Physicochemical properties and issues associated with trypsin hydrolyses of bovine casein-dominant protein ingredients

Milk protein concentrate (MPC) and sodium caseinate (NaCas) were hydrolysed using the enzyme trypsin and the subsequent physical properties of the two ingredients were examined. Trypsin hydrolysis was carried out at pH 7 and at 45 °C on 11.1% (w/w) protein solutions. Heat inactivation of trypsin was carried out when the degree of hydrolysis reached either 10 or 15%. Size-exclusion chromatography and electrophoresis confirmed a significant reduction in protein molecular weight in both ingredients. However, whey proteins in MPC were more resistant to trypsin hydrolysis than casein. Oil-in-water emulsions were prepared using intact or hydrolysed protein, maltodextrin, and sunflower oil. Protein hydrolysis had a negative effect on the subsequent physical properties of emulsions, compared with non-hydrolysed proteins, with a larger particle size (only for NaCas stabilised emulsions), faster creaming rate, lower heat stability, and increased sedimentation observed in hydrolysed protein emulsions.     

Effects of varying time/temperature-conditions of pre-heating and enzymatic cross-linking on techno-functional properties of reconstituted dairy ingredients

The effects of varying time/temperature-conditions of pre-heating and cross-linking with transglutaminase (TG) on the functional properties of reconstituted products from skim milk, WPC and sodium caseinate was analyzed. The degree of cross-linking (DC) of skim milk proteins could be increased from 54.4% to 70.5% by varying process conditions. Thereby the water-holding capacity (WHC) increased from 10% to 20%, while the heat stability decreased. The burning-on was lower than that of the non-treated products at optimum pre-heating conditions (90 °C/30 s). Using sodium caseinate as substrate for TG the DC increased from 39.2% to 100% due to the improvement of the process. As a result the WHC increased by 30% and the heat stability up to 380%. However, the burning-on of casein increased as well. TG-treated sodium caseinate started to gel at 10% protein, whereas untreated sodium caseinate gelled not before 15% protein. The WHC of enzyme-treated whey proteins was lowered. The heat stability of WPC could be doubled by TG-treatment, and the burning-on of the products was, especially at optimum pre-heating conditions, less pronounced. The degree of denaturation of TG-treated whey proteins was 2–5% higher than that of untreated samples.     

Gastric digestion of milk protein ingredients: Study using an in vitro dynamic model

The coagulation behavior and the kinetics of protein hydrolysis of skim milk powder, milk protein concentrate (MPC), calcium-depleted MPC, sodium caseinate, whey protein isolate (WPI), and heated (90°C, 20 min) WPI under gastric conditions were examined using an advanced dynamic digestion model (i.e., a human gastric simulator). During gastric digestion, these protein ingredients exhibited various pH profiles as a function of the digestion time. Skim milk powder and MPC, which contained casein micelles, formed cohesive, ball-like curds with a dense structure after 10 min of digestion; these curds did not disintegrate over 220 min of digestion. Partly calcium-depleted MPC and sodium caseinate, which lacked an intact casein micellar structure, formed curds at approximately 40 min, and a loose, fragmented curd structure was observed after 220 min of digestion. In contrast, no curds were formed in either WPI or heated WPI after 220 min of digestion. In addition, the hydrolysis rates and the compositions of the digesta released from the human gastric simulator were different for the various protein ingredients, as detected by sodium dodecyl sulfate-PAGE. Skim milk powder and MPC exhibited slower hydrolysis rates than calcium-depleted MPC and sodium caseinate. The most rapid hydrolysis occurred in the WPI (with and without heating). This was attributed to the formation of different structured curds under gastric conditions. The results offer novel insights about the coagulation kinetics of proteins from different milk protein ingredients, highlighting the critical role of the food matrix in affecting the course of protein digestion.     

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.     

Improving the structure and rheology of high protein,low fat yoghurt with undenatured whey proteins

The objective of this study was to investigate the effects of whey protein denaturation and whey protein:casein-ratio on the structural, rheological and sensory properties of high protein (8% true protein), low fat (<0.5% fat) yoghurt. Yoghurt milk bases were made by adding undenatured whey proteins from native whey protein concentrate (NWPC) to casein concentrate in different whey protein:casein-ratios. The degree of whey protein denaturation was then controlled by the temperature treatment of the yoghurt milk bases. Addition of NWPC in low (whey protein:casein-ratio 25:75) or medium levels (whey protein:casein-ratio 35:65) in combination with heat treatment at 75 °C for 5 min gave yoghurts with significantly lower firmness, lower storage modulus (G′), and better sensory properties (less coarse and granular and more smooth), compared with corresponding yoghurts produced from yoghurt milk bases heat-treated at 95 °C for 5 min or with control yoghurts (no addition of NWPC).     

