Application of transmission diffusing wave spectroscopy to the study of gelation of milk by acidification and rennet

Transmission diffusing wave spectroscopy has been used to study and compare three milk gelling systems (acid gelation of heated and unheated milks and rennet coagulation of unheated milk). In all cases, DWS was able to demonstrate the point of gelation as indicated by a rapid increase in particle size, as well as the small decreases in casein micelle radius attributed to the collapse or removal of the hairy kappa-casein layer. More importantly, the photon transport mean free path (l(*)) was measured. This parameter is unique to transmission DWS and can potentially give information about developing microstructures and the mechanical properties between different types of gels. The values of l(*) changed during the gelation processes, and these changes were manifested earlier than any change in particle aggregation or rheology of the systems. All three different gelling systems showed different changes in l(*) with time, showing the development of different interactions as the acidification or renneting reactions proceeded. Although a full analysis of the l(*) parameter and its changes cannot be made, it is concluded that they can provide important information on the pre-gelation states of aggregating systems.     

The impact of the concentration of casein micelles and whey protein-stabilized fat globules on the rennet-induced gelation of milk

The rennet-induced aggregation of skim milk recombined with whey protein-stabilized emulsion droplets was studied using diffusing wave spectroscopy (DSW) and small deformation rheology. The effect of different volume fractions of casein micelles and fat globules was investigated by observing changes in turbidity (1/l*), apparent radius, elastic modulus and mean square displacement (MSD), in addition to confocal imaging of the gels.Skim milk containing different concentration of casein micelles showed comparable light-scattering profiles; a higher volume fraction of caseins led to the development of more elastic gels.By following the development of 1/l* in recombined milks, it was possible to describe the behaviour of the fat globules during the initial stages of rennet coagulation. Increasing the volume fraction of fat globules showed a significant increase in gel elasticity, caused by flocculation of the oil droplets. The presence of flocculated oil globules within the gel structure was confirmed by confocal microscopy observations. Moreover, a lower degree of κ-casein hydrolysis was needed to initiate casein micelles aggregation in milk containing whey protein-stabilized oil droplets compared to skim milk.This study for the first time clearly describes the impact of a mixture of casein micelles and whey protein-stabilized fat globules on the pre-gelation stages of rennet coagulation, and further highlights the importance of the flocculation state of the emulsion droplets in affecting the structure formation of the gel.     

Influence of chemical agents on interactions in dairy products: Effect of SDS on casein micelles

The addition of SDS during skim milk reconstitution is an original approach to study the effect of an ionic amphiphilic molecule on the milk system and particularly on the casein micelle component. SDS-induced changes in casein micelles were investigated by turbidimetry, rheology, scanning electron microscopy (SEM) and biochemical measurements (including soluble proteins analysis). This study shows that casein micelles were able to interact together to form micellar aggregates or milk gel without coagulating agents addition, when milk was reconstituted in the presence of SDS. This micellar aggregation, depending on the SDS concentration, is confirmed by SEM observations showing that the general aspect of casein micelles was affected by SDS treatment. Biochemical analysis indicated that SDS induced micellar casein dissociation. SDS-induced milk gel formation required a defined level of casein dissociation which could be also related to a particular micellar state.     

Influence of whey protein denaturation on κ-carrageenan gelation

Response surface methodology was applied to study the effect of different heating temperature/time treatments on whey protein denaturation and its effect on κ-carrageenan gelation in milk. The path of gel formation was followed using small deformation rheology and the extent of whey protein denaturation was determined by gel permeation chromatography. κ-Carrageenan did not influence the rate of whey protein denaturation and it was unlikely that whey protein denaturation played a significant role on κ-carrageenan gelation in milk. In skim milk serum or skim milk ultrafiltrate the path of gel formation and gel strength were not influenced by the severity of heat treatment but increasing the concentration of whey proteins enhanced the gel strength. Heat treatment became important for carrageenan gelation in skim or recombined milks (i.e. in the presence of casein micelles) by influencing the gelation temperature and gel strength. Increasing the concentration of whey proteins in the recombined milks had a beneficial effect on gel strength.     

