Technology and factors influencing Greek-style yogurt - a Review

Methods based on membrane processes. Membrane techniques, especially ultrafiltration (UF), have been successfully used in the yogurt industry for the last 20-25 years [59, 81]. Production of strained yogurt by reverse osmosis (RO) has also been studied. However, previous scientific works revealed that using RO to produce concentrated yogurt created weaker structures which did not give gel properties close to those of concentrated yogurt made by the traditional method [53, 67-70, 78]. Two different systems of UF have been used to produce concentrated yogurt: (a) the fermentation of UF retentate that has the solids content desired in the final product [79, 80, 71], and (b) UF of yogurt at 40-50 °C [10, 66, 77] to produce a concentrated product with the desired total solids content [7]. Several scientific works studied the microstructures and rheological properties of concentrated yogurt obtained by these two UF methods [10, 64, 67- 70, 78]. Greek yoghurts prepared by concentrating a milk base through UF to 13.8% exhibited a hard structure, low syneresis and a high protein and fat content [86]. Applying both, UF and straining, resulted yoghurts with required structural attributes while substantially reducing the generation of AW. Researchers concluded that the concentrated yogurt made from UF milk retentate had much greater firmness than the products manufactured using the traditional method or UF of yogurt [4, 7, 10, 61, 64]. The concentration of milk by UF before yogurt-making carries a risk of bitterness in the final product since the calcium content will be higher [59]. On the other hand, the quality of strained yogurt made by UF of warm yogurt closely resembles the traditional product in terms of elasticity, firmness, and structure [10, 61]. Unfortunately, UF of yogurt affects process efficiency due to permanent membrane fouling [81]. The manufacturing process is as follows: after the fermentation period, the warm yogurt is heated to 58-60 °C for 3 minutes in the plate heater exchanger, to inactivate the culture and control the level of acidity, cooled to 40 °C, concentrated in a two-to-four stage UF plant (depending on the desired degree of concentration), cooled in a plate cooler to about 20 °C and finally packaged [4]. According to Цzer et al. [53] and Tamime et al. [10, 64], UF applications can be used as an industrial alternative to the traditional strained yogurt-making process. Several studies which have investigated the rheology of concentrated yogurt produced by a range of techniques for increasing total solids have concluded that compared to other techniques (such as RO and direct recombination), UF of yogurt gives the gel properties that are closest to those of the traditional product [53, 67, 78]. Other advantages of UF as compared with other conventional methods are: higher yield (10% increase), shortening of processing time (e.g., by 25%), reduced wheying-off, and easy automation and process control [4, 59]. In addition, when using UF instead of the traditional method, the volumes of milk and starter cultures are reduced by around 10% and 80%, respectively [59]. Due to all these advantages, a wide range of UF plants are now available on the market for the production of strained yogurt on a large scale [58, 60, 61].

Acid-induced milk gels are formed by aggregation of casein particles as the pH of milk decreases and the isoelectric point (pH 4.6) of casein is approached [105]. Acid casein gels have a particulate, heterogeneous structure, consisting of fairly large conglomerates and holes (void spaces where the aqueous phase is confined).

These conglomerates are thought to be built of smaller ones, which, in turn, consist of casein particles aggregated in strands and nodes. This heterogeneity, which depends, e.g., on the temperature during gel formation, largely determines the mechanical properties of the gel [38, 106]. Casein gels are very dynamic and rearrangements of the clusters and particles forming the network may occur before or during gel formation [105].

The physical characteristics of these particulate gels are determined by both strong permanent bonds (covalent bonds: SH/S-S exchange) formed during the aggregation, and subsequent rearrangements of protein particles (noncovalent bonds: electrostatic, hydrophobic interactions and, probably, the ever-present Van der Waals attraction, as well as steric and entropic effects related to protein conformation).

The balance between these strong and weak bonds controls the rheology of yogurt gels [53, 70, 106]. Milk protein gels are irreversible, in contrast to many other food gels. Although milk gels are usually classified as particle gels, it is now recognized that they are not simple particle gels because the internal structure of the casein particle plays an important role in the rheological properties of milk gels [37]. Casein micelle structure. At least three types of models for the structure of casein micelles have been proposed. Schmidt [107] and Walstra [108] suggested a model that proposes that the micelle core is divided into discrete sub-units [sub-micelles] with distinctly different properties [37]. In this model, the individual caseins come together in their appropriate portions to form internal sub-micelles, if depleted in к-casein, or external sub-units rich in к-casein, colloidal calcium phosphate (CCP) is regarded as the cement which links these discrete sub-units together. Another model, proposed by Holt [109], regards the micelle as a mineralized, cross-linked protein gel in which the CCP nanoclusters are the agents responsible for cross-linking the proteins and holding the network together [110]. A major failing of these two models is their lack of a plausible mechanism for assembly, growth and, more importantly, termination of growth of the casein micelles. All such elements are in place in a recent model, proposed by Horne [110], which suggests a dual-binding (polycondensation-type) mechanism for gel assembly [37, 111]. In the dual-binding model, micellar assembly and growth take place by a polymerization process involving, as the name suggests, two distinct forms of bonding: crosslinking through hydrophobic regions of the caseins or bridging across CCP nanoclusters. Central to the model is the concept that micellar integrity and hence stability is maintained by a localized excess of hydrophobic attraction over electrostatic repulsion [111]. The energy of interaction between molecules present inside the micelle is calculated as the sum of electrostatic repulsion and hydrophobic attraction as