酪蛋白种类和二次均质工艺对再制稀奶油搅打特性的影响

本研究旨在分析3 种常见的酪蛋白产品,胶束酪蛋白浓缩物（micellar casein concentrate,MCC）、酪蛋白酸钙（calcium caseinate,CaC）及酪蛋白酸钠（sodium caseinate,NaC）对再制稀奶油搅打特性的影响,以及再制稀奶油进行二次均质后其搅打特性的变化。结果表明：MCC、CaC及NaC再制稀奶油的搅打特性受酪蛋白质量分数的影响,且其对二次均质工艺的敏感性不同。MCC和CaC的质量分数较高（1.5%和2.5%）时,制备的稀奶油具有良好的搅打特性,最大起泡率在170%～200%范围内,泄漏率在0～1.5%范围内;进行二次均质后最大起泡率和泄漏率的变化较小。而NaC在较低的质量分数（0.5%）条件下制备的稀奶油才可具有较好的搅打特性,最大起泡率为（198.2±4.0）%;当NaC质量分数增至1.5%时,最大起泡率下降至（119.0±15.4）%。二次均质后NaC再制稀奶油的最大起泡率下降,泄漏率增加。研究认为,以MCC和CaC为原料制备的稀奶油无论是否进行二次均质,均有良好的搅打特性。     

Incorporation of dairy ingredients into wheat bread: effects on dough rheology and bread quality

 Dairy ingredients are used in breadmaking for their nutritional benefits and functional properties. The effects of the traditionally-used whole and skimmed milk powder, sodium caseinate, casein hydrolysate and three whey protein concentrates on dough rheology and bread quality were studied. Whole and skimmed milk powders improved sensory characteristics. Sodium caseinate and hydrolysed casein displayed beneficial functional properties in breadmaking including low proof time, high volume and low firmness. Both ingredients increased dough height measured with the rheofermentometer. Bread with 2% or 4% sodium caseinate added was rated highly in sensory evaluation. Incorporation of whey protein concentrates generally increased proof time, decreased loaf volume and decreased dough height measured with the rheofermentometer. Received: 6 April 1999 / Revised version: 13 July 1999     

Physico-chemical factors controlling the foamability and foam stability of milk proteins: Sodium caseinate and whey protein concentrates

We explored the foaming behavior of the two main types of milk proteins: flexible caseins and globular whey proteins. Direct foam comparison was complemented with measurements in model experiments such as thin foam films, dynamic surface tension, and protein adsorption. Foaming was studied as a function of pH (from below to above isoelectric point, pI) and range of ionic strengths. Maximum foamability was observed near pI ≈ 4.2 for WPC in contrast to sodium caseinate which had minimum foaming near pI = 4.6. Good foamability behavior correlated well with an increased adsorption, faster dynamic surface tension decrease and increased film lifetime. Differences in the stability of the foams and foam were explained with the different molecular structure and different aggregation behavior of the two protein types. Far from its isoelectric pI, casein adsorption layers are denser and thicker thus ensuring better stabilization. Added electrolyte increased further the adsorption and the repulsion between the surfaces (probably by steric and/or osmotic mechanism). In contrast the globular molecules of WPC probably could not compact well to ensure the necessary films and foams stabilization far from pI, even after electrolyte addition.     

Effect of Ultra-high Temperature Treatment on the Enzymatic Cross-linking of Micellar Casein and Sodium Caseinate by Transglutaminase

ABSTRACT: It was found that ultra-high temperature (UHT) treatment of sodium caseinate and native whey protein-depleted micellar casein drastically increases the protein polymerization effect of an enzymatic treatment by microbial transglutaminase (TG). As a result the concentration of the isopeptide ε-(γ-glutamyl)lysine was increased significantly in UHT-treated micellar casein solutions after TG incubation compared with the unheated casein solution. Sodium caseinate was more susceptible to the cross-linking reaction as compared with the native casein micelles. The results demonstrate that the protein structure significantly affects the TG cross-linking reaction. The effect of an UHT treatment was considered to be related to a better TG accessibility due to a more open casein micelle structure and to the inactivation of a TG inhibitor substance. The results demonstrate that an unidentified component in the natural milk serum inhibits the TG reaction. The thermal inactivation of a TG inhibitor is the dominant effect explaining the improved cross-linking of UHT-treated casein micelles as well as sodium caseinate.     

