High-pressure-induced changes in the rennet coagulation properties of bovine milk

The mechanism of high-pressure (HP)-induced changes in rennet coagulation properties of milk, particularly the role of whey protein-casein micelle associations, was studied. Treatment at 100 or 250 MPa reduced the rennet coagulation time (RCT) of raw skimmed bovine milk, compared with untreated milk. Treatment at 400 MPa had little effect, but at 600 MPa, RCT increased considerably. HP-induced increases in RCT did not occur in serum protein-free milk or milk treated with the sulphydryl-oxidising agent KIO₃, which prevents association of denatured β-lactoglobulin with casein micelles. Treatment at 5 or 10 °C at 250–600 MPa resulted in shorter RCT than treatment at 20 °C. In milk without KIO₃, coagulum strength was highest after treatment at 250 or 400 MPa, whereas in milk with KIO₃ it was highest after treatment at 400 MPa. These results indicate the significance of HP-induced association of whey proteins with casein micelles for rennet coagulation properties of milk.     

Fractionation of pasteurized skim milk proteins by dynamic filtration

This paper describes a two-stage process for separating milk proteins from pasteurized skim milk in three fractions: casein micelles, β-Lactoglobulin (β-Lg) and other large whey proteins, and α-Lactalbumin (α-La). Casein micelles were extracted in the retentate of a microfiltration using rotating ceramic disk membranes. α-La and β-Lg transmissions remained between 0.8 and 0.98. Their yields in permeate reached 81% for α-La and 76.6% for β-Lg at a VRR of 5.4. The separation between β-Lg and α-La was carried out by UF using a rotating disk module equipped with a 50 kDa PES circular membrane. Permeate fluxes were very high, remaining above 340 L h^?1 m^?2 at VRR = 5 and 40 °C. α-La transmission remained generally between 0.2 and 0.13 giving yields from 28% to 34%. β-Lg rejection was above 0.94, giving a maximum selectivity of 4.2. These data confirm the potential of dynamic membrane filtration for separating α-La and β-Lg proteins from skim milk.     

The prediction of moisture sorption isotherms for dairy powders

Moisture sorption isotherms were measured for whey protein isolate, high micellar casein and a milk protein concentrate powder. No temperature dependence was observed over the temperature range of 4–37 °C. At 50 °C the powders absorbed less moisture than observed at the lower temperatures. These isotherms were used to predict the isotherms for freeze-dried amorphous lactose/casein/whey protein powders. An isotherm for micellar casein was predicted using a simple additive isotherm model and was used along with isotherms for whey protein and amorphous lactose to predict moisture sorption isotherms for commercial dairy powders. Predicted isotherms compared well with measured isotherms indicating that this simple additive isotherm model is suitable for predicting moisture sorption isotherms of dairy powders. Delayed lactose crystallisation was observed in lactose/whey protein powders when compared to lactose/casein powders over the same water activity range.     

Effect of milk fat concentration and gel firmness on syneresis during curd stirring in cheese-making

An experiment was undertaken to investigate the effect of milk fat level (0%, 2.5% and 5.0% w/w) and gel firmness level at cutting (5, 35 and 65 Pa) on indices of syneresis, while curd was undergoing stirring. The curd moisture content, yield of whey, fat in whey and casein fines in whey were measured at fixed intervals between 5 and 75 min after cutting the gel. The casein level in milk and clotting conditions was kept constant in all trials. The trials were carried out using recombined whole milk in an 11 L cheese vat. The fat level in milk had a large negative effect on the yield of whey. A clear effect of gel firmness on casein fines was observed. The best overall prediction, in terms of coefficient of determination, was for curd moisture content using milk fat concentration, time after gel cutting and set-to-cut time (R² = 0.95).     

Effect of hydrolysis of casein by plasmin on the heat stability of milk

This study examined the effect of hydrolysis of casein by added plasmin (6 mg L⁻¹) on the heat stability of raw, pre-heated, serum protein-free or concentrated skim milk. Plasmin activity markedly affected the heat stability–pH profile of skim milk and serum protein-free milk, apparently by altering the properties of the casein micelles. It is probable that changes in the surface charge of the micelles, as a result of the hydrolysis of caseins, contributed to this effect. Hydrolysis by plasmin reduced the zeta-potential of the casein micelles from ∼−19 to ∼−16 mV. The effect of hydrolysis of casein by plasmin on the heat stability of pre-heated milk was less pronounced, shifting the heat stability–pH profile to more alkaline values; the heat stability of concentrated milk was unaffected by plasmin. A very high (50 mg L⁻¹) level of added plasmin resulted in clearing of the skim milk; the L^* value decreased from ∼75 to ∼35 after 24 h incubation at 37 °C. Clearing was correlated with a change in casein micelle diameter from an initial value of ∼175 to ∼250 nm. It is suggested that plasmin-induced changes in zeta-potential may promote micellar aggregation or changes in micelle stucture.     