Acid-induced gelation of heat-treated milk studied by diffusing wave spectroscopy

Fresh skim milk is a stable colloidal system containing casein micelles and whey proteins. By decreasing the pH, the casein micelles become unstable and a gel is formed. During heat treatment at temperatures higher than 70 degrees C, the major whey proteins, e.g. alpha-lactalbumin and beta-lactoglobulin denature and start to interact with each other and with casein micelles. This changes the colloidal properties of the casein micelles. In this article, the pH-induced gel formation of heat-treated milk and the role of whey proteins was studied. Heat treatment in the range 70-90 degrees C induced a shift in gelation pH of skim milk to more alkaline pH values. This shift was directly related to whey protein denaturation. By using WPF milk it was shown that beta-lactoglobulin is principally responsible for the shift in gelation pH. alpha-lactalbumin caused neither alone nor in combination with beta-lg, an effect on the gelation pH. Heat treatment of milk for 10 min at 90 degrees C resulted in complete denaturation of the beta-lg present in skim milk but it is estimated that the casein micelles are coated only up to 40% by whey proteins when compared with pure whey protein aggregates.     

Hydrophilic lecithins protect milk proteins against heat-induced aggregation

In the present study, the heat-induced interaction between whey proteins and casein micelles was studied. To that end, the particle size distribution of 5.5% (w/w) casein micellar dispersions was determined by photon correlation spectroscopy as a function of both the whey protein concentration and heating time at 80 °C. The results clearly indicated that heat-induced aggregation of the casein micelles only occurred in the presence of whey proteins.

In an effort to overcome the heat-induced interactions between whey proteins and casein micelles, the influence of different soybean lecithins was investigated. Comparing native to hydrolysed, as well as hydroxylated soybean lecithin, it was observed that the heat-stabilising effect of the lecithins was directly related to their hydrophilicity: whereas native soybean lecithin had hardly any beneficial effect, highly hydrolysed as well as hydroxylated soybean lecithin largely prevented heat-induced casein micelle aggregation in the presence of whey proteins.

From experimental observations on the heat-induced decrease of whey protein solubility both in the absence and presence of hydrolysed lecithin, it was deduced that the latter may stabilise the exposed hydrophobic surface sites of heat-denatured whey proteins. Dynamic surface tension measurements indicated that the heat-stabilising properties of lecithins were mainly determined by their critical aggregation concentration.     

Effect of size, proteic composition, and heat treatment on the colloidal stability of proteolyzed bovine casein micelles

The casein micelles of reconstituted nonfat milk that have been fractionated by controlled pore glass chromatography showed a relationship between their size and their proteic composition: The fractions containing the smaller particles were richer in κ-casein than the fractions containing the bigger ones, in accordance with the casein micelle model of submicelles. The initial aggregation rate of micelles of different sizes, partially proteolyzed with chymosin (para-casein micelles), was measured in conditions of enzyme excess in which aggregation is the rate-limiting step of enzymatic coagulation, showing higher rates for the smaller micelles with the production of less compact para-casein micelle networks. This behavior could be explained in terms of electrostatic and steric colloidal stabilization due to their lower negative net charge and size and to a higher surface density of hydrophobic “patches” of proteolyzed κ-casein related to a higher probability of effective collisions between particles. Differences in the β-casein content did not seem to affect the initial aggregation rate of the micelles. On the contrary, the modifications of the micelle surface by heating affected the colloidal stability of the hydrolyzed micelles in different ways. The denaturation of the whey proteins and the formation of covalent complexes with κ-casein modify the micelle surface, increasing specially the steric stabilization, and produces a diminution in the number of hydrophobic sites that could be able to give interparticle hydrophobic interactions.     

Stability against aggregation of bovine casein micelles in the presence of acidic and alkaline gelatin

The effect of the presence of colloidal dispersed and molecular dispersed acidic (type A) and alkaline (type B) gelatins with similar molecular weight and size but different isoelectric points (7.9 and 4.9) on the stability against aggregation of bovine casein micelles was investigated by turbidimetric titration and laser techniques, over a wide range of biopolymers concentrations, gelatin/casein ratio in the initial mixture (0.03–20), pH (4.9–6.7) and ionic strength (10⁻³(milk salts)–1.0 NaCl), using glucono-δ-lactone (GL) as acidifier. Aggregates of acid gelatin A interact with the oppositely charged micellar casein at an ionic strength of around 10⁻³(milk salts) and pH 6.7 resulting in the formation of an electroneutral complex by ionic bonds between the carboxyl groups of casein and the amino groups of the gelatin molecules. The complexes obtained are polynuclear, the aggregation of which is not as sensitive to pH as that of free casein micelles. Aggregation of such complexes is the result of bridging flocculation. The “molar” ratio gelatin aggregates/casein micelles in the mixed aggregates is 4/1. The complexes are formed and stabilised via electrostatic interaction rather than through hydrogen bonds or hydrophobic interaction. In the presence of an excess of gelatin molecules in the initial mixture a charged gelatin–casein complex forms and some dissociation of casein micelles occurs and, as a consequence, soluble complexes are obtained. During the addition of alkaline gelatin B aggregates to the micellar casein solution and subsequent acidification of the mixture by GL, no effect of the presence of gelatin B on the stability of micellar casein was observed. Received: 28 March 2000 Accepted: 5 October 2000     

