Role of the soluble and micelle-bound heat-induced protein aggregates on network formation in acid skim milk gels

Gel formation was monitored by low amplitude rheometry during acidification at 40 degrees C with 1.5% glucono-delta-lactone in combined milk systems containing soluble and/or micelle-bound heat-induced (95 degrees C/10 min) aggregates of denatured whey proteins and kappa-casein and in heated dairy mixes with varying micellar casein/whey protein ratio (CN/WP). Both soluble and micelle-bound aggregates increased gelation pH and gel strength. Micelle-bound aggregates seemed to modify the micelle surface so that micelles were destabilized at a pH of 5.1 (instead of 4.7), while soluble aggregates precipitated at their calculated pI of approximately 5.3, and initiated an early gelation by interacting with the micelles. Decreasing the CN/WP ratio produced larger aggregates with higher whey protein: kappa-casein ratio, which gave more elastic gels. The specific effects of the micellar and soluble aggregates on gel strength are discussed with respect to their relative proportions in the heated milk.     

Formation of soluble and micelle-bound protein aggregates in heated milk

The formation of heat-induced aggregates of kappa-casein and denatured whey proteins was investigated in milk-based dairy mixtures containing casein micelles and serum proteins in different ratios. Both soluble and micelle-bound aggregates were isolated from the mixtures heated at 95 degrees C for 10 min, using size exclusion chromatography. Quantitative analysis of the protein composition of the aggregates by reverse phase high-performance liquid chromatography strongly suggested that primary aggregates of beta-lactoglobulin and alpha-lactalbumin in a 3 to 1 ratio were involved as well as kappa-casein, and alpha(s2)-casein in micellar aggregates. The results gave evidence that heat-induced dissociation of micellar kappa-casein was implicated in the formation of the soluble aggregates and indicated that a significant amount of kappa-casein was left unreacted after heating. The average size of the aggregates was 3.5-5.5 million Da, depending on the available kappa-casein or the casein:whey protein ratio in the mixtures. The size and density of these aggregates relative to those of casein micelles were discussed.     

Role of kappa-casein in the association of denatured whey proteins with casein micelles in heated reconstituted skim milk

Reconstituted skim milk at pH from 6.5 to 7.1 was unheated, preheated (68 degrees C/20 min), or heated at 90 degrees C for 20-30 min. On preheating, the size of the casein micelles decreased by about 5-20 nm, with a greater effect at higher pH. The casein micelle size of the heated milk at pH 6.5 increased by about 30 nm when compared to that of the unheated or preheated milk. As the pH was increased before heating, the particle size gradually decreased so that, at pH 7.1, the size was markedly smaller than that for the unheated milk and slightly smaller than that for the preheated milk. High levels (about 85%) of denatured whey protein associated with the casein micelles at pH 6.5, and this level decreased as the pH increased so that, at pH 7.1, low levels (about 15%) were associated with the micelles. Low levels of alphaS-casein and beta-casein were found in the serum regardless of the heat treatment or the pH of the milk. At pH 6.5, low levels (about 10%) of kappa-casein were also found in the milk serum. In the unheated milk, the level of serum kappa-casein increased slightly with increasing pH; in the heated samples, the level of serum kappa-casein increased markedly and linearly with increasing pH so that, at pH 7.1, about 70% of the kappa-casein was in the serum phase. The results of this study indicate that the pH dependence of the levels of serum phase kappa-casein may be responsible for the change in distribution of the whey proteins between the colloidal and serum phases. This is the first report to demonstrate significant levels of dissociation of kappa-casein from the micelles at pH between 6.5 and 6.7, although this dissociation phenomenon is well known on heating milk at high temperatures at pH above 6.7.     

Limited enzymatic treatment of skim milk using chymosin affects the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates

The effects of heat treatment and limited kappa-casein hydrolysis on the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates were investigated as a possible explanation for the gelation properties of combined rennet and acid gels. Reconstituted skim milk was submitted to combinations of 0-67% hydrolysis of the kappa-casein at 5 degrees C and heat treatment at 90 degrees C for 10 min. The protein composition of the ultracentrifugal fractions was obtained by reverse-phase high-performance liquid chromatography (RP-HPLC). The aggregates contained in each phase were isolated by size-exclusion chromatography and analyzed by RP-HPLC and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Upon heating only, 20-30% of the total kappa-casein dissociated, while 20-30% of the total whey protein attached to the micelles. When heated milk was renneted, little changes were observed in the distribution and composition of the aggregates. Conversely, the heat treatment of partially renneted milk induced the formation of essentially micelle-bound aggregates. The results were discussed in terms of the preferred interaction between hydrophobic para-kappa-casein and denatured whey proteins.     