Baroprotection of vegetative bacteria by milk constituents: A study of Listeria innocua

The baroprotective effect of milk constituents on Listeria innocua 4202 treated at 350 or 500 MPa for 5 min was examined. High-pressure (HP) treatment of L. innocua 4202 (1×10⁹ cfu mL⁻¹) resulted in complete inactivation in simulated milk ultra-filtrate (SMUF), a ∼5.0 log reduction in phosphate-buffered saline and a ∼2.9 log reduction in milk. The addition of micellar casein to SMUF increased survival of the bacterium by 3 logs, compared with SMUF alone, but the protective effect was negated if the minerals associated with the casein micelles were removed. The colloidal minerals calcium (30 mm), magnesium (5 mm), citrate (10 mm) and phosphate (20 mm), suspended in SMUF increased survival by ∼3.3, ∼1.7, ∼3.3 and ∼3.5 logs, respectively. The buffering capacity of the suspending medium was found to be a key factor in microbial baroresistance. Buffering by phosphate and citrate in milk may protect microorganisms against changes in pH during HP treatment, whereas the divalent cations calcium and magnesium may protect cell membranes against HP.     

Ethanol stability of casein micelles cross-linked with transglutaminase

The influence of transglutaminase (TGase)-induced cross-linking on the ethanol stability of skimmed milk was investigated. The stability of milk against ethanol-induced coagulation increased in sigmoidal fashion with milk pH (5.0–7.5) for all samples; ethanol stability also increased upon incubation (0–24 h) with 0.05 g L⁻¹ TGase at 30 °C. In untreated milk, addition of ethanol induced a collapse of the polyelectrolyte brush of κ-casein on the micelle surface, thereby facilitating micellar aggregation. Dynamic and static light scattering measurements indicated that in TGase-treated milk, the ethanol-induced collapse of the polyelectrolyte brush was far less than in untreated milk, suggesting that the increased ethanol stability of TGase-treated casein micelles is caused by the cross-linking of the polyelectrolyte brush on the micellar surface.     

Formation of soluble protein complexes and yoghurt properties influenced by the addition of whey protein concentrate

The effects of whey protein concentrate (WPC) on the formation of soluble protein complexes and yoghurt texture were evaluated. Skim milk (SM) and skim milk enriched with 1% WPC (SM + 1%WPC) or 2% WPC (SM + 2%WPC) were left unheated or heated and then made into yoghurt gels. Yoghurt prepared from heated SM + 2%WPC had significantly higher storage modulus, water holding capacity and firmness values and a denser microstructure than those prepared only from skim milk. Electrophoretic analysis of the milk showed that the level of β-lactoglobulin and κ-casein in the serum phase increased with increasing WPC concentration, indicating that the content of disulfide-linked β-lactoglobulin and κ-casein was higher in SM + 2%WPC than in SM, suggesting that more soluble protein complexes had been formed. Consequently, yoghurt prepared from heated SM enriched with WPC may have more bonds and more protein complexes in the protein network than yoghurt prepared only from SM, thus resulting in firmer gels.Practical applicationsYoghurt, one of the most popular fermented milk products, is of high economic importance to the dairy industry worldwide. In particular, high-protein yoghurt, such as Greek-style or set-type yoghurt, has been driving its ongoing popularity over recent years. In current industrial production of high-protein yoghurt, protein fortification and heat treatment of milk are two of the most important processing parameters affecting yoghurt texture. Whey protein concentrate has been added to milk to reduce whey separation and to increase the firmness of the yoghurt. From a technological point of view, the interaction of the denatured whey proteins with casein micelles or with κ-casein in the serum phases is regarded as responsible for obtaining a good yoghurt structure. The present research has shown that it is possible to produce yoghurt with a range of textural properties by precisely controlling the rate of whey protein fortification during its manufacture. Therefore, this study provides a better understanding of the effect of WPC fortification and aims to extend this insight for the production of good-quality yoghurt.     