Diffusing wave spectroscopy investigations of acid milk gels containing pectin

The influence of the polysaccharide pectin on the gelation of acidified milk is studied in concentrated, undiluted, quiescent systems, primarily using diffusing wave spectroscopy. For pectins with a low degree of methylesterification (DM), interactions with milk-serum calcium yielded precipitated polysaccharide aggregates, even without acidification, that subsequently did not interact with casein micelles. However, high DM fine structures do not interact significantly with serum-calcium and absorb onto casein micelles as the pH is reduced below 5. A limited surface coverage of high DM pectin facilitates efficient bridging which enhances the rate of micelle aggregation and subsequent gelation and produces a clear signature in the shape of the measured MSD. The work highlights the fact that the behaviour of pectin in milk systems depends not only on the interaction of different polymeric fine structures with casein micelles, but also to a large extent on the interactions with calcium.     

Effect of pH and ionic strength on competitive protein adsorption to air/water interfaces in aqueous foams made with mixed milk proteins

Quantitative analysis of competitive milk protein adsorption to air/water interfaces in aqueous foam was performed by capillary electrophoresis (CE). Foams were made by whipping protein solutions, in which skim milk powder (SMP) and whey protein isolate (WPI) were mixed at 0.5% protein in different proportions at different pH values and NaCl concentrations. Preferential adsorption of beta-casein into foam phases occurred under most solution conditions, if partial dissociation of the casein micelles had occurred. Preferential adsorption of beta-casein was not observed with added Ca2+, due to the re-association of casein micelles. Enrichment of caseins into the foam phase was more apparent than that of whey proteins. The foamability of SMP demonstrated a continuous improvement due to the gradually increasing dissociation of casein micelles when the concentration of NaCl increased from 0 to 0.8 M. The foamability of WPI increased when NaCl concentration rose from 0 to 0.1 M, and decreased with further increase in NaCl concentration. NaCl at low concentration (I < or = 0.4) did not show a significant effect on the competitive adsorption among milk proteins, indicating that electrostatic interactions do not play a key role in competitive adsorption. NaCl at higher concentration, e.g., 0.6 M, caused less whey protein to be adsorbed to the air/water interfaces. The whippability of WPI was highest at pH 4.5 and lowest at pH 3, and that of SMP was the opposite. The proportions of beta-lactoglobulin and alpha-lactalbumin in the foam phase were lower at acidic pH and higher at basic pH, compared with that at natural pH of WPI.     

The effect of the melting of the collagen-like gelatin aggregates on the stability against aggregation of the bovine casein micelles

The effect of the melting of the collagen-like acid and alkaline gelatin aggregates on the stability against aggregation of bovine casein micelles was investigated by turbidimetry, DSC and circular dichroism in the wide range of biopolymers concentrations, gelatin/casein ratio (R) in initial mixture (R=0.03–20), pH (4.9–6.7), ionic strength (I=10⁻³ to 1.0/NaCl/), and temperature (10°–70 °C), using glucono-δ-lactone (GL) as acidifier. At low ionic strength (10⁻³/milk salts/) and neutral pH interaction between gelatin molecules and casein micelles is suppressed significantly above 36 °C. The melting of the collagen-like acidic gelatin above this temperature shifts the pH at which the complex formation is maximal to the acidic range. The cause may be that some of the functional ionized groups of gelatin molecules are inaccessible due to the conformational changes. The presence of gelatin B molecules had no effect on the aggregation stability of micellar casein in the range 0.03<R<20. At very high total protein concentration (above 10%) depletion flocculation of casein micelles was observed. The reason for the very high stability of casein micelles in this case cannot be explained by volume exclusion. Received: 28 March 2000 Accepted: 5 October 2000     

