Effects of added whey protein aggregates on textural and microstructural properties of acidified milk model systems

Non-fat milk model systems containing 5% total protein were investigated with addition of micro- or nanoparticulated whey protein at two levels of casein (2.5% and 3.5%, w/w). The systems were subjected to homogenisation (20 MPa), heat treatment (90 °C for 5 min) and chemical (glucono-delta-lactone) acidification to pH 4.6 and characterised in terms of denaturation degree of whey protein, particle size, textural properties, rheology and microstructure. The model systems with nanoparticulated whey protein exhibited significant larger particle size after heating and provided acid gels with higher firmness and viscosity, faster gelation and lower syneresis and a denser microstructure. In contrast, microparticulated whey protein appeared to only weakly interact with other proteins present and resulted in a protein network with low connectivity in the resulting gels. Increasing the casein/whey protein ratio did not decrease the gel strength in the acidified milk model systems with added whey protein aggregates.     

Interactions in heated milk model systems with different ratios of nanoparticulated whey protein at varying pH

To better understand the interactions between nanoparticulated whey protein (NWP) and other milk proteins during acidification, milk model systems were diluted to 0.5% protein concentration and adjusted to pH of 6.0–4.5 following homogenisation and heat treatment. The diluted systems with different concentrations of NWP (0–0.5%) were characterised in terms of particle size, viscosity, surface charge and hydrophobicity. When pH was adjusted to 5.5, aggregation was initiated at levels of NWP (0.25–0.5%) leading to significant increase in particle size and viscosity. Pure NWP (0.5%) showed largest initial surface charge (−27 mv) and higher surface hydrophobicity than the other systems. The results indicated that NWP could self-associate above pH 5.5 and not only the decrease of electrostatic repulsion but also other interactions, such as hydrophobic interaction, play an important role in contributing to the early self-association of NWP.     

Rheological and structural properties of differently acidified and renneted milk gels

In this study we assessed the rheological and structural properties of differently acidified and renneted milk gels by controlling pH value and renneting extent. Skim milk were exactly renneted to 4 extents (20, 35, 55, and 74%) and then direct acidified to the desired pH (4.8, 5.0, 5.2, 5.5, 5.8, and 6.2), respectively. Rheological properties were assessed by dynamic rheological measurements, structural properties were studied by spontaneous whey separation and confocal laser scanning micrograph, and protein interactions were studied by dissociation test. Results showed that minimally renneted milk samples (20 and 35%) formed weak gels with low storage modulus, and the acidification range within which gels could form was narrow (pH ≤5.2). Highly renneted milk samples formed more gels with high storage modulus. The results of this study revealed that acidification determined the structural properties of highly renneted milk gels. As pH increased from 5.0 to 6.2, highly renneted milk gels had lower loss tangent, decreased spontaneous syneresis, and smaller pores. For both the low and high rennetings, divalent calcium bonds contributed less at low pH than at high pH. In conclusion, renneting increased the pH range suitable for gel formation; acidification determined the spontaneous syneresis and microstructure of highly renneted milk gels.     

Are disulphide bonds formed during acid gelation of preheated milk?

The role of thiol/disulphide exchanges during acid gelation of preheated milk was studied with milk samples with or without N‐ethylmaleimide (NEM), a thiol‐blocking agent, and acidified to pH 4 by the addition of glucono‐delta‐lactone at 20 °C. Active or total thiol groups, particle size with light scattering measurements in a dissociating solvent or by SDS‐agarose electrophoresis were determined on acidified milk samples. Diffusing wave spectroscopy and rheology in low strain were applied during acidification of sample, while rheology in large strain was applied on final acid gels. The only effect of the presence of NEM was a reduced firmness of acid gels as measured at large strain and a reduced tendency to form large aggregates at pH<5.5. In conclusions, thiol/disulphide exchanges during acidification of milk played only a minor role in the building of acid gel networks from heated milk.     