Improvement of enzymatic cross-linking of casein micelles with transglutaminase by glutathione addition

The impact of glutathione (GSH) on cross-linking of micellar casein and sodium caseinate by microbial transglutaminase (TG) was investigated. Micellar casein was obtained by removing whey proteins from skim milk by means of membrane separation techniques. The addition of GSH (0.05–0.1 mm) was found to enhance the cross-linking of micellar casein suspended in milk serum. Cross-linking of sodium caseinate remained unaffected by GSH addition. Mixing TG and milk serum before addition of substrate proteins, however, resulted in an almost complete inhibition of the enzyme. When GSH and TG were present in the system before substrate addition, the reactivity of TG can be maintained. Hence, it was concluded that the addition of GSH mainly affected interactions between TG and an indigenous TG inhibitor present in milk serum. The addition of GSH allows cross-linking in milk products by TG without requiring a prior heat treatment of the milk beyond pasteurisation conditions.     

Inactivation of an indigenous transglutaminase inhibitor in milk serum by means of UHT-treatment and membrane separation techniques

An indigenous inhibitor in raw milk inhibits cross-linking by transglutaminase (TG). The enzymatic cross-linking of micellar casein, compared with sodium caseinate, taking thermal inactivation of the TG inhibitor in the milk serum into consideration, was investigated. Inhibitor-free micellar casein was prepared by membrane separation combined with heat treatment of the UF permeate. The inhibitor permeated through MF (nominal pore size 0.1 μm) and UF (cutoff 25 kDa) membranes. TG-catalyzed cross-linking of casein micelles was clearly enhanced by UHT-treatment of UF permeate. Variation of the enzyme concentration showed that the inhibitory effect could not be compensated by higher enzyme concentrations when the casein micelles were suspended in unheated milk serum. Sodium caseinate, however, underwent high degrees of cross-linking even in unheated milk serum. By mixing an unheated milk serum and a UHT-treated milk serum at different ratios, the relative TG inhibitor activity was analysed. High inactivation (>80%) of the TG inhibitor is necessary to achieve high degrees of protein cross-linking.     

Formation of whey protein/κ-casein complexes in heated milk: Preferential reaction of whey protein with κ-casein in the casein micelles

Studies of the formation of soluble κ-casein/whey protein (WP) complexes in heated (90 °C 10 min⁻¹) milk and related mixtures of proteins have been made. The use of milk samples containing different genetic variants, and having different compositions, allowed the effects of changing the natural protein balance on the formation of particles to be investigated. In addition, studies were made of the effects of addition of WP or of purified κ-casein to the milk samples. The addition of WP caused an increase in the amount and the size of the complexes, but addition of κ-casein to the milk had little or no effect on the complex formation, nor did it seem that the added κ-casein could react with the WP in the milk. Conversely, in systems where the casein micelles were absent, the purified κ-casein reacted well with WP, suggesting that in milk heated at its normal pH the WP react preferentially with the κ-casein on the casein micelles.     

Comparison of rheological properties of acid gels made from heated casein combined with β-lactoglobulin or egg ovalbumin

Gelation of heat-treated milk protein solutions by acid fermentation was performed using a model system of micellar casein alone (systR), micellar casein and β-lactoglobulin (systB) or micellar casein and egg ovalbumin (systO) dissolved in a milk ultrafiltrate and heat-treated at 90°C for 24 min. Solubility of globular proteins at given pH values and their interactions with casein were determined. Particle size and ζ-potential of the protein systems were measured before and after heat treatment. Acid gelation of the heated systems was monitored using dynamic low-amplitude strain oscillation. Ovalbumin alone or with casein produced larger particles than casein alone or with β-lactoglobulin upon heat treatment. The systems gelled at different pH values, i.e. 4.88, 5.47 and 5.88 in systR, systB and systO, respectively. The latter two pH values were clearly related to the pH of the loss of solubility of the heated globular proteins. While the gelation pattern for systR resembled unheated milk, the pattern for systB and systO showed the usual maximum for tan δ of heated milk.     

Effect of chemical phosphorylation on solubility of buffalo milk proteins

Buffalo milk proteins (casein, co-precipitate or whey protein concentrate) were phosphorylated with phosphorus oxychloride (POCl₃) at three different pH values (5.0, 7.0 and 9.0). The solubilities of phosphorylated milk proteins were examined over the pH range 3.0–9.0 in water and ionic (0.1 m NaCl or 10–70 mm Ca²⁺) systems. The solubilities of buffalo milk proteins decreased at pH 3.0, while there was an increase in the solubilities of casein and co-precipitate near their isoelectric points upon phosphorylation. Solubilities of these phosphorylated milk proteins were pH dependent in 0.1 m NaCl but there was a decrease in their solubilities with increase in calcium ion concentration. This alteration could be due to the shifting of isoionic points of phosphorylated buffalo milk proteins towards acidic pH.     