Effects of biopolymer ratio and heat treatment on the complex formation between whey protein isolate and soluble fraction of Persian gum

Proteins and polysaccharides are the two most important natural ingredients with unique functional properties. Their interactions can be considered as a route to produce new materials of various technological applications. In this study, the effect of pH (3–7) as well as the mixing ratio of whey protein isolate (WPI)/soluble part of Persian gum (PG) (1:3, 1:1, 3:1, 6:1, and 9:1% w/w WPI/PG) on the complex formation behavior of the two polymers were studied using spectrophotometric and dynamic light scattering techniques, soluble protein measurement, and microscopic observations. Investigating the curves of turbidity versus pH showed that pHc and pH_?1, which are associated with the formation of soluble and insoluble complexes, moved toward higher pH values by increasing WPI:PG ratio. Increased solubility was observed at pHc?>?pH?>?pH_?1 for all biopolymers mixtures. In addition, lowering the pH toward pH_?1 resulted in a decrease in the size of complexes, while further decrease of pH beyond this point led to larger particles. No significant difference was noticed between pH_c and pH_?1 of heated and unheated WPI/PG mixtures, although the turbidity of heated samples increased due to the formation of larger aggregates.     

Distribution of strontium in milk component

The distribution of strontium between the milk components, i.e., serum, casein micelles, whey and hydroxyapatite was determined. The sorption on hydroxyapatite was investigated using batch method and radiotracer technique. The aqueous phase comprised of either milk or whey. The sorption of strontium on hydroxyapatite depended on the method of its preparation and on the composition of the aqueous phase. The sorption of strontium was increased with an increase of pH. The presence of citrate species resulted in decrease of the sorption of strontium on hydroxyapatite. The sorption of ⁸⁵Sr on hydroxyapatite decreased with the increasing concentration of Ca²⁺ ions. Addition of Ca²⁺ ions to milk resulted in milk pH decrease. The decrease in pH value after calcium addition to milk is related to exchanges between added calcium and micellar H⁺. The average value of strontium sorption on casein micelles in milk with presence of hydroxyapatite was (47.3 ± 5.6) %. The average value of sorption of ⁸⁵Sr on casein micelles in milk without the addition of hydroxyapatite was (68.9 ± 2.2) %.     

Diffusing wave spectroscopy study of the colloidal interactions occurring between casein micelles and emulsion droplets: comparison to hard-sphere behavior

Understanding the underlying processes that govern interparticle interactions in colloidal systems is fundamental to predicting changes in their bulk properties. In this paper we discuss the colloidal behavior of casein micelles and protein-stabilized fat globules individually and in a mixture. The colloidal interactions were observed by transmission diffusing wave spectroscopy. The turbidity parameter, l*, and the diffusion coefficients of the samples studied were measured experimentally and compared to the theoretically calculated parameters for a hard-sphere system. The light scattering properties of casein micelles (volume fraction phi = 0.1-0.2) dispersed in milk permeate showed no deviation from the theoretically predicted model. Whey protein isolate (WPI)-stabilized emulsions (phi = 0.025-0.1) prepared either in milk permeate or in 5 mM imidazole buffer at pH 6.8 showed a behavior identical to that of the hard-sphere model. Similarly to the WPI-stabilized fat globules, the sodium caseinate (NaCas)-stabilized emulsions (phi = 0.025-0.1) prepared in milk permeate also showed resemblance to the theory. In contrast, NaCas-stabilized emulsions prepared in 5 mM imidazole buffer exhibited some discrepancy from the theoretically calculated parameters. The deviation from theory is attributed to the enhanced steric stabilization properties of these droplets in a low ionic strength environment. When recombined milks made from concentrated milk and WPI- and NaCas-stabilized droplets prepared in permeate (phi = 0.125-0.2) were studied, the experimental data showed a significant deviation from the theoretical behavior of a hard-sphere model due to mixing of two different species.     

Variation of the calcium content in skim milk by diafiltration and ion exchange – Effects on permeation rate and structure of deposited layers in the RO

The effect of different calcium levels on the permeation rate and on the structure of deposits formed in reverse osmosis (RO) was studied as a function of the transmembrane pressure difference. An almost complete removal of the soluble calcium together with the other low molecular constituents by diafiltration significantly changed the deposited protein layer compared with untreated skim milk. By determination of the flow resistance of the deposited layer Δp_L it was concluded that by the accumulation of individual micellar casein particles a loose structure was formed. The removal of the total calcium, i.e. both soluble and the calcium bound in the casein micelles, by ion exchange resulted in the disintegration of the micelle structure, causing a very dense deposited protein layer consisting of casein submicelles and a significant reduction of the permeation rate compared with untreated skim milk.     

