Coagulation properties of ultrafiltered milk retentates measured using rheology and diffusing wave spectroscopy

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Abstract

The effect of concentration of milk by ultrafiltration on renneting has been widely studied as it is of great interest in dairy technology. Although a number of reports are available on the texture and microstructure of the milk gels formed at various concentrations, very little is understood on the effect of concentration on the stages preceding aggregation, or how concentration may affect the interactions between micelles. This study aims to investigate the renneting behavior of milk concentrated by ultrafiltration (without diafiltration) to 3× and 5× (v/v) and compare it to that of skim milk. The scattering properties of the casein micelles under quiescent conditions suggest that they deviate from hard-sphere behavior at 5× concentration (micelle volume fraction, ϕ = 0.5). The release of the caseinomacropeptide during renneting was not significantly different amongst the three different casein concentrations tested (1×, 3×, and 5×). No significant differences were also noted in the rennet coagulation time as detected by both diffusing wave spectroscopy and rheology. Concentrated milk samples formed significantly (p-value < 0.05) stiffer gels than regular milk due to an increased number of bonds in the network. The level of milk concentration also accelerated a change in the spatial distribution and rate of change of turbidity of the micelles because of a decrease in the overall inter-particle distance and increased collision frequencies. This in situ investigation of concentrated milk samples suggested that the changes in rennet coagulation with concentration are merely a cause of crowding effects.

Introduction

Skim milk is a dilute suspension of proteins, minerals and lactose. Amongst the protein fraction, caseins constitute about 80% of the total protein and they are of special interest as they play a major role in the processing functionality of milk. The casein proteins are organized into casein micelles that are polydispersed in size (between 50 and 600 nm) and are highly hydrated (Fox, Guinee, Cogan, & McSweeney, 2000). The casein micelle is held together internally by colloidal calcium phosphate and hydrophobic interactions. The κ-casein contributes to the colloidal stability of the casein micelles. This protein is present mostly on the outside of the protein assembly and provides electrostatic and steric repulsion (De Kruif, 1999). During renneting, this salted polyelectrolyte brush is gradually cleaved by the enzymatic action of chymosin and the micelles, once sufficiently destabilized, aggregate and form a gel network. This mechanism of destabilization of the casein micelles is often exploited in cheese manufacture (De Kruif, 1998, De Kruif, 1999, Fox et al., 2000).

The rennet reaction involves two stages (Fox et al., 2000). The primary stage is the enzymatic cleavage of κ-casein, and this is overlapping with a second stage, which involves aggregation of the destabilized micelles. Rearrangements of the gel network then occur. In untreated skim milk at native pH, the second stage of the reaction occurs when at least 85–90% of the casein-macropeptide is released into solution (Sandra, Alexander, & Dalgleish, 2007). Although reports are available on the gelation behavior of milk concentrated by UF, very little is known about the preceding stages of gelation, as not very many tools are available to follow the changes in the dynamics of the casein micelles in situ, without dilution or disruption of the sample. It has been suggested that the initial rate of the enzymatic reaction increases with increasing protein concentration (Lucisano, Peri, & Donati, 1985), as the amount of substrate (i.e. κ-casein) available is the limiting factor in the reaction.

Concentration of skim milk by ultrafiltration (UF) changes the composition of milk by increasing the proteins and colloidal minerals in the retentate while decreasing the water, soluble minerals, lactose and non-protein nitrogen, which are transmitted through the membrane and found in the permeate (Mistry & Maubois, 2004). UF has become an increasingly important processing step for manufacturing cheese and dairy ingredients or for milk standardization. The properties of casein micelles in milk concentrates are of interest to better learn how to use UF retentates in various processes, and for a more fundamental understanding of the colloidal stability of the casein micelles in milk. A better understanding of the effect of UF-concentration on micelle interactions and gelation properties could lead to improved UF-cheese manufacturing.

It is generally accepted that gel stiffness increases with the level of concentration in UF retentates (Karlsson et al., 2007, Waungana et al., 1998). However, some disagreement still exists on whether rennet-induced gelation of UF retentates shows different gelation times or if concentration does not affect it. Mehaia and Cheryan (1983) provided a summary table of some studies on the effect of ultrafiltration on milk clotting time (up to the year 1983). It appears that an effect of concentration may be present at reduced pH but not at the natural milk pH (Lucisano et al., 1985, Waungana et al., 1998). Guinee, O'Callaghan, Pudja, and O'Brien (1996) found that concentration by ultrafiltration decreases gelation time and causes a faster firming rate. Karlsson et al. (2007) investigated differences between rennet coagulation of highly concentrated milk (i.e. 19% casein) and regular skim milk at pH 5.8 and found that when the same amount of rennet is added to the UF retentate or the skim milk, the concentrated milk has a longer coagulation time and higher gel firming rate than non-concentrated milk. Dalgleish, 1980, Lucisano et al., 1985, Waungana et al., 1998 showed similar coagulation times between concentrated and regular milk at neutral pH. The discrepancies in the results could be partially attributed to the different methods employed for following κ-casein hydrolysis, clotting or aggregation time, the different casein sources and processing history of the concentrated milk, as well as the definition of aggregation or coagulation time.

The main objective of this work was to investigate the rennet-induced gelation of the casein micelles in skim milk at different concentrations (1×, 3× and 5×, based on volume concentration) obtained by ultrafiltration, observing the formation of structure, in situ, without sample dilution. In the present work, the rennet gelation of UF-retentates was followed using rheology and diffusing wave spectroscopy. Prior to renneting, the stability of casein micelles in UF retentates was tested and the experimental parameters were compared to those predicted by hard-sphere theory.

Section snippets

Skim milk

Whole milk was collected from the Ponsonby Dairy Research Station of the University of Guelph. Immediately after collection, 0.02% (w/v) sodium azide was added as a bacteriostatic agent. The milk was skimmed by centrifugation at 5000 g for 20 min at 4 °C with a Beckman J2-21 centrifuge and JA-10 rotor and filtering four times through Whatman glass fiber filters (Fisher Scientific, Whitby, ON, Canada). The skim milk was kept refrigerated until use. All experiments were performed on two different

Milk composition as a function of concentration

The pH values of milk were not significantly different from the original milk after concentration (Table 1). Culioli and Sherman, 1978, Waungana et al., 1998, Karlsson et al., 2007 reported a decrease in pH for UF concentrated milk when compared to non-concentrated milk; however much higher levels of protein concentration were achieved, most likely by employing also some diafiltration (i.e. addition of water during membrane filtration). The decrease of pH with increasing volume fraction has

Conclusions

Dairy-gel products such as cheese made from UF milk retentate may have different properties than those made from non-concentrated milk because UF affects coagulation properties and final gel characteristics. However, concentrating milk by UF did not significantly affect the primary stages of rennet gelation and neither did it affect the rennet coagulation time (RCT) for 3× and 5× concentrated milks, as measured by DWS and by rheology. On the other hand, increasing concentration by UF did cause

Acknowledgments

This work was funded by the Ontario Dairy Council and the Natural Sciences and Engineering Council of Canada, through the Industrial Chair program.

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