Effects of high hydrostatic pressure treatments on haemagglutination activity and structural conformations of phytohemagglutinin from red kidney bean (Phaseolus vulgaris)

Red kidney beans were subjected to high hydrostatic pressure (HHP) treatment (50, 150, 250, 350, 450 MPa) and phytohaemagglutinin (PHA) was then extracted by affinity chromatography. It appeared that HHP treatment could increase crude extract yield and decrease its haemagglutination activity. For purified samples, PHA yield was not affected at pressures <450 MPa while the haemagglutination activity was noticeably reduced at 450 MPa. The structural changes were investigated using electrophoresis, size exclusion chromatography (SEC), Fourier transform infrared (FTIR) and differencial scanning calorimetry (DSC). Electrophoresis and SEC profiles revealed a new high molecular weight polymer after 450 MPa treatment. At pressures <450 MPa, FTIR showed an increase in β-sheet structure and a decrease in α-helix. At 450 MPa, the bands at 1688 cm^?1, representing aggregate strands and random coils, increased. The conclusions are that pressures <450 MPa can cause PHA unfolding and induce PHA aggregation at 450 MPa.     

Effects of combined high pressure and enzymatic treatments on the hydrolysis and immunoreactivity of dairy whey proteins

The effect of high-pressure (HP) treatment on the hydrolysis of dairy whey proteins by trypsin, chymotrypsin and pepsin was analysed. Isostatic pressure (100–300 MPa for 15 min at 37 °C) was applied to the protein substrate prior to its enzymatic hydrolysis. Digestion was also conducted at atmospheric pressure (0.1 MPa) and under high pressure. The extent of hydrolysis was measured by the o-phthaldialdehyde method, the peptide profile was analysed by reverse-phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and the residual immunochemical reactivity was assessed by an ELISA test using a pool of seven sera from children allergic to bovine milk, an individual serum also positive (positive control) and two sera from non-allergic children (negative controls). The high pressure increased the degree of hydrolysis by the three enzymes used. Chymotrypsin and trypsin showed the highest proteolysis at 100 and 200 MPa followed by pepsin at 300 MPa. The β-lactoglobulin was hydrolysed by trypsin and chymotrypsin at atmospheric and at high pressures, whereas the pepsin only hydrolysed this protein under high pressure. Pepsin and trypsin hydrolysed α-lactalbumin in all cases. In contrast, this protein was not digested by chymotrypsin, irrespective of the pressure applied. An important decrease of immunochemical reactivity was found for pepsin and trypsin hydrolysates obtained under high pressure. The pool of seven sera detected immunoreactivity in the products of chymotrypsin hydrolysis under high pressure, which was not detected when the serum of one patient was used. The results suggest that dairy whey hydrolysates obtained by pepsin and trypsin in combination with HP treatment could be used as a source of peptides in hypo-allergenic infant formulae.     

Influence of processing conditions on reducing γ-aminobutyric acid content during fortified milk production

The work studied the effects of processing conditions on the γ-aminobutyric acid (GABA) loss during fortified milk production. Bovine milk or their proteins/lactose fractions (0.66% whey protein and 2.6% casein or 4.9% lactose, w/v) containing 0.05–1.0% added γ-aminobutyric acid (w/w, based on bulk milk or these fractions) were subjected to a simulated milk technological process as following the sequential preheating (25–60 °C), homogenization (0–20 MPa), and pasteurization (62 °C/30 min, 72 °C/15 s, 95 °C/5 min, and 138 °C/2 s) or their unit processes to treat GABA. The resulting samples were characterized through GABA and lactose concentrations under various processing conditions. The amine and carboxyl groups and the structural characteristics of the resulting protein (lactose) were also examined through their concentrations (for lactose) and mass/spectral analyses, respectively. The results showed that the increase in temperature significantly promoted a reduction in GABA content. Whey protein fractions than caseins were primarily responsible for inducing GABA, whereas lactose had no remarkable effect on it. The rationale for GABA reduction is potential reactions with milk proteins/lactose, which preliminarily confirmed by the measurement of protein modification and lactose mass spectrometry.     