Casein precipitation equilibria in the presence of calcium ions and phosphates

Calcium ion induced aggregation and precipitation equilibria of β-casein were studied in the presence or absence of mono- and polyphosphates at pH 5.5 and 7.5. We analyze the data by assuming ion binding at a well-defined stoichiometric ratio, leading to protein charge neutralization and formation of slightly soluble complexes. The precipitation curves are in good agreement with the model, showing the expected larger induction region and steeper slope at the lower pH. The addition of phosphates leads to better precipitation of the protein, which can be explained by a mechanism involving the formation of calcium phosphate microcrystals. These crystals provide a substrate for protein adsorption, with subsequent cross-binding of the casein micelles and formation of sturdy aggregates of co-precipitated calcium phosphate and casein. The crystallite formation leads to effective separations in commercial caseinates, which would be impossible with Ca²⁺ alone.     

Effect of temperature and pH on the aggregation and the surface hydrophobicity of bovine κ-casein

κ-Casein (κ-CN) aggregation by heating has been studied at pH 7.2 and 5.2 using UV-visible spectrophotometry, sodium dodecyl sulfate polyacrylamide gel electrophoresis, spectrofluorometric study of the 1–8 aniline naphtalene sulfonate (ANS)–κ-CN binding and circular dichroism (CD) spectroscopy. The aggregation process to form aggregates like micelles or submicelles and the structural characteristics of these aggregates were pH dependent. Far-UV CD showed that the aggregates obtained by heating presented changes in the κ-CN secondary structure. Near-UV CD spectra showed a certain degree of tertiary organization in the Tyr environment for the protein heated or unheated, only at pH 5.2. ANS binding at both pH was quite different and depends on the self-association process. Heating produced exposition of hydrophobic binding sites only at pH 7.2, including those in the neighborhood of the κ-CN Trp residue.     

The rennet coagulation mechanism of skim milk as observed by transmission diffusing wave spectroscopy

The technique of forward-scattering diffusing wave spectroscopy has been used to study the rennet-induced gelation of skim milk. The results allow the comparison of a colloidal suspension at a realistic concentration (Phi approximately 10%) compared with well-established measurements made on highly-diluted milk samples. It is shown that the partially renneted casein micelles do not begin to approach one another until the extent of breakdown of kappa-casein has reached about 70%; above this point, they interact increasingly strongly with the extent of proteolysis. This interaction initially restricts the diffusive motion of the particles rather than causing true aggregation. Only after more extensive removal of the protective kappa-casein does true aggregation occur, with the appearance of a space-filling gel (defined by rheology as having a value of tandelta<1). The results show in greater detail than hitherto the progress of interactions between the particles in a system where the steric stabilization is progressively destroyed, and suggest that the renneting of milk at its normal concentration cannot be described simply by reactions between freely-diffusing particles.     

Bound water measurements for aqueous protein solutions and food gels

The role of aqueous media in the stabilization of globular proteins and formation of gels was studied by absorption millimeter spectroscopy. This method allowed to measure bound water, the fraction of water which had decreased rotational mobility owing to the presence of solute. Hydration data for globular proteins were compared with data obtained previously for low-weight molecules and groups. It was found that rotational mobility of water molecules in the hydration shells of various kinds of solutes (groups) decreased in the following order: water structure breaking compounds>polar groups>unfolded proteins>globular proteins>non-polar groups. Time courses of the storage modulus were determined for the chemical acidification by glucono-δ-lactone (GDL) of milk samples prepared from skimmed milk powder (SMP). Gelation of unheated milk was a monotonous process that started at pH 4.9. Heat-treated milk from SMP (16 and 14 g per 100 ml) acidified by GDL (3 g per 100 ml) at 43 °C gave non-monotonous kinetics of gelation with two phases corresponding to the mechanisms induced by denatured whey proteins at pH>5 and by casein–casein interactions at pH 4.8–4.9. For heat-treated milk, measurement of bound water gave two stages of decrease in water mobility. Additional hydration of SMP during acidification gave 0.15–0.2 g and 0.8 g bound H₂O per gram of SMP for unheated and heat-treated milk, respectively.