Rennet coagulation properties of UHT‐treated phosphocasein dispersions as a function of casein and NaCl concentrations

The renneting properties of whey protein‐free, UHT‐heated (140 °C/10 s) casein dispersions were investigated as a function of casein and NaCl concentration. It was found that the rennet coagulation time and gel firmness can be optimised when the whey protein‐free casein concentration is increased, while the added NaCl concentration is kept low. The strongest gel firmness occurs at 0.05 and 0.08 m NaCl addition and at a micellar casein concentration between 6.0 and 6.6 g/100 mL. Weak rennet gels were formed at 3.0–3.6 g/100 mL casein at all NaCl concentrations tested.     

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.     

Isolation and characterisation of κ-casein/whey protein particles from heated milk protein concentrate and role of κ-casein in whey protein aggregation

Milk protein concentrate (79% protein) reconstituted at 13.5% (w/v) protein was heated (90 °C, 25 min, pH 7.2) with or without added calcium chloride. After fractionation of the casein and whey protein aggregates by fast protein liquid chromatography, the heat stability (90 °C, up to 1 h) of the fractions (0.25%, w/v, protein) was assessed. The heat-induced aggregates were composed of whey protein and casein, in whey protein:casein ratios ranging from 1:0.5 to 1:9. The heat stability was positively correlated with the casein concentration in the samples. The samples containing the highest proportion of caseins were the most heat-stable, and close to 100% (w/w) of the aggregates were recovered post-heat treatment in the supernatant of such samples (centrifugation for 30 min at 10,000 × g). κ-Casein appeared to act as a chaperone controlling the aggregation of whey proteins, and this effect was stronger in the presence of α_S- and β-casein.     

Small and large deformation rheology and microstructure of κ-carrageenan gels containing commercial milk protein products

The rheological behaviour of commercial milk protein/κ-carrageenan mixtures in aqueous solutions was studied at neutral pH. Four milk protein ingredients; skim milk powder, milk protein concentrate, sodium caseinate, and whey protein isolate were considered. As seen by confocal laser microscopy, mixtures of κ-carrageenan with skim milk powder, milk protein concentrate, and sodium caseinate showed phase separation, but no phase separation was observed in mixtures containing whey protein isolate. For κ-carrageenan concentrations up to 0.5 wt%, the viscosity of the mixtures at low shear rates increased markedly in the case of skim milk powder and milk protein concentrate addition, but did not change by the addition of sodium caseinate or whey protein isolate. For κ-carrageenan concentrations from 1 to 2.5 wt%, small and large deformation rheological measurements, performed on the milk protein/κ-carrageenan gels, showed that skim milk powder, milk protein concentrate or sodium caseinate markedly improved the strength of the resulting gels, but whey protein isolate had no effect on the gel stength.     

Pilot-scale β-casein depletion from micellar casein via cold microfiltration in the diafiltration mode

The aim of this study was to produce pilot scale batches of β-casein concentrate and micellar casein concentrate with reduced β-casein level. The isolation of β-casein was done using membrane filtration at cold temperatures (≤5 °C). A micellar casein concentrate was obtained from skim milk by means of warm microfiltration (MF) at 50 °C (0.1 μm pore size, ceramic membranes). The concentrate was stored at 2–3 °C for approximately 40 h to induce temperature-dependent dissociation of β-casein from casein micelles. β-casein was separated from the cold-stored concentrate using MF (0.3 μm pore size, organic membranes) at ≤5 °C. β-Casein permeate was warmed up to 50 °C to lead self-association of β-casein micelles before ultrafiltration at 50 °C (10 kDa cut-off, organic membranes). Two streams, a β-casein-depleted and a β-casein concentrate, were generated. A purity of 92.64% and a yield of up to 18.07% were achieved for β-casein.     

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.     