Effects of added sodium caseinate on the formation of particles in heated milk

The effects of heat at temperatures in the range of 80-90 degrees C on mixtures of reconstituted skim milk powder (RSMP) and sodium caseinate have been determined. In the absence of caseinate, the action of heat on RSMP produces soluble complexes of whey proteins and kappa-casein, as well as complexes of whey protein with the casein micelles. When sodium caseinate was added to RSMP at levels of 0.5 and 1.0%, the denaturation of the whey protein and the production of the soluble complexes in the serum were hardly affected, either in rate or in amount. However, during the heating, the caseinate disappeared from the serum. Further studies on model mixtures of the different components showed that it was probable that the bulk of the caseinate associated with the casein micelles during heating, probably by binding inside the surface layer of kappa-casein, because no increase in the diameters of the casein micelles could be observed.     

Interaction between casein micelles and whey protein/κ-casein complexes during renneting of heat-treated reconstituted skim milk powder and casein micelle/serum mixtures

Casein micelles were separated from unheated reconstituted skim milk powder (RSMP) and were resuspended in the serum of RSMP that had been heated, with and without dialysis of this serum against unheated RSMP. Using size-exclusion chromatography, it was found that the soluble complexes of whey protein (WP) with κ-casein in the serum of the heated milk bind progressively to unheated casein micelles during renneting, even prior to the onset of clotting. Similar trends were noted when casein micelles from RSMP heated at pH values of 6.7, 7.1, or 6.3, each with different amounts of WP coating the micelles, were renneted in the presence of soluble WP/κ-casein complexes. No matter what was the initial load of micelle-bound WP complexes, all micelle types were capable of binding additional serum protein complexes during renneting. However, it is not clear that this binding of WP/κ-casein complexes to the micellar surface is a direct cause of the impaired rennet clotting of the RSMP.     

Rheological properties of acid gels prepared from heated pH-adjusted skim milk

Reconstituted skim milk was adjusted to pH values between 6.5 and 7.1 and heated (90 degrees C) for up to 30 min. The skim milk samples were then readjusted to pH 6.7. Acid gels prepared from heated milk had markedly higher G ' values, a reduced gelation time, and an increased gelation pH than those prepared from unheated milk. An increased pH at heating decreased the gelation time, increased the gelation pH, and increased the final G ' of acid set gels prepared from the heated milk samples. There were only small differences in the level of whey protein denaturation in the samples at different pH values, and these differences could not account for the differences in the G ' of the acid gels. The levels of denatured whey protein associated with the casein micelles decreased and the levels of soluble denatured whey proteins increased as the pH at heating was increased. The results indicated that the soluble denatured whey proteins had a greater effect on the final G ' of the acid gels than the denatured whey proteins associated with the casein micelles.     

Acid gelation properties of heated skim milk as a result of enzymatically induced changes in the micelle/serum distribution of the whey protein/kappa-casein aggregates

Changes in the acid gelation properties of skim milk as a result of variations in the micelle/serum distribution of the heat-induced whey protein/kappa-casein aggregates, induced by the combination of heat treatment and limited renneting, were investigated. No dramatic change in the zeta potential or the isoelectric point of the casein micelles was suggested, whether the aggregates were all attached to the casein micelle or not. Fluorescence intensity measurement using 8-anilino-1-naphthalenesulfonic acid (ANS) showed that the heat-induced aggregates were highly hydrophobic. Dynamic oscillation viscosimetry showed that acid gelation using glucono-delta-lactone (GDL) started at a higher pH value in prerenneted milk. However, no change in the gelation profile of skim milk could be related to the proportion of aggregates bound to the surface of the casein micelles. The results support the idea of an early interaction between the serum aggregates and the casein micelles on acidification.     