A novel technique for differentiation of proteins in the development of acid gel structure from control and heat treated milk using confocal scanning laser microscopy

The incorporation of caseins and whey proteins into acid gels produced from unheated and heat treated skimmed milk was studied by confocal scanning laser microscopy (CSLM) using fluorescent labelled proteins. Bovine casein micelles were labelled using Alexa Fluor 594, while whey proteins were labelled using Alexa Fluor 488. Samples of the labelled protein solutions were introduced into aliquots of pasteurised skim milk, and skim milk heated to 90 degrees C for 2 min and 95 degrees C for 8 min. The milk was acidified at 40 degrees C to a final pH of 4.4 using 20 g glucono-delta-lactone/l (GDL). The formation of gels was observed with CSLM at two wavelengths (488 nm and 594 nm), and also by visual and rheological methods. In the control milk, as pH decreased distinct casein aggregates appeared, and as further pH reduction occurred, the whey proteins could be seen to coat the casein aggregates. With the heated milks, the gel structure was formed of continuous strands consisting of both casein and whey protein. The formation of the gel network was correlated with an increase in the elastic modulus for all three treatments, in relation to the severity of heat treatment. This model system allows the separate observation of the caseins and whey proteins, and the study of the interactions between the two protein fractions during the formation of the acid gel structure, on a real-time basis. The system could therefore be a valuable tool in the study of structure formation in yoghurt and other dairy protein systems.     

The relationship between rheological parameters and whey separation in milk gels

The relation between whey separation of rennet-induced gels and rheological properties of those gels is reasonably well understood. A low fracture stress and a high value for the loss tangent at low frequencies have been correlated with a tendency to exhibit syneresis in rennet gels. In contrast, little is known about the relationship between mechanical properties of gels and whey separation in acid-induced milk gels, such as yoghurt, although this continues to be a major defect. In recent work, it has been found that conditions such as high milk heat treatment, fast rates of acidification and high incubation temperatures all gave high levels of whey separation compared with gels made from unheated milk that were incubated at low temperatures and where the rate of acidification was slow (i.e. when bacterial cultures were used instead of the acidogen, glucono-δ-lactone). The tendency to exhibit whey separation in acid gels made from heated milk was related to a low fracture strain and an increase in the loss tangent (observed even at high frequencies) during the gelation process (a high value indicates conditions favouring relaxation of bonds). Excessive rearrangements of particles in the gel network before and during gelation were implicated as being responsible for whey separation and rheological conditions that appeared to indicate this defect are described. It was also concluded that techniques that measure the spontaneous formation of surface whey should be distinguished from those that measure the expression of whey from networks under pressure as the latter tests only measure gel rigidity.     

Comparison of micellar casein isolate and nonfat dry milk for use in the production of high-protein cultured milk products

High protein levels in yogurt, as well as the presence of denatured whey proteins in the milk, lead to the development of firm gels that can make it difficult to formulate a fluid beverage. We wanted to prepare high-protein yogurts and explore the effects of using micellar casein isolate (MCI), which was significantly depleted in whey protein by microfiltration. Little is known about the use of whey protein-depleted milk protein powders for high-protein yogurt products. Microfiltration also depletes soluble ions, in addition to whey proteins, and so alterations to the ionic strength of rehydrated MCI dispersions were also explored, to understand their effects on a high-protein yogurt gel system. Yogurts were prepared at 8% protein (wt/wt) from MCI or nonfat dry milk (NDM). The NDM was dispersed in water, and MCI powders were dispersed in water (with either low levels of added lactose to allow fermentation to achieve the target pH, or a high level to match the lactose content of the NDM sample) or in ultrafiltered (UF) milk permeate to align its ionic strength with that of the NDM dispersion. Dispersions were then heated at 85°C for 30 min while stirring, cooled to 40°C in an ice bath, and fermented with yogurt cultures to a final pH of 4.3. The stiffness of set-style yogurt gels, as determined by the storage modulus, was lowest in whey protein-depleted milk (i.e., MCI) prepared with a high ionic strength (UF permeate). Confocal laser scanning microscopy and permeability measurements revealed no large differences in the gel microstructure of MCI samples prepared in various dispersants. Stirred yogurt made from MCI that was prepared with low ionic strength showed slow rates of elastic bond reformation after stirring, as well as slower increases in cluster particle size throughout the ambient storage period. Both the presence of denatured whey proteins and the ionic strength of milk dispersions significantly affected the properties of set and stirred-style yogurt gels. Results from this study showed that the ionic strength of the heated milk dispersion before fermentation had a large influence on the gelation pH and strength of acid milk gels, but only when prepared at high (8%) protein levels. Results also showed that depleting milk of whey proteins before fermentation led to the development of weak yogurt gels, which were slow to rebody and may be better suited for preparing cultured milk beverages where low viscosities are desirable.     