Impact of cryoconcentration on casein micelle size distribution,micelles inter-distance,and flow behavior of skim milk during refrigerated storage

Cryoconcentration combined with a cascade effect was used to concentrate skim milk up to 25.12% total dry matter. Size, shape, and inter-micellar distance of casein micelles were characterized by ZetasizerNano-ZS, transmission electron microscopy, and ImageJ analyses. Flow properties of the cryoconcentrated skim milk were evaluated during 5 weeks of storage under refrigerated condition at 4 °C. Milk color was also evaluated according to the L*, a*, and b* system. The cryoconcentrated skim milk obtained after three cryoconcentration cycles was characterized by a monomodal distribution of its micelles with a tendency to smaller casein micelles. Approximately 60% of the total micellar volume was occupied by the casein micelles with a size of 100–200 nm, less than 18% of the volume with a size of 50–100 nm and only less than 1% was occupied by micelles with a size > 350 nm. This result shows that cryoconcentration changed the distribution of the mean size of the casein micelles to smaller units. No significant difference was observed on the inter-micellar distance. Cryoconcentration significantly improved the color of skim milk by increasing the L* value up to 67 which was similar to that of whole milk. Transition from a Newtonian to a non-Newtonian behavior was observed from the fourth week storage with a slight increase of casein micelle size.Industrial relevanceA concentration procedure of skim milk based on a complete block cryoconcentration technique was proposed. Application of this sub-zero technology permitted the concentration of skim milk total dry matter up to 25%. The casein micelle size was positively affected by moving the major part of the micelles toward the smaller size, whereas the inter-micellar distance was not affected. This new knowledge can be exploited in milk-based products to enhance the product stability. The cryoconcentrated skim milk color was positively affected since its L* value, which represents the milk whiteness, was significantly improved. The flow behavior of the cryoconcentrated milk was of Newtonian type up to 4 weeks of storage at 4 °C. The generated knowledge in this study can be easily used by the milk processing industry in order to make stable milk product with high dry matter content without adding milk powder, which negatively affects the product sensory properties (floury consistency).     

Effect of heat treatment and casein to whey protein ratio of skim milk on graininess and roughness of stirred yoghurt

The aim of this work was to study how heat treatment and casein (CN) to whey protein (WP) ratio of skim milk affect physical characteristics of stirred yoghurt. Different heat treatments (95 °C/256 s, 110 °C/180 s and 130 °C/80 s) were applied to the yoghurt milk with the CN to WP ratios of 1.5:1, 2:1, 3:1 and 4:1. Physical properties, including graininess and roughness, of stirred yoghurt were determined during storage at 4 °C for 15 days. Visual roughness, number of grains, perimeter of grains, storage modulus, and yield stress decreased, when heating temperature or CN to WP ratio increased.     

Modifying the microstructure of low-fat yoghurt by microfluidisation of milk at different pressures to enhance rheological and sensory properties

The effects of microfluidisation of milk at different pressures, prior to heat treatment, on structural and sensory properties of low-fat stirred yoghurt, were investigated. Low-fat yoghurts prepared from microfluidised milk were compared with low-fat (1.5%) and full-fat (3.5%) control yoghurts made with homogenised (20/5 MPa) milk. The microstructure of low-fat yoghurts prepared with microfluidised milk consisted of smaller and more uniform fat globules, well incorporated into more interconnected fat-protein gel networks, compared with those of control yoghurts. This modification in microstructure caused significant changes in gel particle size, sensory profile and rheological behaviour. Microfluidisation increased the gel particle size, gel strength and viscosity; marked beneficial effects were found at higher pressures (50–150 MPa). Microfluidising milk at 50–150 MPa increased the gel strength by 171–195% and viscosity by 98–103%, creating low-fat yoghurts with creaminess and desirable texture properties similar to, or better than, full-fat conventional yoghurt.     

Structures and some properties of soluble protein complexes formed by the heating of reconstituted skim milk powder

The effects of heat on reconstituted skim milk powder (RSMP) at a temperature of 90 °C have been determined, with respect to the formation of soluble protein complexes. Heating at the natural pH of the RSMP forms soluble complexes in the milk serum, which can be separated by size exclusion chromatography. The diameters of the particles were found to be up to 50 nm, as shown by light scattering and by scanning electron microscopy. The formation of the soluble complexes was strongly affected by the treatment of the RSMP before heating. Treatment of the milk with small amounts of glutaraldehyde, to fix the casein micellar structure, severely inhibited the formation of the soluble complexes. The pH at which the milk was heated (in the range 6.5–7.2) also had an effect on the amounts and the sizes of the complexes, as determined by size exclusion chromatography. The amount of soluble complex present in the RSMP affected the strength of the acid gels produced from the different milks, as was shown by exchanging the sera of milks treated differently with glutaraldehyde, and by following the gelation of milks heated at different pH values. Increased amounts of soluble complexes gave stronger acid gels. However, the relationship appeared to break down if milk was heated at pH 7.2, where the soluble material appeared to change and the acid gel was much weaker.