Casein proteins as molecular chaperones

Under conditions of stress, such as elevated temperature, molecular chaperones stabilize proteins from unfolding, aggregating, and precipitating. We have investigated the chaperone activity of the major milk proteins alpha(S)-, beta-, and kappa-casein with reduced insulin and the milk whey proteins, alpha-lactalbumin and beta-lactoglobulin, and compared it with that of the mammalian small heat shock protein (sHsp), alpha-crystallin, and clusterin. alpha(S)-Casein exhibited different chaperone behavior under reduction and heat stresses, i.e., chaperone activity increased with increasing temperature (as observed with alpha-crystallin), but under reduction stress, its chaperone activity increased at lower temperatures. beta- and kappa-casein had comparable chaperone ability with each other but were less effective than alpha(S)-casein. Under molecular crowding conditions, precipitation of stressed protein was accelerated, and alpha(S)-casein was a poorer chaperone. Furthermore, at slightly alkaline pH values, alpha(S)-casein was a less effective chaperone than at neutral pH. Detailed fluorescence, size exclusion chromatography, and real-time NMR studies studies indicated that the casein proteins underwent conformational changes and stabilized the partially unfolded whey proteins prior to formation of high molecular weight soluble complexes. These results are consistent with casein proteins acting as molecular chaperones in a manner similar to sHsps and clusterin.     

Glucose slows down the heat-induced aggregation of β-lactoglobulin at neutral pH

The behavior of β-lactoglobulin (β-Lg) during heat treatments depends on the environmental conditions. The influence of the presence or absence of a reducing sugar, namely, glucose, on the modification of the protein during heating has been studied using fluorescence, polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC), and transmission electron microscopy. Glycated products were formed during heating 24 h at 90 °C and pH 7. The fluorescence results revealed an accumulation of the advanced Maillard products and the formation of aggregates during heating. PAGE and SEC data suggested that the products in the control samples were essentially composed of covalently linked fibrillar aggregates and that their formation was faster than that for glycated samples. We showed that glucose affected the growing step of covalent aggregates but not the initial denaturation/aggregation step of native protein. Glucose-modified proteins formed a mixture of short fibrils and polydisperse aggregates. Our results revealed that β-Lg forms fibrils at neutral pH after heating and that glucose slows the formation of these fibrils.     

Controlled proteolysis and the properties of milk gels

Milk gels induced by partial proteolysis of the kappa-casein followed by acidification were studied, and their gelation behavior was compared to that of milk gels induced by simultaneous acidification and renneting, using dynamic rheology. There were generally two stages (at pH values below and above 5.0) in the gelation of the milk whose kappa-casein had been partially proteolyzed and acidified. The onset of gelation was at higher pH as the degree of kappa-casein proteolysis increased. The development of G' immediately after the onset of gelation was faster in the milk gels induced by simultaneous acidification and renneting, because of the continuing kappa-casein proteolysis. Preheat treatment caused the onset of gelation to occur at higher pH than for unheated milk. However, the maximum tan delta during gelation always occurred at the same pH (for a given concentration of acidulant), and its value and position were independent of the extent of renneting and whether the milks had been heat treated. The results are discussed in terms of the interactions between casein micelles occurring during gelation.     

Complexation of whey proteins with carrageenan

The formation of electrostatic complexes of whey protein (WP) and a nongelling carrageenan (CG) was investigated as a function of pH, ionic strength, temperature, and protein-to-polysaccharide (Pr:Ps) ratio. On lowering the pH, the formation of soluble WP/CG complexes was initiated at pH(c) and insoluble complexes at pH(phi), below which precipitation occurred. The values of the transition pH varied as a function of the ionic strength. It was shown that at [NaCl] = 45 mM, the value of pH(phi) was the highest, showing that the presence of monovalent ions was favorable to the formation of complexes by screening the residual negative charges of the CG. When CaCl(2) was added to the mixtures, complexes of WP/CG were formed up to pH 8 via calcium bridging. The electrostatic nature of the primary interaction was confirmed from the slight effect of temperature on the pH(phi). Increasing the Pr:Ps ratio led to an increase of the pH(phi) until a ratio of 30:1 (w/w), at which saturation of the CG chain seemed to be reached. The behavior of WP/CG complexes was investigated at a low Pr:Ps ratio, when the biopolymers were mixed directly at low pH. It resulted in an increase of the pH of the mixture, as compared to the initial pH of the separate WP and CG solutions. The pH increase was accompanied by a decrease in conductivity. The trapping of protons inside the complex probably resulted from a residual negative charge on the CG. If NaCl was present in the mixture, the complex took up the Na(+) ions instead of the H(+) ions.     