Power ultrasound as a tool to improve the processability of protein-enriched fermented milk gels for Greek yogurt manufacture

One approach to avoid production of acid whey during the manufacture of high-protein yogurt and related products is to concentrate the milk before fermentation. However, the resultant gels are firm so that stirring in the tank and further processing are difficult on an industrial scale. We hypothesize that power ultrasound (US) during fermentation softens the gel because sound waves cause cavitation and strong shear forces in the fluid. Skim milk was standardized to different protein contents up to 12%, heated (85°C, 30 min), and acidified with thermophilic or mesophilic starter cultures. An excessive increase in gel firmness as a function of protein content was detected. In the next series of experiments, US was applied during fermentation. Milks (10% protein) were acidified at 43.5°C and sonicated from pH 5.8 to 5.1 with a sonotrode (20 kHz, 20 W). Immediately after fermentation, gels were agitated using a rheometer with a vane geometry. The maximum torque required to break the gel was reduced by 75% following US, and gel firmness was reduced by 80%. Gels were then processed into stirred yogurt and analyzed. Sonicated samples were smoother with fewer large aggregates. Confocal laser scanning microscopy images suggested a less cohesive structure and more compact microgel particles, resulting in reduced viscosity. We concluded that US is a promising tool to weaken the gel and facilitate further processing. This enables new approaches for the manufacture of Greek yogurt, particularly in regard to avoiding production of acid whey and developing products with novel textures.     

Preliminary studies on the gelation processes of fermented and GDL-acidified bovine and caprine milk systems

Bovine and caprine milk gels were made by GDL-acidification and yogurt fermentation (using 'ropy' and 'non-ropy' starter cultures). The respective gelation processes were monitored rheologically using dynamic oscillatory testing. The bovine fermented systems produced gel structures with about half the strength of the equivalent chemically acidified gels. The fermented caprine milk systems produced gel structures some eight to 10 times weaker than the equivalent acidified systems. In all cases the caprine systems were weaker than the bovine gels despite having higher protein contents. In all cases the 'ropy' milk systems followed somewhat different gelation patterns and formed weaker gels than the equivalent 'non-ropy' and GDL-acidified systems. These data suggested that the starter culture material (biomass and extracellular polysaccharides) may have interfered with the protein–protein interactions during yogurt fermentation. This produced weaker gel structures, possibly by a modified gelation mechanism .     

Effect of Trichoderma reesei tyrosinase on rheology and microstructure of acidified milk gels

The aim of this work was to study the potential of tyrosinase enzymes in structural engineering of acid-induced milk protein gels. Fat free raw milk, heated milk or a sodium caseinate solution were treated with tyrosinases from Trichoderma reesei (TrTyr) and Agaricus bisporus (AbTyr) and the reference enzyme transglutaminase (TG) prior to acid-induced gelation. TrTyr treatment increased the firmness of raw milk and sodium caseinate gels, but not that of heated milk gels, even though protein cross-linking was detected in heated milk. AbTyr did not cross-link proteins in any of the studied milk protein systems. TG was superior to TrTyr in gels prepared of heated milk. In acidified heated milk and sodium caseinate, TrTyr and TG treatment resulted in a decrease of the pore size. Scanning electron microscopy revealed more extensive particle interactions in the heated milk gels with TG than with TrTyr.     

