bind to the exposed hydrophobic site of b-Lg. The heat stability of the denatured b-Lg was significantly improved (pl0.05) by the presence of biotin or lecithin upon heating to 70oC or 75oC. As expected, biotin and lecithin were able to bind with the exposed hydrophobic surfaces of b-Lg heated at 70oC; hence formation of aggregates was significantly reduced (pl0.05) as compared to the non-modified b-Lg system. Nevertheless, upon further heating to 75oC the ability of both reagents to reduce aggregation was gradually decreased and was not seen at 85oC. These results indicate the involvement of non-covalent interactions in aggregation of b-Lg during mild heating (at ~70oC) but further heating (l70oC) mainly involved covalent interactions. The non-covalent interactions are thought to facilitate -SH/S-S interchange reactions in the later stages of aggregation. Thus, covalent interactions appear to play an important role in the formation of high-Mw aggregates. In contrast to its use in the single b-Lg system, DHLA enhanced polymerization reaction when added to a mixed whey protein system, whey protein isolate (WPI), in this study. Although intermediate aggregates were significantly reduced (pl0.05) compared with heating the mixture alone, DHLA was very reactive towards the S-S bond of a-La, thus, initiating the formation of high-Mw aggregates. Similarly, GSH was reactive towards both the -SH group of b-Lg and S-S bond of a-La, leading to aggregation in the heated WPI system. Nevertheless, the formation of high-Mw aggregates was significantly less (pl0.05) than in DHLA-modified system indicating that GSH is less reactive towards S-S bonds of a-La than is DHLA. The presence of DTNB or NEM, on the other hand, considerably improved the heat stability of major whey proteins. The low (or no) reactivity of DTNB and NEM towards the S-S bond of a-La was the main reason for the improved heat stability of whey proteins in the presence of these reagents. It can be concluded that this study demonstrated the possibility of improving the thermal stability of whey proteins by adding -SH-specific reagents and the ability of these reagents was strongly influenced by the presence of all whey proteins and not determined by any single whey protein. A mechanism describing the pathways of inhibition by these reagents was established.