Effect of pH on the association of denatured whey proteins with casein micelles in heated reconstituted skim milk

Skim milk was adjusted to pH values between 6.5 and 6.7 and heated (80, 90, and 100 degrees C) for up to 60 min. Changes in casein micelle size, level of whey protein denaturation, and level of whey protein association with the micelles were monitored for each milk sample. Changes in casein micelle size were markedly affected by the pH at heating. At low pH (6.5-6.55), the casein micelle size increased markedly during the early stages of heating, and the size plateaued on prolonged heating. The maximum increase in size was approximately 30-35 nm. In contrast, at high pH (6.7), much smaller changes in size were observed on heating and the maximum increase in size was only approximately 10 nm. An intermediate behavior was observed at pH values between these two extremes. The rate of denaturation of the major whey proteins, alpha-lactalbumin and beta-lactoglobulin, was essentially unaffected by the pH at heating for the small pH changes involved in this study, and the changes in casein micelle size were poorly related to the level of whey protein denaturation. In contrast, the level of denatured whey proteins associating with the micelles was markedly dependent on the pH at heating, with high levels of association at pH 6.5-6.55 and low levels of association at pH 6.7. Changes in casein micelle size were related to the levels of denatured whey proteins that were associated with the casein micelles, although there was a small deviation from linearity at low levels of association (<15%). Further studies on reconstituted and fresh milk samples at smaller pH steps confirmed that the association of whey proteins with the casein micelles was markedly affected by the pH at heating. These results indicate that the changes in casein micelle size induced by the heat treatment of skim milk were a consequence of the whey proteins associating with the casein micelles and that the level of association was markedly influenced by small pH changes of the milk. It was not possible to determine whether the association itself influenced the casein micelle size or whether parallel reactions involving micellar aggregation caused the increase in micelle size as whey protein association progressed.     

Heat-induced aggregation of whey proteins: comparison of cheese WPC with acid WPC and relevance of mineral composition

Heat-induced aggregation of whey proteins in solutions made from two commercial whey protein concentrates (WPCs), one derived from mineral acid whey (acid WPC) and the other from cheese whey (cheese WPC), was studied using polyacrylamide gel electrophoresis (PAGE), size exclusion chromatography (SEC), and transmission electron microscopy (TEM). Heat treatment (75 degrees C) of acid WPC solutions (12.0%, w/w, pH 6.9) resulted in formation of relatively small "soluble" aggregates that were predominantly disulfide-linked. By contrast, heat treatment of the cheese WPC solutions (under the same conditions) caused formation of relatively large aggregates, containing high proportions of aggregates linked by noncovalent associations. The rate of aggregation of both beta-lactoglobulin and alpha-lactalbumin at 75 degrees C, measured as the loss of native proteins by PAGE, was higher in the cheese WPC solution than in the acid WPC solution. Cross dialysis of the two WPC solutions resulted in alteration of the mineral composition of each WPC solution and reversing their heat-induced aggregation behavior. The results demonstrated that the mineral composition is very important in controlling the aggregation behavior of WPC products.     

Behavior of protein in the presence of calcium during heating of whey protein concentrate solutions

The effect of added CaCl(2) on heat-induced changes in whey protein (WP) solutions prepared from whey protein isolate (WP1), acid whey protein concentrate (WP2), and cheese whey protein concentrate (WP3) was investigated. The loss of native-like, proteins, aggregation, and gel firmness of WP were maximum at certain levels of added CaCl(2). These levels were different for different WP products. The effect of added CaCl(2) on these changes appeared to be related to the initial calcium concentrations of these solutions. The higher the calcium content of the product, the less available sites for added CaCl(2) to bind. It was considered that addition of CaCl(2) changed the types of protein interactions that formed the protein aggregates during heating. Added calcium caused dramatic decreases in fracture stress of WP gels due to the formation of large protein aggregates.     