Interactions between milk proteins and gellan gum in acidified gels

The mechanical properties, microstructure and water holding capacity of systems formed from whey protein concentrate (0–3% WPC w/w), sodium caseinate (0–2% w/w), and gellan gum (0.1–0.3% w/w) in the coil or helix conformational state (Coil/Helix), were investigated. This polymer combination resulted in bi-polymeric or tri-polymeric systems, which were slowly acidified to pH 4.0 by the addition of GDL in order to favor electrostatic protein–polysaccharide interactions. The properties of the tri-polymeric systems differed considerably from the bi-polymeric ones. At high polymer concentrations the WPC-gellan samples showed incompatibility and microphase separation, which resulted in weaker and less deformable gels. However, in systems with coil gellan the incompatibility was less intense, which was attributed to the formation of electrostatic complexes between the protein and the polysaccharide during the mixing process. In caseinate–gellan systems, complex formation was observed and an increase in the gel mechanical properties as the caseinate concentration rose, although the water holding capacity decreased at higher gellan concentrations. The caseinate–gellan coacervate was not visualized in the tri-polymeric systems and the incompatibility between the biopolymers was intensified, although the mechanical properties were considerably higher than in the bi-polymeric gels.     

Functional properties of whey proteins microparticulated at low pH

The main aim of the study was to assess the effect of microparticulation at low pH on the functionality of heat-denatured whey proteins (WP). Spray-dried, microparticulated WP (MWP) powders were produced from 7% (wt/wt) WP dispersions at pH 3, acidified with citric or lactic acid, and microfluidized with or without heat denaturation. Nonmicroparticulated, spray-dried powders produced at neutral pH or pH 3 served as controls. The powders were examined for their functional and physicochemical properties. Denatured MWP had an approximately 2 orders of magnitude reduction in particle size compared with those produced at neutral pH, with high colloidal stability indicated by substantially improved solubility. The detection of monomeric forms of WP in PAGE also confirmed the particle size reduction. Microparticulated WP exhibited enhanced heat stability, as indicated by thermograms, along with better emulsifying properties compared with those produced at neutral pH. However, MWP powders created weaker heat-induced gels at neutral pH compared with controls. However, they created comparatively strong cold acid-set gels. At low pH, a combination of heat and high hydrodynamic pressure produced WP micro-aggregates with improved colloidal stability that affects other functionalities.     

Acid gelation properties of fibrillated model milk protein concentrate dispersions

Whey proteins in milk are globular proteins that can be converted into fibrils to enhance functional properties such gelation, emulsification, and foaming. A model fibrillated milk protein concentrate (MPC) was developed by mixing micellar casein concentrate (MCC) with fibrillated milk whey proteins. Similarly, a control model MPC was obtained by mixing MCC with milk whey proteins. The resulting fibrillated model MPC and control model MPC contained 5% protein and a ratio of casein to whey proteins similar to milk. The objective of the current study was to understand the rheological characteristics of fibrillated and control model MPC during acid gelation, using Förster resonance energy transfer (FRET) to assess small amplitude oscillation and casein–whey protein interaction. The results from the FRET index images showed greater interactions between caseins and whey proteins in fibrillated model MPC compared with the moderate and uniform interactions in control model MPC gels. Rheological study showed that the maximum storage modulus of acid gel of fibrillated model MPC was 546.9 ± 15.5 Pa, which was significantly higher than acid gel made from control model MPC (336.9 ± 11.3 Pa), indicating that fibrillated model MPC produced a firmer gel. Therefore, it can be concluded that acid gel produced from fibrillated model MPC was stronger than control model MPC. Selective fibrillation of the whey protein fraction in MPC can be used to improve gelation characteristics of acid gel type products.     