IE = ER + HI(1)

where, IE: interaction energy; ER: electrostatic repulsion; HI: hydrophobic interaction [110]. This model sees the micellar CCP not just as cross-links but also as neutralizing agents which, being positively charged, bind to negatively charged phosphoserine clusters to reduce the protein charge to the level where the attractive interactions between the hydrophobic regions of the caseins can be allowed to dominate [110]. Figure 2 illustrates the structure of the casein micelle according to the dual-binding model and can be used to explain the two types of linkage postulated between protein molecules. The first linkage is hydrophobic, where two or more hydrophobic regions from different molecules form a bonded cluster.

Figure 2. Dual-binding model of structure of casein micelle

Bonding occurs between the hydrophobic regions, shown as rectangular bars, and by linkage of hydrophilic regions containing phosphoserine clusters to CCP clusters. Molecules of к-casein limit further growth and are labeled with the letter `к'. Source: Adapted from Horne (1998) [110].

The growth of these polymers is inhibited by the protein-charged residues whose repulsion pushes up the interaction free energy. Neutralization of the phosphoserine clusters by incorporation into the CCP diminishes that free energy as well as producing the second type of cross-linking bridge, since it is considered that up to four or more phosphoserine clusters from different casein molecules can be accommodated at each CCP nanocluster [Horne, 1998] [110]. Although the к-casein molecules can interact via their hydrophobic domains with the hydrophobic regions of the other caseins, further growth beyond the к- casein is not possible because it possesses neither a phosphoserine cluster for linkage via CCP [the only phosphoserine residue in к-casein lies in the macropeptide which forms the putative hairy layer deemed essential for micellar stability in all accepted models, thus, this residue cannot be involved in any cross-linking via CCP], nor another hydrophobic anchor point to extend the chain via this route. к-Casein acts as a terminator for both types of growth. Unless circumvented by the growing network, it will become part of the surface structure of the micelle.

Hence its surface location, a prime requirement for any structural model, arises naturally in this model [110]. This concept of a localized excess of hydrophobic attraction over electrostatic repulsion allows the visualization of micellar growth and successfully accommodates the response of the micelles to changes in pH, temperature, urea addition or removal of CCP by sequestrants, all in accordance with experimental observations [112]. Urea does not rupture the CCP linkages but disrupts the hydrophobic bonds, bringing about micellar disintegration.

Further, micellar integrity is largely maintained when the CCP is dissolved out by acidification because the phosphoserine negative charges are neutralized by the acid medium. If the milk is dialyzed and the pH is then restored to that of the original milk, dissociation of the micelle complex is observed as the negative charges of the phosphoserine residues are not neutralized and the electrostatic repulsion effect predominates over the hydrophobic attraction. The same dissociation is observed at natural pH when the CCP is removed by sequestration with EDTA. Increasing pH from the natural value in milk leads to dissociation of the micelles. Whether this is due to conversion of the phosphoserine residues from singly to doubly negatively charged units which are no longer capable of linking to the CCP nanoclusters, or whether the increase in charge itself is sufficient to upset the balance of electrostatic repulsion and hydrophobic attraction in favour of electrostatic repulsion and the micelles dissociate. Decreasing the temperature decreases the level of hydrophobic attraction and any Я-casein not linked through its phosphoserine cluster could then be released into the serum phase [110-112]. These facts suggests that CCP does not cement the micelle together, as described by the earlier models, but rather it helps to control and modulate the effects of calcium and charged groups on caseins. It is also clear that hydrophobic interactions and hydrogen bonding are important for micelle integrity [37].

Formation of acid milk gels. As the pH of milk is reduced, CCP is dissolved, the micelle structure is altered (the charge on individual caseins is altered and the ionic strength of the solution increased) and caseins are liberated into the serum phase [38, 113]. The extent of liberation of caseins depends on the temperature at acidification (low temperatures results in a decrease in the level of hydrophobic attractions inside the casein micelle); at fermentation temperatures commonly used for yogurt manufacture (>30^oC), no dissociation of casein likely occurs. When the isoelectric point of caseins (pH ~ 4.6) is approached, aggregation occurs and low-energy bonds, mainly hydrophobic, are progressively established between proteins. Three pH regions in the acidification of milk from pH 6.7 to 4.6 can be distinguished:

Source: Adapted from Vasbinderef al. (2003) [118].