Plasmin activity and proteolysis in milk protein ingredients

Despite the widespread use of milk protein ingredients and the well-known detrimental effects of plasmin-induced casein hydrolysis on product flavour and stability, little research has been carried out on the occurrence and activity of plasmin in milk protein ingredients. In this study, 19 sodium caseinate (NaCas), 13 calcium caseinate (CaCas), 2 micellar casein isolate (MCI) and 14 milk protein concentrate (MPC) samples were studied for plasmin and plasminogen-derived activity and proteolysis after reconstitution. Results indicated a higher occurrence and activity of plasmin in MPC and MCI than in NaCas and CaCas. During storage, activation of plasminogen to plasmin and autolysis of plasmin were observed. Furthermore, extensive plasmin-induced casein hydrolysis was observed. The specificity of casein hydrolysis was similar in all samples and proteolysis per unit plasmin activity was most extensive in MPC. These results indicate that residual plasmin activity should be a factor of consideration in milk protein ingredient selection.     

Effect of Transglutaminase on Physicochemical Properties of Set-style Yogurt

In this study, the effect of some ingredients such as skimmed milk powder, whey, sodium caseinate, calcium caseinate, whey protein concentrate (35, 60 kg/100 kg dry solids), whole milk powder, condensed milk and transglutaminase (TGase) on the properties of set-style yogurt was investigated. These protein and dry matter sources (2%) and TGase (1 U/g milk protein) were added into pasteurized milk and incubated prior to fermentation for 2 h at 40°C. After fermentation, enzyme action was stopped by heating for 1 min at 80°C. The control groups were conducted with addition of these materials into milk without TGase. All of the milk samples were inoculated with yogurt cultures at 45°C, until the pH was dropped to 4.4. Syneresis, gel-strength, acetaldehyde amounts, and the degree of TGase reaction were determined. As a result, yogurt products made from enzyme-treated milk showed increased gel strength and less syneresis. SDS-PAGE results showed that the enzyme TGase produced crosslink formation between different protein fractions of milk. In addition, it was also determined that TGase application caused a decrease in acetaldehyde amounts.     

Heat-induced aggregation of whey proteins in the presence of κ-casein or sodium caseinate

Aggregates were formed by heating mixtures of whey protein isolate (WPI) and pure κ-casein or sodium caseinate at pH 7 and 0.1 M NaCl. The aggregates were characterized by static and dynamic light scattering and size exclusion chromatography. After extensive heat-treatment at 80 °C for 24 h, almost all whey proteins and κ-casein formed mixed aggregates, but a large proportion of the sodium caseinate did not aggregate. At a given WPI concentration the size of the aggregates decreased with increasing κ-casein or sodium caseinate concentration, but the overall self-similar structure of the aggregates was the same. The presence of κ-casein or caseinate therefore inhibited growth of the heat-induced whey protein aggregates. The results were discussed relative to the reported chaperone-like activity of casein molecules towards heat aggregation of globular proteins.     

EFFECT OF ENZYMATIC HYDROLYSIS ON SOME FUNCTIONAL PROPERTIES OF WHEY PROTEIN

A study was undertaken to further elucidate the functional properties of whey protein with respect to foaming and emulsifying capacities and to observe the effect of enzymatic hydrolysis on these properties. Emulsion capacity decreased as proteolysis continued suggesting there is an optimum mean molecular size of the proteins involved which is lower than that of casein. Heat treatment of the reconstituted protein concentrate was necessary for foam stability; specific volume and foam stability increased directly with temperature of heating. Re effect of pH on whippability, data indicate that the greater the net charge the greater the tendency to foam. A limited amount of hydrolysis appears desirable to increase foaming but greatly decreases foam stability.     

Degradation of milk proteins by extracellular proteinase from brevibacterium linens flk‐61

The objective of this study was to investigate the specificity of an extracellular proteinase from Brevibacterium linens FLK‐61 on the degradation of milk proteins (α_s1, ß‐casein, sodium caseinate and whey proteins). Each protein and the enzyme solution were incubated at 45° C for “0”;, 15, and 30 min and 1, 2, 3, 4, and 5 h. After that, hydrolysis of the proteins was evaluated by urea‐PAGE electrophoresis and densitometry. It was found that the enzymatic degradation of α_st casein occured much faster and it was more complete (P<0.01) than degradation of β‐casein. No degradation of whey proteins was found during 5 h incubation.