Tribo-rheological properties of acid milk gels with different types of gelatin: Effect of concentration

We investigated the effect of low concentrations (0.1 to 1%, wt/wt) of gelatin (types A and B) on the properties of acid milk gels in terms of rheology, tribology, texture, and water-holding capacity to better understand the role of gelatin in yogurt. The 2 types of gelatin showed similar effects on the properties of milk gels, with some minor differences, such as lubrication behavior at low concentrations. During acidification, gelatin at ≤0.4% caused an increase in the gel strength, and at higher concentrations it showed a negative effect. However, during cooling and annealing, we observed a positive effect on gel strength with 0.8 and 1% gelatin. Gelling and melting occurred at 0.8 and 1% concentrations of both types of gelatin. The addition of gelatin tended to decrease the storage modulus of milk gels and increase the apparent viscosity, pseudoplasticity, consistency, and yield stress. The firmness of the gels was decreased by gelatin at medium concentrations, but increased at high concentrations. Gelatin significantly enhanced the water-holding capacity of the gels; we observed no serum at concentrations ≥0.4%. With the addition of gelatin at concentrations ≥0.4%, the particle size of gels was greatly reduced, and their lubrication properties were significantly improved. This study showed that 0.4% was an effective concentration in acid milk gel; above this concentration, the properties of the milk gels were greatly changed. Tribology provided important information for understanding the role of gelatin in milk gels.     

Effects of starter culture and its exopolysaccharides on the gelation of glucono-δ-lactone-acidified bovine and caprine milk

The effects of the starter culture biomass material and its exopolysaccharides (EPS) on the structure of bovine and caprine acid gels were investigated. Inactivated cell material and/or extracellular polysaccharide, which had been separated from yogurt starter cultures, were added to both types of milk. The milk systems were acidified using glucono-δ-lactone (GDL) and the gelation processes were monitored using dynamic (oscillatory) rheological testing. The addition of cell material had no effect on the bovine acid gel, whereas the addition of polysaccharide produced a weaker gel structure. The addition of cell material (> 5 × 10⁸ cells/mL) and polysaccharide (0.35% w/v) weakened the caprine acid gel system. The treated gels were some three to eight times weaker than the untreated samples, suggesting that the starter culture metabolites and especially the EPS may act by interfering with the gel structures, particularly in the cases of milk systems such as caprine yogurt or low protein content bovine yogurt.     

Structural mechanisms leading to improved water retention in acid milk gels by use of transglutaminase

Water retention in transglutaminase (TG)-treated acid milk gels was studied and linked with the gel formation dynamics. Heat-treated skim milk with and without pre-treatment by TG was acidified at 20 °C, 30 °C and 40 °C at constant glucono-δ-lactone (GDL) level to obtain different acidification rates. Formation dynamics and structural properties of acid-induced gels were followed by rheological and near-infrared light backscattering measurements as well as microscopy. TG-treated gels showed decreased tan δ values all through the acidification, which was pronounced around the gelation point. Backscattered light intensity was lowered in TG-treated gels compared to the controls indicating that TG-treated gels were comprised of smaller aggregates. Water holding capacity (WHC) was measured by using centrifugation at selected pH points (pH 5.2, 5.0, 4.8 and 4.6) during acidification. Both acidification temperature and TG treatment had significant effects on the water retention properties of the gels. Spontaneous syneresis observed at high acidification temperatures (≥30 °C) was prevented upon TG-treatment. WHC of TG-treated gels was significantly higher compared to the control gels at all pH points. TG-treated milk gels showed a homogeneous network formed of smaller aggregate and pore sizes at the gelation point and did not show any large-scale re-organisation thereafter. Transglutaminase is likely to act as a fixative of the protein network at an early stage of gelation and thereby limiting network rearrangements that take place in acid milk gels formed at high acidification temperatures leading to contraction and subsequent wheying off.     