The extent and rate of denaturation of whey proteins are determined by a number of factors. Amongst these are the pH value, the ionic strength and the ionic composition, the protein concentration and casein to whey protein ratio of the heat treated whey protein solution, and the duration and temperature of the heat treatment [121].Increasing the pH above the natural pH of milk markedly accelerates the rate of denaturation of Я-lactoglobulin. Generally a decrease in the pH of milk systems prior to heating results in an increased association between the denatured whey proteins and the casein micelle. Even small changes in pH can shift the distribution of the association of the denatured whey proteins with the casein micelle. For example, at a level of 95% whey protein denaturation, approximately 70% of denatured whey proteins are associated with the casein micelle at pH 6.55. This decrease to approximately 30% when the pH of milk prior to heating is 6.7. The difference in association level is reflected in the increase in the casein micelle size when milk is heated at the lower pH [117]. According to Vasbinder et al. [118], the denatured whey protein aggregates that form contain a ratio of a-lactalbumin to Я-lactoglobulin which is representative of the ratio of total denatured whey proteins in milk. a-lactalbumin is more easily incorporated in aggregates than it is involved in coating of micelles, while the whey protein coating of the casein micelles clearly contains more Я-lactoglobulin. Vasbinder and de Kruif [122] stated that at high pH, Я-lactoglobulin-Я-lactoglobulin interactions causing whey protein aggregates are favoured over к-casein-Я-lactoglobulin interactions, while k- casein-Я-lactoglobulin-Я-lactoglobulin reactions hardly take place. At lower pH, formation of separate whey protein aggregates hardly occurs, but clusters of whey proteins are formed on the surface of the casein micelle. Apparently, at these conditions K-casein-(Я- lactoglobulin)n interactions are favoured over к-casein-Я-lactoglobulin interactions. Figure 5 summarizes the different interactions that take place between casein micelles and denatured whey proteins when different pH mediums are considered prior to heating.

Figure 5. Schematic representation of the interactions between casein micelles and whey proteins occurring in milk during heat treatment for 10 min at 80 °C at pH values ranging from 6.35 to 6.9.

Native whey proteins are not included in the figure.

Source: Adapted from Vasbinderef al. (2003) [118].

Anema [40] explains this phenomenon by stating that as the pH of the milk is increased from about pH 6.5 to pH 7.1 before heating, к-casein progressively dissociates from the casein micelles so that, at pH 6.5, the majority of the к-casein is associated with the casein micelles, whereas at pH 7.1, about 60-70% of the к-casein is found in the milk serum. As the denatured whey proteins interact with the casein micelles via disulfide bonding with the к-casein, this dissociation of к-casein probably explains why the association of the whey proteins with the casein micelles is pH-dependent. It is important to note that a more severe heat treatment at a constant pH will cause more denaturation of whey proteins, but the ratio of denatured whey proteins associated with the casein micelle and present in aggregates will remain constant [118]. Heat-induced interactions of casein micelles and whey proteins are also affected by the casein to whey protein ratio of the milk base. It is believed that к-casein presents limited number of available binding sites for Я-lactoglobulin association. Thus, when these sites are saturated, denatured whey proteins will interact with each other, increasing the amounts of whey protein aggregates in the system. According to Cho et al. [123], after a heat treatment at pH 6.7, a maximum number of disulfide bonds between k- casein and whey proteins is formed when using a casein to whey protein ratio of 4:1 [124]. Calcium ions promote the association of Я-lactoglobulin with casein micelles, perhaps due to the ability of ions to influence the degree of electrostatic attraction or repulsion between Я- lactoglobulin and к-casein by providing an ionic environment around the interacting molecules. Additionally, salts could be affecting the reactivity of thiol groups. Furthermore, lactose concentration is a limiting factor for the whey protein denaturation. The glucosyl residues are bound to Я-lactoglobulin via gluconic acid or melibionic acid, making this whey protein fraction stable against heat treatment. Lactose concentrations of milk with normal chemical composition do not have any negative effect on the rate of whey protein denaturation. However, if the lactose level of yogurt milk is increased during standardization, the rate of whey protein denaturation is likely reduced. In order to overcome this handicap, milk should exposed to a higher heat treatment (at >90 °C for 10-15 min) [41]. The association of denatured whey proteins with micellar caseins on heating gives improved yogurt texture and gel strength [125]. Yoghurt prepared from heated skim milk and 2% protein from whey protein concentrate had higher storage modulus, firmness, water holding capacity and a denser microstructure than those prepared only from skim milk [126]. On the other hand, it is thought that native whey proteins do not interact with casein micelles during the acidification of unheated milk and act as a destructive filler, or a structure breaker, in acid milk gels [51]. Lee and Lucey [127] reported that yogurt gels made from milk heated at high temperatures [>80°C] presented a higher cross-linked and branched protein structure with smaller pores than gels made from milk heated at low temperatures [31]. This branched microstructure increases the elasticity, gel strength and water binding capacity of the final gel [38, 121, 127-129]. Therefore, yogurts produced from heated milks will have greater firmness and lower susceptibility to syneresis. According to Sodini et al. [51], a heating that ensures 60 to 90% of Я-lactoglobulin denaturation generally optimizes both the WHC and the rheological properties of the final gel. On the other hand, a too severe heating generally (above 90% Я-lactoglobulin denaturation) has a slightly detrimental effect on yogurt's physical properties.