糖接枝处理改善大豆蛋白纤维聚集体泡沫稳定性

为了探究糖接枝对大豆蛋白纤维聚集行为和泡沫性质的影响,明确蛋白质结构与功能的关系,该研究以大豆蛋白(soy protein isolation,SPI)和乳糖(lactose)为原料,通过干热法制备糖接枝大豆蛋白(SPI-lactose conjugate,SPI-Lac),以及在酸性条件下加热诱导其形成纤维聚集体(p H值2.0),制备了一种糖接枝大豆蛋白纤维聚集体(SPI-lactose conjugate fibillar aggregates),并考察了糖接枝对大豆蛋白的纤维聚集行为及泡沫性质的影响。研究结果表明:大豆蛋白在酸性条件下(p H值2.0)经加热后会发生水解,同时水解产物不断聚集形成大分子的纤维聚集体。糖接枝导致大豆蛋白的水解速度下降,但荧光光强和粒径的结果表明糖接枝能增强纤维聚集能力。SPI-Lac在中性条件下的溶解度(p H值5.0—7.0)显著高于SPI(P0.05),且不同时间处理的SPI-Lac纤维聚集体均能改善SPI在酸性条件下的溶解度(p H值2.0—5.0)。此外,不同时间处理的SPI-Lac纤维聚集体在酸性条件下的起泡能力均高于SPI纤维聚集体。SPI和SPI-Lac纤维聚集体的形成会导致SPI起泡能力的下降,但是短时间酸热处理形成的纤维聚集体泡沫稳定性得到显著改善。因此,糖接枝结合短时间酸热处理制备的糖接枝大豆蛋白纤维聚集体在中性条件下的泡沫稳定性显著提高(P0.05),是合理有效的蛋白质改性方法。     

适宜含水率保持油茶籽贮藏品质

为了确定油茶籽贮藏适宜的含水率,研究了在4℃,不同含水率(7%、10%、13%、16%、20%)油茶籽贮藏期间的品质变化。结果表明,较低的含水率能较好保持油茶籽的贮藏特性及营养品质。其中,含水率为7%的油茶籽贮藏效果较好,但与10%处理效果差异不明显(P>0.05)。在整个贮藏期,含水率为7%时油茶籽可溶性蛋白下降了13.05 mg/g,油酸含量下降了2.38%,酸值、过氧化值等品质指标上升速率较慢,同时能较好保持β-谷甾醇和角鲨烯等生物活性成分;其次是10%的含水率处理。而含水率为20%的油茶籽贮藏期间可溶性蛋白下降较快,贮藏结束时为25.47 mg/g,油茶籽劣变严重,所提取的油样品质变差,营养物质含量较少,因此含水率20%的油茶籽不适宜长期贮藏。综合考虑油茶籽品质因素和处理成本,认为控制含水率在10%以下能较好保持油茶籽的贮藏品质。该研究可为科学合理地贮藏油茶籽提供参考。     

Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 degrees C). The pasteurizing heat treatment (72 degrees C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 degrees C for 120 s, 120 degrees C for 120 s, and 140 degrees C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and kappa-casein (kappa-CN) and a proportion of the alpha(s2)-CN. Pressure treatment of milk at 200 MPa caused beta-lactoglobulin (beta-LG) to form disulfide-bonded dimers and incorporated beta-LG into aggregates, probably disulfide-bonded to kappa-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated beta-LG and most of the minor whey proteins, as well as kappa-CN and much of the alpha(s2)-CN, into aggregates. The accessibility of alpha(s2)-CN and formation of complexes involving alpha(s2)-CN, kappa-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the alpha-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities.     

Study of thermal aggregation of oat globulin by laser light scattering

The heat-induced aggregation of oat globulin was studied using size-exclusion chromatography (SEC) combined with on-line multiangle laser light scattering (MALLS) and quasi-elastic light scattering (QELS). The unheated oat globulin exists as a mixture of hexamer (>95%), trimer, and dimer forms of hexamer. The molecular weight of the hexamer was estimated by MALLS to be 330 000, close to that deduced from the genomic cloned data of the acidic and basic polypeptides of oat globulin. From QELS measurements, it can be predicted that the hexamer exists as two annular trimeric rings, with a diameter of 11.8 nm, placed on top of each other, forming an oblate cylinder with a height of about 8.5 nm. Upon heating at 100 degrees C, the oat globulin hexamers and trimers were dissociated into monomers. The heat-denatured monomers, probably assuming an extended structure, were associated to form small aggregates, which were further aggregated to high molecular weight complexes. Upon further heating (60 min), the soluble aggregates were associated to form insoluble aggregates. Aggregation of oat globulin occurred at a much faster rate at 110 degrees C. The results indicate that the SEC-MALLS-QELS system is suitable for studying thermal aggregation of food proteins.