Formulation,process conditions,and biological evaluation of dairy mixed gels containing fava bean and milk proteins: Effect on protein retention in growing young rats

Food formulation and process conditions can indirectly influence AA digestibility and bioavailability. Here we investigated the effects of formulation and process conditions used in the manufacture of novel blended dairy gels (called “mixed gels” here) containing fava bean (Vicia faba) globular proteins on both protein composition and metabolism when given to young rats. Three mixed dairy gels containing casein micelles and fava bean proteins were produced either by chemical acidification (A) with glucono-δ-lactone (GDL) or by lactic acid fermentation. Fermented gels containing casein and fava bean proteins were produced without (F) or with (FW) whey proteins. The AA composition of mixed gels was evaluated. The electrophoretic patterns of mixed protein gels analyzed by densitometry evidenced heat denaturation and aggregation via disulfide bonds of fava bean 11S legumin that could aggregate upon heating of the mixtures before gelation. Moreover, fermented gels showed no particular protein proteolysis compared with gel obtained by GDL-induced acidification. Kinetics of acidification were also evaluated. The pH decreased rapidly during gelation of GDL-induced acid gel compared with fermented gel. Freeze-dried F, A, and FW mixed gels were then fed to 30 young (1 mo old) male Wistar rats for 21 d (n = 10/diet). Fermented mixed gels significantly increased protein efficiency ratio (+58%) and lean mass (+26%), particularly muscle mass (+9%), and muscle protein content (+15%) compared with GDL-induced acid gel. Furthermore, F and FW formulas led to significantly higher apparent digestibility and true digestibility (+7%) than A formula. Blending fava bean, casein, and whey proteins in the fermented gel FW resulted in 10% higher leucine content and significantly higher protein retention in young rats (+7% and +28%) than the F and A mixed gels, respectively. Based on protein gain in young rats, the fermented fava bean, casein, and whey mixed proteins gel was the most promising candidate for further development of mixed protein gels with enhanced nutritional benefits.     

Effect of different growth conditions on biomass increase in kefir grains

Kefir is a functional dairy product and the effects of kefir consumption on health have been well documented. Kefir grains have naturally high numbers of lactic acid bacteria and yeasts and are used in manufacturing kefir. The biomass of kefir grains slowly increases after successive fermentations. The effects of adding whey protein isolate, modified whey protein (MWP, fat replacer; Carbery Inc., Cork, Ireland), or inulin to milk and different atmospheric conditions (ambient or 6% CO₂) during fermentation on the increase in biomass of kefir grains were investigated. Reconstituted milks (10% milk powder) enriched with whey protein isolate (2%), MWP (2%), and inulin (2%) were inoculated with kefir grains and fermented in ambient and 6% CO₂ incubators at 25°C until a final pH of 4.6 was reached. Biomass increments of kefir grains were determined weekly over 30 d. Lactic acid bacteria and yeast contents of kefir grains were also determined. The highest biomass increase (392%) was found in kefir grains grown in milk supplemented with whey protein isolate under ambient atmospheric conditions. Application of CO₂ did not provide a significant supporting effect on the biomass of kefir grains. Addition of MWP significantly accelerated the formation of kefir grain biomass (223%). The use of whey protein isolate, MWP, or inulin in milk did not cause any adverse effects on the microbial flora of kefir grains.     

Characterisation of heat-set milk protein gels

Milk protein solutions [10% protein, 40/60 whey protein/casein ratio containing whey protein concentrate (WPC) and low-heat or high-heat milk protein concentrate (MPC)] containing fat (4% or 14%) and 70–80% water, form gels with interesting textural and functional properties if heated at high temperatures (90 °C, 15 min; 110 °C, 20 min) without stirring. Adjustment of pH before heating (HCl or glucono-δ-lactone) produces soft, spoonable gels at pH 6.25–6.6, but very firm, cuttable gels at pH 5.25–6.0. Gels made with low-heat MPC, WPC and low fat gave some syneresis; high-fat gels were slightly firmer than low-fat gels. Citrate markedly reduced gel firmness; adding calcium had little effect on firmness, but increased syneresis of low-heat MPC/WPC gels. The gels showed resistance to melting, and could be boiled or fried without flowing. Microstructural analysis indicated a network structure of casein micelles and fat globules interlinked by denatured whey proteins.